Distribution and transport of sediment-bound metal contaminants in the rio grande de tarcoles, costa rica (Central America)

USGS Publications Warehouse

Fuller, C.C.; Davis, J.A.; Cain, D.J.; Lamothe, P.J.; Fries Fernandez, T.L.G.; Vargas, J.A.; Murillo, M.M.

1990-01-01

A reconnaissance survey of the extent of metal contamination in the Rio Grande de Tarcoles river system of Costa Rica indicated high levels of chromium (Cr) in the fine-grain bed sediments (83 times Cr background or 3000->5000 ??g/g). In the main channel of the river downstream of the San Jose urban area, Cr contamination in sediments was 4-6 times background and remained relatively constant over 50 km to the mouth of the river. Sediment from a mangrove swamp at the river mouth had Cr levels 2-3 times above background. Similar patterns of dilution were observed for lead (Pb) and zinc (Zn) sediment contamination, although the contamination levels were lower. The high affinity of Cr towards particulate phases, probably as Cr(III), allows the use of Cr contamination levels for delineating regions of deposition of fine-grained sediments and dilution of particle associated contaminants during transport and deposition.A reconnaissance survey of the extent of metal contamination in the Rio Grande de Tarcoles river system of Costa Rica indicated high levels of chromium (Cr) in the fine-grain bed sediments (83 times Cr background or 3000->5000 ??g/g). In the main channel of the river downstream of the San Jose urban area, Cr contamination in sediments was 4-6 times background and remained relatively constant over 50 km to the mouth of the river. Sediments from a mangrove swamp at the river mouth had Cr levels 2-3 times above background. Similar patterns of dilution were observed for lead (Pb) and zinc (Zn) sediment contamination, although the contamination levels were lower. The high affinity of Cr towards particulate phases, probably as Cr(III), allows the use of Cr contamination levels for delineating regions of deposition of fine-grained sediments and dilution of particle associated contaminants during transport and deposition.

Characterization of two ammonia-oxidizing bacteria isolated from reactors operated with low dissolved oxygen concentrations.

PubMed

Park, H-D; Noguera, D R

2007-05-01

To obtain ammonia-oxidizing bacterial (AOB) strains inhabiting low dissolved oxygen (DO) environments and to characterize them to better understand their function and ecology. Using a serial dilution method, two AOB strains (ML1 and NL7) were isolated from chemostat reactors operated with low DO concentrations (0.12-0.24 mg l(-1)). Phylogenetically, strains ML1 and NL7 are affiliated to AOB within the Nitrosomonas europaea and Nitrosomonas oligotropha lineages, respectively. Kinetically, strain ML1 had high affinity for oxygen (0.24 +/- 0.13 mg l(-1)) and low affinity for ammonia (1.62 +/- 0.97 mg N l(-1)), while strain NL7 had high affinity for ammonia (0.48 +/- 0.35 mg l(-1)), but a surprisingly low affinity for oxygen (1.22 +/- 0.43 mg l(-1)). A co-culture experiment was used to iteratively estimate decay constants for both strains. The results indicated that AOB without high affinity for oxygen may have other mechanisms to persist in low DO environments, with high affinity for ammonia being important. This study provides a method to determine AOB growth kinetic parameters without assuming or neglecting decay constant. And, this is the first report on oxygen affinity constant of a N. oligotropha strain.

The role of CYP2D6 in primary and secondary oxidative metabolism of dextromethorphan: in vitro studies using human liver microsomes.

PubMed Central

Kerry, N L; Somogyi, A A; Bochner, F; Mikus, G

1994-01-01

1. The enzyme kinetics of dextromethorphan O-demethylation in liver microsomes from three extensive metabolisers (EM) with respect to CYP2D6 indicated high (Km1 2.2-9.4 microM) and low (Km2 55.5-307.3 microM) affinity sites whereas microsomes from two poor metabolisers (PM) indicated a single site (Km 560 and 157 microM). Similar differences were shown for 3-methoxymorphinan O-demethylation to 3-hydroxymorphinan (Km 6.9-9.6 microM in EM subjects; Km 307 and 213 microM in PM subjects). 2. Dextromethorphan O-demethylation was inhibited competitively by quinidine (Ki 0.1 microM), rac-perhexiline (Ki 0.4 microM), dextropropoxyphene (Ki 6 microM), rac-methadone (Ki 8 microM), and 3-methoxymorphinan (Ki 15 microM). These compounds were also potent inhibitors of 3-methoxymorphinan O-demethylation with IC50 values ranging from 0.02-12 microM. Anti-LKM1 serum inhibited both dextromethorphan and 3-methoxymorphinan O-demethylations in a titre-dependent manner. 3. The Michaelis-Menten constant for dextromethorphan N-demethylation to 3-methoxymorphinan (Km 632-977 microM) and dextrorphan N-demethylation to 3-hydroxymorphinan (Km 1571-4286 microM) did not differ between EM and PM microsomes. These N-demethylation reactions were not inhibited by quinidine and rac-methadone or LKM1 antibodies. 4. Dextromethorphan and 3-methoxymorphinan are metabolised by the same P450 isoform, CYP2D6, whereas the N-demethylation reactions are not carried out by CYP2D6. PMID:7826826

Magnetic mesoporous silica nanoparticles: fabrication and their laccase immobilization performance.

PubMed

Wang, Feng; Guo, Chen; Yang, Liang-rong; Liu, Chun-Zhao

2010-12-01

Newly large-pore magnetic mesoporous silica nanoparticles (MMSNPs) with wormhole framework structures were synthesized for the first time by using tetraethyl orthosilicate as the silica source and amine-terminated Jeffamine surfactants as template. Iminodiacerate was attached on these MMSNPs through a silane-coupling agent and chelated with Cu(2+). The Cu(2+)-chelated MMSNPs (MMSNPs-CPTS-IDA-Cu(2+)) showed higher adsorption capacity of 98.1 mg g(-1)-particles and activity recovery of 92.5% for laccase via metal affinity adsorption in comparison with MMSNPs via physical adsorption. The Michaelis constant (K(m)) and catalytic constant (k(cat)) of laccase immobilized on the MMSNPs-CPTS-IDA-Cu(2+) were 3.28 mM and 155.4 min(-1), respectively. Storage stability and temperature endurance of the immobilized laccase on MMSNPs-CPTS-IDA-Cu(2+) increased significantly, and the immobilized laccase retained 86.6% of its initial activity after 10 successive batch reactions operated with magnetic separation. 2010 Elsevier Ltd. All rights reserved.

A dye-binding assay for measurement of the binding of Cu(II) to proteins.

PubMed

Wilkinson-White, Lorna E; Easterbrook-Smith, Simon B

2008-10-01

We analysed the theory of the coupled equilibria between a metal ion, a metal ion-binding dye and a metal ion-binding protein in order to develop a procedure for estimating the apparent affinity constant of a metal ion:protein complex. This can be done by analysing from measurements of the change in the concentration of the metal ion:dye complex with variation in the concentration of either the metal ion or the protein. Using experimentally determined values for the affinity constant of Cu(II) for the dye, 2-(5-bromo-2-pyridylaxo)-5-(N-propyl-N-sulfopropylamino) aniline (5-Br-PSAA), this procedure was used to estimate the apparent affinity constants for formation of Cu(II):transthyretin, yielding values which were in agreement with literature values. An apparent affinity constant for Cu(II) binding to alpha-synuclein of approximately 1 x 10(9)M(-1) was obtained from measurements of tyrosine fluorescence quenching by Cu(II). This value was in good agreement with that obtained using 5-Br-PSAA. Our analysis and data therefore show that measurement of changes in the equilibria between Cu(II) and 5-Br-PSAA by Cu(II)-binding proteins provides a general procedure for estimating the affinities of proteins for Cu(II).

Quantitative Comparison of Human Parainfluenza Virus Hemagglutinin-Neuraminidase Receptor Binding and Receptor Cleavage

PubMed Central

Tappert, Mary M.; Porterfield, J. Zachary; Mehta-D'Souza, Padmaja; Gulati, Shelly

2013-01-01

The human parainfluenza virus (hPIV) hemagglutinin-neuraminidase (HN) protein binds (H) oligosaccharide receptors that contain N-acetylneuraminic acid (Neu5Ac) and cleaves (N) Neu5Ac from these oligosaccharides. In order to determine if one of HN′s two functions is predominant, we measured the affinity of H for its ligands by a solid-phase binding assay with two glycoprotein substrates and by surface plasmon resonance with three monovalent glycans. We compared the dissociation constant (Kd) values from these experiments with previously determined Michaelis-Menten constants (Kms) for the enzyme activity. We found that glycoprotein substrates and monovalent glycans containing Neu5Acα2-3Galβ1-4GlcNAc bind HN with Kd values in the 10 to 100 μM range. Km values for HN were previously determined to be on the order of 1 mM (M. M. Tappert, D. F. Smith, and G. M. Air, J. Virol. 85:12146–12159, 2011). A Km value greater than the Kd value indicates that cleavage occurs faster than the dissociation of binding and will dominate under N-permissive conditions. We propose, therefore, that HN is a neuraminidase that can hold its substrate long enough to act as a binding protein. The N activity can therefore regulate binding by reducing virus-receptor interactions when the concentration of receptor is high. PMID:23740997

Real-time and label-free analysis of binding thermodynamics of carbohydrate-protein interactions on unfixed cancer cell surfaces using a QCM biosensor

PubMed Central

Li, Xueming; Song, Siyu; Shuai, Qi; Pei, Yihan; Aastrup, Teodor; Pei, Yuxin; Pei, Zhichao

2015-01-01

A novel approach to the study of binding thermodynamics and kinetics of carbohydrate-protein interactions on unfixed cancer cell surfaces using a quartz crystal microbalance (QCM) biosensor was developed, in which binding events take place at the cell surface, more closely mimicking a biologically relevant environment. In this study, colon adenocarcinoma cells (KM-12) and ovary adenocarcinoma cells (SKOV-3) grew on the optimized polystyrene-coated biosensor chip without fixation. The association and dissociation between the cell surface carbohydrates and a range of lectins, including WGA, Con A, UEA-I, GS-II, PNA and SBA, were monitored in real time and without label for evaluation of cell surface glycosylation. Furthermore, the thermodynamic and kinetic parameters of the interaction between lectins and cell surface glycan were studied, providing detailed information about the interactions, such as the association rate constant, dissociation rate constant, affinity constant, as well as the changes of entropy, enthalpy and Gibbs free energy. This application provides an insight into the cell surface glycosylation and the complex molecular recognition on the intact cell surface, which may have impacts on disease diagnosis and drug discovery. PMID:26369583

Binding affinities of anti-acetylcholine receptor autoantibodies in myasthenia gravis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bray, J.J.; Drachman, D.B.

1982-01-01

Antibodies directed against acetylcholine (ACh) receptors are present in the sera of nearly 90% of patients with myasthenia gravis (MG), and are involved in the pathogenesis of this autoimmune disease. However, the antibody titers measured by the standard radioimmunoassay correspond poorly with the clinical severity of the disease. To determine whether this disparity could be accounted for by differences in the binding affinities of anti-ACh receptor antibodies in different patients, we have measured the binding affinities of these autoantibodies in 15 sera from MG patients. The affinity constants (K/sub o/), as determined by Scatchard analysis, were all in the rangemore » of 10/sup 10/ M/sup -1/, comparable to the highest values reported in immunized animals. The affinity constants were truly representative of the population of autoantibodies detected by the radioimmunoassay, as shown by the remarkable linearity of the Scatchard plots (r/sup 2/>0.90) and the close correlation between the antibody titers determined by extrapolation of the Scatchard plots and by saturation analysis (r = 0.99; p < 0.001). There was only a 6-fold variation in affinity constants measured in this series of patients despite widely differing antibody titers and severity of the disease. Factors other than the titer and affinity of anti-ACh receptor antibodies may correlate better with the clinical manifestations of MG.« less

Synthesis, biological evaluation, and docking studies of gigantol analogs as calmodulin inhibitors.

PubMed

Reyes-Ramírez, Adelfo; Leyte-Lugo, Martha; Figueroa, Mario; Serrano-Alba, Trinidad; González-Andrade, Martín; Mata, Rachel

2011-07-01

Several analogs of gigantol (1) were synthesized to evaluate their effect on the complexes Ca(2+)-calmodulin (CaM) and Ca(2+)-CaM-CaM sensitive phosphodiesterase 1 (PDE1). The compounds belong to four structural groups including, 1,2-diphenylethanes (2-11), diphenylmethanes (13-15), 1,3-diphenylpropenones (16-18), and 1,3-diphenylpropanes (20-22). In vitro enzymatic studies showed that all compounds except 11 inhibited the complex Ca(2+)-CaM-PDE1 with IC(50) values ranging from 9 to 146 μM. On the other hand, all analogs but 11, 12 and 15 quenched the extrinsic fluorescence of the CaM biosensor hCaM-M124C-mBBr to different extent, then revealing different affinities to CaM; their affinity constants (K(m)) values were in the range of 3-80 μM. Molecular modeling studies indicated that all these compounds bound to CaM at the same site that the classical inhibitors trifluoperazine (TFP) and chlorpromazine (CPZ). Some of these analogs could be worthy candidates for developing new anti-tumor, local anesthetics, antidepressants, antipsychotic, or smooth muscle relaxant drugs, with anti-CaM properties due to their good affinity to CaM and the straightforwardness of their synthesis. In addition they could be valuable tools for the study of Ca(2+)-CaM functions. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

A Chlorogenic Acid Esterase with a Unique Substrate Specificity from Ustilago maydis

PubMed Central

Haase-Aschoff, Paul; Kelle, Sebastian; Linke, Diana; Krings, Ulrich; Popper, Lutz; Berger, Ralf G.

2014-01-01

An extracellular chlorogenic acid esterase from Ustilago maydis (UmChlE) was purified to homogeneity by using three separation steps, including anion-exchange chromatography on a Q Sepharose FF column, preparative isoelectric focusing (IEF), and, finally, a combination of affinity chromatography and hydrophobic interaction chromatography on polyamide. SDS-PAGE analysis suggested a monomeric protein of ∼71 kDa. The purified enzyme showed maximal activity at pH 7.5 and at 37°C and was active over a wide pH range (3.5 to 9.5). Previously described chlorogenic acid esterases exhibited a comparable affinity for chlorogenic acid, but the enzyme from Ustilago was also active on typical feruloyl esterase substrates. Kinetic constants for chlorogenic acid, methyl p-coumarate, methyl caffeate, and methyl ferulate were as follows: Km values of 19.6 μM, 64.1 μM, 72.5 μM, and 101.8 μM, respectively, and kcat/Km values of 25.83 mM−1 s−1, 7.63 mM−1 s−1, 3.83 mM−1 s−1 and 3.75 mM−1 s−1, respectively. UmChlE released ferulic, p-coumaric, and caffeic acids from natural substrates such as destarched wheat bran (DSWB) and coffee pulp (CP), confirming activity on complex plant biomass. The full-length gene encoding UmChlE consisted of 1,758 bp, corresponding to a protein of 585 amino acids, and was functionally produced in Pichia pastoris GS115. Sequence alignments with annotated chlorogenic acid and feruloyl esterases underlined the uniqueness of this enzyme. PMID:25548041

Integration of biotic ligand models (BLM) and bioaccumulation kinetics into a mechanistic framework for metal uptake in aquatic organisms.

PubMed

Veltman, Karin; Huijbregts, Mark A J; Hendriks, A Jan

2010-07-01

Both biotic ligand models (BLM) and bioaccumulation models aim to quantify metal exposure based on mechanistic knowledge, but key factors included in the description of metal uptake differ between the two approaches. Here, we present a quantitative comparison of both approaches and show that BLM and bioaccumulation kinetics can be merged into a common mechanistic framework for metal uptake in aquatic organisms. Our results show that metal-specific absorption efficiencies calculated from BLM-parameters for freshwater fish are highly comparable, i.e. within a factor of 2.4 for silver, cadmium, copper, and zinc, to bioaccumulation-absorption efficiencies for predominantly marine fish. Conditional affinity constants are significantly related to the metal-specific covalent index. Additionally, the affinity constants of calcium, cadmium, copper, sodium, and zinc are significantly comparable across aquatic species, including molluscs, daphnids, and fish. This suggests that affinity constants can be estimated from the covalent index, and constants can be extrapolated across species. A new model is proposed that integrates the combined effect of metal chemodynamics, as speciation, competition, and ligand affinity, and species characteristics, as size, on metal uptake by aquatic organisms. An important direction for further research is the quantitative comparison of the proposed model with acute toxicity values for organisms belonging to different size classes.

Characterization of solution-phase drug-protein interactions by ultrafast affinity extraction.

PubMed

Beeram, Sandya R; Zheng, Xiwei; Suh, Kyungah; Hage, David S

2018-03-03

A number of tools based on high-performance affinity separations have been developed for studying drug-protein interactions. An example of one recent approach is ultrafast affinity extraction. This method has been employed to examine the free (or non-bound) fractions of drugs and other solutes in simple or complex samples that contain soluble binding agents. These free fractions have also been used to determine the binding constants and rate constants for the interactions of drugs with these soluble agents. This report describes the general principles of ultrafast affinity extraction and the experimental conditions under which it can be used to characterize such interactions. This method will be illustrated by utilizing data that have been obtained when using this approach to measure the binding and dissociation of various drugs with the serum transport proteins human serum albumin and alpha 1 -acid glycoprotein. A number of practical factors will be discussed that should be considered in the design and optimization of this approach for use with single-column or multi-column systems. Techniques will also be described for analyzing the resulting data for the determination of free fractions, rate constants and binding constants. In addition, the extension of this method to complex samples, such as clinical specimens, will be considered. Copyright © 2018 Elsevier Inc. All rights reserved.

Analysis of the activation of acetylcholinesterase by carbon nanoparticles using a monolithic immobilized enzyme microreactor: role of the water molecules in the active site gorge.

PubMed

Ibrahim, Firas; Andre, Claire; Iutzeler, Anne; Guillaume, Yves Claude

2013-10-01

A biochromatographic system was used to study the direct effect of carbon nanoparticles (CNPs) on the acetylcholinesterase (AChE) activity. The AChE enzyme was covalently immobilized on a monolithic CIM-disk via its NH2 residues. Our results showed an increase in the AChE activity in presence of CNPs. The catalytic constant (k(cat)) was increased while the Michaelis constant (K(m)) was slightly decreased. This indicated an increase in the enzyme efficiency with increase of the substrate affinity to the active site. The thermodynamic data of the activation mechanism of the enzyme, i.e. ΔH* and ΔS*, showed no change in the substrate interaction mechanism with the anionic binding site. The increase of the enthalpy (ΔH*) and the entropy (ΔS*) with decrease in the free energy of activation (Ea) was related to structural conformation change in the active site gorge. This affected the stability of water molecules in the active site gorge and facilitated water displacement by substrate for entering to the active site of the enzyme.

Kinetic study of the effects of calcium ions on cationic artichoke (Cynara scolymus L.) peroxidase: calcium binding, steady-state kinetics and reactions with hydrogen peroxide.

PubMed

Hiner, Alexander N P; Sidrach, Lara; Chazarra, Soledad; Varón, Ramón; Tudela, José; García-Cánovas, Francisco; Rodríguez-López, José Neptuno

2004-01-01

The apparent catalytic constant (k(cat)) of artichoke (Cynara scolymus L.) peroxidase (AKPC) with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) increased 130-fold in the presence of calcium ions (Ca2+) but the affinity (K(m)) of the enzyme for ABTS was 500 times lower than for Ca2+-free AKPC. AKPC is known to exhibit an equilibrium between 6-aquo hexa-coordinate and penta-coordinate forms of the haem iron that is modulated by Ca2+ and affects compound I formation. Measurements of the Ca2+ dissociation constant (K(D)) were complicated by the water-association/dissociation equilibrium yielding a global value more than 1000 times too high. The value for the Ca2+ binding step alone has now been determined to be K(D) approximately 10 nM. AKPC-Ca2+ was more resistant to inactivation by hydrogen peroxide (H(2)O(2)) and exhibited increased catalase activity. An analysis of the complex H(2)O(2) concentration dependent kinetics of Ca2+-free AKPC is presented.

Function of muscle-type lactate dehydrogenase and citrate synthase of the Galápagos marine iguana, Amblyrhynchus cristatus, in relation to temperature.

PubMed

Fields, Peter A; Strothers, Chad M; Mitchell, Mark A

2008-05-01

The Galápagos marine iguana, Amblyrhynchus cristatus, is unique among lizards in foraging subtidally, leading to activity across a broad range of ambient temperatures ( approximately 14-40 degrees C). To determine whether the marine iguana shows any biochemical changes consistent with maintaining enzyme function at both warm and cold body temperatures, we examined the function of the aerobic enzyme citrate synthase (CS) and the muscle isoform of the anaerobic enzyme lactate dehydrogenase (A(4)-LDH) in A. cristatus and a confamilial species, Iguana iguana, from 14 to 46 degrees C. We also deduced amino acid sequences from cDNA of each enzyme. In CS, despite two amino acid substitutions, we found no difference in the apparent Michaelis-Menten constant K(m) of oxaloacetate at any temperature, indicating that the substrate affinity of CS in A. cristatus has not adapted to changes in thermal environment. In A(4)-LDH, we used site-directed mutagenesis to show that the substitutions T9A and I283V (A. cristatus --> I. iguana) individually have no effect on kinetics, but together significantly decrease the K(m) of pyruvate and catalytic rate constant (k(cat)) of the A. cristatus ortholog. Thus, our data show that A. cristatus A(4)-LDH has not become cold adapted in response to this species' aquatic foraging behavior, and instead may be consistent with moderate warm adaptation with respect to the I. iguana ortholog.

Comparative Study of Three Methods for Affinity Measurements: Capillary Electrophoresis Coupled with UV Detection and Mass Spectrometry, and Direct Infusion Mass Spectrometry

NASA Astrophysics Data System (ADS)

Mironov, Gleb G.; Logie, Jennifer; Okhonin, Victor; Renaud, Justin B.; Mayer, Paul M.; Berezovski, Maxim V.

2012-07-01

We present affinity capillary electrophoresis and mass spectrometry (ACE-MS) as a comprehensive separation technique for label-free solution-based affinity analysis. The application of ACE-MS for measuring affinity constants between eight small molecule drugs [ibuprofen, s-flurbiprofen, diclofenac, phenylbutazone, naproxen, folic acid, resveratrol, and 4,4'-(propane-1,3-diyl) dibenzoic acid] and β-cyclodextrin is described. We couple on-line ACE with MS to combine the separation and kinetic capability of ACE together with the molecular weight and structural elucidation of MS in one system. To understand the full potential of ACE-MS, we compare it with two other methods: Direct infusion mass spectrometry (DIMS) and ACE with UV detection (ACE-UV). After the evaluation, DIMS provides less reliable equilibrium dissociation constants than separation-based ACE-UV and ACE-MS, and cannot be used solely for the study of noncovalent interactions. ACE-MS determines apparent dissociation constants for all reacting small molecules in a mixture, even in cases when drugs overlap with each other during separation. The ability of ACE-MS to interact, separate, and rapidly scan through m/z can facilitate the simultaneous affinity analysis of multiple interacting pairs, potentially leading to the high-throughput screening of drug candidates.

Transient times in linear metabolic pathways under constant affinity constraints.

PubMed

Lloréns, M; Nuño, J C; Montero, F

1997-10-15

In the early seventies, Easterby began the analytical study of transition times for linear reaction schemes [Easterby (1973) Biochim. Biophys. Acta 293, 552-558]. In this pioneer work and in subsequent papers, a state function (the transient time) was used to measure the period before the stationary state, for systems constrained to work under both constant and variable input flux, was reached. Despite the undoubted usefulness of this quantity to describe the time-dependent features of these kinds of systems, its application to the study of chemical reactions under other constraints is questionable. In the present work, a generalization of these magnitudes to linear metabolic pathways functioning under a constant-affinity constraint is carried out. It is proved that classical definitions of transient times do not reflect the actual properties of the transition to the steady state in systems evolving under this restriction. Alternatively, a more adequate framework for interpretation of the transient times for systems with both constant and variable input flux is suggested. Within this context, new definitions that reflect more accurately the transient characteristics of constant affinity systems are stated. Finally, the meaning of these transient times is discussed.

Hit-Validation Methodologies for Ligands Isolated from DNA-Encoded Chemical Libraries.

PubMed

Zimmermann, Gunther; Li, Yizhou; Rieder, Ulrike; Mattarella, Martin; Neri, Dario; Scheuermann, Jörg

2017-05-04

DNA-encoded chemical libraries (DECLs) are large collections of compounds linked to DNA fragments, serving as amplifiable barcodes, which can be screened on target proteins of interest. In typical DECL selections, preferential binders are identified by high-throughput DNA sequencing, by comparing their frequency before and after the affinity capture step. Hits identified in this procedure need to be confirmed, by resynthesis and by performing affinity measurements. In this article we present new methods based on hybridization of oligonucleotide conjugates with fluorescently labeled complementary oligonucleotides; these facilitate the determination of affinity constants and kinetic dissociation constants. The experimental procedures were demonstrated with acetazolamide, a binder to carbonic anhydrase IX with a dissociation constant in the nanomolar range. The detection of binding events was compatible not only with fluorescence polarization methodologies, but also with Alphascreen technology and with microscale thermophoresis. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

High-Affinity Binding of Remyelinating Natural Autoantibodies to Myelin-Mimicking Lipid Bilayers Revealed by Nanohole Surface Plasmon Resonance

PubMed Central

Wittenberg, Nathan J.; Im, Hyungsoon; Xu, Xiaohua; Wootla, Bharath; Watzlawik, Jens; Warrington, Arthur E.; Rodriguez, Moses; Oh, Sang-Hyun

2012-01-01

Multiple sclerosis is a progressive neurological disorder that results in the degradation of myelin sheaths that insulate axons in the central nervous system. Therefore promotion of myelin repair is a major thrust of multiple sclerosis treatment research. Two mouse monoclonal natural autoantibodies, O1 and O4, promote myelin repair in several mouse models of multiple sclerosis. Natural autoantibodies are generally polyreactive and predominantly of the IgM isotype. The prevailing paradigm is that because they are polyreactive, these antibodies bind antigens with low affinities. Despite their wide use in neuroscience and glial cell research, however, the affinities and kinetic constants of O1 and O4 antibodies have not been measured to date. In this work, we developed a membrane biosensing platform based on surface plasmon resonance in gold nanohole arrays with a series of surface modification techniques to form myelin-mimicking lipid bilayer membranes to measure both the association and dissociation rate constants for O1 and O4 antibodies binding to their myelin lipid antigens. The ratio of rate constants shows that O1 and O4 bind to galactocerebroside and sulfated galactocerebroside, respectively, with unusually small apparent dissociation constants (KD ~0.9 nM) for natural autoantibodies. This is approximately one to two orders of magnitude lower than typically observed for the highest affinity natural autoantibodies. We propose that the unusually high affinity of O1 and O4 to their targets in myelin contributes to the mechanism by which they signal oligodendrocytes and induce central nervous system repair. PMID:22762372

Determine equilibrium dissociation constant of drug-membrane receptor affinity using the cell membrane chromatography relative standard method.

PubMed

Ma, Weina; Yang, Liu; Lv, Yanni; Fu, Jia; Zhang, Yanmin; He, Langchong

2017-06-23

The equilibrium dissociation constant (K D ) of drug-membrane receptor affinity is the basic parameter that reflects the strength of interaction. The cell membrane chromatography (CMC) method is an effective technique to study the characteristics of drug-membrane receptor affinity. In this study, the K D value of CMC relative standard method for the determination of drug-membrane receptor affinity was established to analyze the relative K D values of drugs binding to the membrane receptors (Epidermal growth factor receptor and angiotensin II receptor). The K D values obtained by the CMC relative standard method had a strong correlation with those obtained by the frontal analysis method. Additionally, the K D values obtained by CMC relative standard method correlated with pharmacological activity of the drug being evaluated. The CMC relative standard method is a convenient and effective method to evaluate drug-membrane receptor affinity. Copyright © 2017 Elsevier B.V. All rights reserved.

Statistical theory of chromatography: new outlooks for affinity chromatography.

PubMed Central

Denizot, F C; Delaage, M A

1975-01-01

We have developed further the statistical approach to chromatography initiated by Giddings and Eyring, and applied it to affinity chromatography. By means of a convenient expression of moments the convergence towards the Laplace-Gauss distribution has been established. The Gaussian character is not preserved if other causes of dispersion are taken into account, but expressions of moments can be obtained in a generalized form. A simple procedure is deduced for expressing the fundamental constants of the model in terms of purely experimental quantities. Thus, affinity chromatography can be used to determine rate constants of association and dissociation in a range considered as the domain of the stopped-flow methods. PMID:1061072

The constant region affects antigen binding of antibodies to DNA by altering secondary structure.

PubMed

Xia, Yumin; Janda, Alena; Eryilmaz, Ertan; Casadevall, Arturo; Putterman, Chaim

2013-11-01

We previously demonstrated an important role of the constant region in the pathogenicity of anti-DNA antibodies. To determine the mechanisms by which the constant region affects autoantibody binding, a panel of isotype-switch variants (IgG1, IgG2a, IgG2b) was generated from the murine PL9-11 IgG3 autoantibody. The affinity of the PL9-11 antibody panel for histone was measured by surface plasmon resonance (SPR). Tryptophan fluorescence was used to determine wavelength shifts of the antibody panel upon binding to DNA and histone. Finally, circular dichroism spectroscopy was used to measure changes in secondary structure. SPR analysis revealed significant differences in histone binding affinity between members of the PL9-11 panel. The wavelength shifts of tryptophan fluorescence emission were found to be dependent on the antibody isotype, while circular dichroism analysis determined that changes in antibody secondary structure content differed between isotypes upon antigen binding. Thus, the antigen binding affinity is dependent on the particular constant region expressed. Moreover, the effects of antibody binding to antigen were also constant region dependent. Alteration of secondary structures influenced by constant regions may explain differences in fine specificity of anti-DNA antibodies between antibodies with similar variable regions, as well as cross-reactivity of anti-DNA antibodies with non-DNA antigens. Copyright © 2013 Elsevier Ltd. All rights reserved.

Comparison of Oxidation Kinetics of Nitrite-Oxidizing Bacteria: Nitrite Availability as a Key Factor in Niche Differentiation

PubMed Central

Nowka, Boris; Daims, Holger

2014-01-01

Nitrification has an immense impact on nitrogen cycling in natural ecosystems and in wastewater treatment plants. Mathematical models function as tools to capture the complexity of these biological systems, but kinetic parameters especially of nitrite-oxidizing bacteria (NOB) are lacking because of a limited number of pure cultures until recently. In this study, we compared the nitrite oxidation kinetics of six pure cultures and one enrichment culture representing three genera of NOB (Nitrobacter, Nitrospira, Nitrotoga). With half-saturation constants (Km) between 9 and 27 μM nitrite, Nitrospira bacteria are adapted to live under significant substrate limitation. Nitrobacter showed a wide range of lower substrate affinities, with Km values between 49 and 544 μM nitrite. However, the advantage of Nitrobacter emerged under excess nitrite supply, sustaining high maximum specific activities (Vmax) of 64 to 164 μmol nitrite/mg protein/h, contrary to the lower activities of Nitrospira of 18 to 48 μmol nitrite/mg protein/h. The Vmax (26 μmol nitrite/mg protein/h) and Km (58 μM nitrite) of “Candidatus Nitrotoga arctica” measured at a low temperature of 17°C suggest that Nitrotoga can advantageously compete with other NOB, especially in cold habitats. The kinetic parameters determined represent improved basis values for nitrifying models and will support predictions of community structure and nitrification rates in natural and engineered ecosystems. PMID:25398863

Temperature Dependence of Inorganic Nitrogen Uptake: Reduced Affinity for Nitrate at Suboptimal Temperatures in Both Algae and Bacteria

PubMed Central

Reay, David S.; Nedwell, David B.; Priddle, Julian; Ellis-Evans, J. Cynan

1999-01-01

Nitrate utilization and ammonium utilization were studied by using three algal isolates, six bacterial isolates, and a range of temperatures in chemostat and batch cultures. We quantified affinities for both substrates by determining specific affinities (specific affinity = maximum growth rate/half-saturation constant) based on estimates of kinetic parameters obtained from chemostat experiments. At suboptimal temperatures, the residual concentrations of nitrate in batch cultures and the steady-state concentrations of nitrate in chemostat cultures both increased. The specific affinity for nitrate was strongly dependent on temperature (Q10 ≈ 3, where Q10 is the proportional change with a 10°C temperature increase) and consistently decreased at temperatures below the optimum temperature. In contrast, the steady-state concentrations of ammonium remained relatively constant over the same temperature range, and the specific affinity for ammonium exhibited no clear temperature dependence. This is the first time that a consistent effect of low temperature on affinity for nitrate has been identified for psychrophilic, mesophilic, and thermophilic bacteria and algae. The different responses of nitrate uptake and ammonium uptake to temperature imply that there is increasing dependence on ammonium as an inorganic nitrogen source at low temperatures. PMID:10347046

Increased Antibody Affinity Confers Broad In Vitro Protection against Escape Mutants of Severe Acute Respiratory Syndrome Coronavirus

PubMed Central

Rani, Mridula; Bolles, Meagan; Donaldson, Eric F.; Van Blarcom, Thomas; Baric, Ralph; Iverson, Brent

2012-01-01

Even though the effect of antibody affinity on neutralization potency is well documented, surprisingly, its impact on neutralization breadth and escape has not been systematically determined. Here, random mutagenesis and DNA shuffling of the single-chain variable fragment of the neutralizing antibody 80R followed by bacterial display screening using anchored periplasmic expression (APEx) were used to generate a number of higher-affinity variants of the severe acute respiratory syndrome coronavirus (SARS-CoV)-neutralizing antibody 80R with equilibrium dissociation constants (KD) as low as 37 pM, a >270-fold improvement relative to that of the parental 80R single-chain variable fragment (scFv). As expected, antigen affinity was shown to correlate directly with neutralization potency toward the icUrbani strain of SARS-CoV. Additionally, the highest-affinity antibody fragment displayed 10-fold-increased broad neutralization in vitro and completely protected against several SARS-CoV strains containing substitutions associated with antibody escape. Importantly, higher affinity also led to the suppression of viral escape mutants in vitro. Escape from the highest-affinity variant required reduced selective pressure and multiple substitutions in the binding epitope. Collectively, these results support the hypothesis that engineered antibodies with picomolar dissociation constants for a neutralizing epitope can confer escape-resistant protection. PMID:22696652

A chlorogenic acid esterase with a unique substrate specificity from Ustilago maydis.

PubMed

Nieter, Annabel; Haase-Aschoff, Paul; Kelle, Sebastian; Linke, Diana; Krings, Ulrich; Popper, Lutz; Berger, Ralf G

2015-03-01

An extracellular chlorogenic acid esterase from Ustilago maydis (UmChlE) was purified to homogeneity by using three separation steps, including anion-exchange chromatography on a Q Sepharose FF column, preparative isoelectric focusing (IEF), and, finally, a combination of affinity chromatography and hydrophobic interaction chromatography on polyamide. SDS-PAGE analysis suggested a monomeric protein of ∼71 kDa. The purified enzyme showed maximal activity at pH 7.5 and at 37°C and was active over a wide pH range (3.5 to 9.5). Previously described chlorogenic acid esterases exhibited a comparable affinity for chlorogenic acid, but the enzyme from Ustilago was also active on typical feruloyl esterase substrates. Kinetic constants for chlorogenic acid, methyl p-coumarate, methyl caffeate, and methyl ferulate were as follows: Km values of 19.6 μM, 64.1 μM, 72.5 μM, and 101.8 μM, respectively, and kcat/Km values of 25.83 mM(-1) s(-1), 7.63 mM(-1) s(-1), 3.83 mM(-1) s(-1) and 3.75 mM(-1) s(-1), respectively. UmChlE released ferulic, p-coumaric, and caffeic acids from natural substrates such as destarched wheat bran (DSWB) and coffee pulp (CP), confirming activity on complex plant biomass. The full-length gene encoding UmChlE consisted of 1,758 bp, corresponding to a protein of 585 amino acids, and was functionally produced in Pichia pastoris GS115. Sequence alignments with annotated chlorogenic acid and feruloyl esterases underlined the uniqueness of this enzyme. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Extracellular enzyme kinetics scale with resource availability

USGS Publications Warehouse

Sinsabaugh, Robert L.; Belnap, Jayne; Findlay, Stuart G.; Follstad Shah, Jennifer J.; Hill, Brian H.; Kuehn, Kevin A.; Kuske, Cheryl; Litvak, Marcy E.; Martinez, Noelle G.; Moorhead, Daryl L.; Warnock, Daniel D.

2014-01-01

Microbial community metabolism relies on external digestion, mediated by extracellular enzymes that break down complex organic matter into molecules small enough for cells to assimilate. We analyzed the kinetics of 40 extracellular enzymes that mediate the degradation and assimilation of carbon, nitrogen and phosphorus by diverse aquatic and terrestrial microbial communities (1160 cases). Regression analyses were conducted by habitat (aquatic and terrestrial), enzyme class (hydrolases and oxidoreductases) and assay methodology (low affinity and high affinity substrates) to relate potential reaction rates to substrate availability. Across enzyme classes and habitats, the scaling relationships between apparent Vmax and apparent Km followed similar power laws with exponents of 0.44 to 0.67. These exponents, called elasticities, were not statistically distinct from a central value of 0.50, which occurs when the Km of an enzyme equals substrate concentration, a condition optimal for maintenance of steady state. We also conducted an ecosystem scale analysis of ten extracellular hydrolase activities in relation to soil and sediment organic carbon (2,000–5,000 cases/enzyme) that yielded elasticities near 1.0 (0.9 ± 0.2, n = 36). At the metabolomic scale, the elasticity of extracellular enzymatic reactions is the proportionality constant that connects the C:N:P stoichiometries of organic matter and ecoenzymatic activities. At the ecosystem scale, the elasticity of extracellular enzymatic reactions shows that organic matter ultimately limits effective enzyme binding sites. Our findings suggest that one mechanism by which microbial communities maintain homeostasis is regulating extracellular enzyme expression to optimize the short-term responsiveness of substrate acquisition. The analyses also show that, like elemental stoichiometry, the fundamental attributes of enzymatic reactions can be extrapolated from biochemical to community and ecosystem scales.

All human Na(+)-K(+)-ATPase alpha-subunit isoforms have a similar affinity for cardiac glycosides.

PubMed

Wang, J; Velotta, J B; McDonough, A A; Farley, R A

2001-10-01

Three alpha-subunit isoforms of the sodium pump, which is the receptor for cardiac glycosides, are expressed in human heart. The aim of this study was to determine whether these isoforms have distinct affinities for the cardiac glycoside ouabain. Equilibrium ouabain binding to membranes from a panel of different human tissues and cell lines derived from human tissues was compared by an F statistic to determine whether a single population of binding sites or two populations of sites with different affinities would better fit the data. For all tissues, the single-site model fit the data as well as the two-site model. The mean equilibrium dissociation constant (K(d)) for all samples calculated using the single-site model was 18 +/- 6 nM (mean +/- SD). No difference in K(d) was found between nonfailing and failing human heart samples, although the maximum number of binding sites in failing heart was only approximately 50% of the number of sites in nonfailing heart. Measurement of association rate constants and dissociation rate constants confirmed that the binding affinities of the different human alpha-isoforms are similar to each other, although calculated K(d) values were lower than those determined by equilibrium binding. These results indicate both that the affinity of all human alpha-subunit isoforms for ouabain is similar and that the increased sensitivity of failing human heart to cardiac glycosides is probably due to a reduction in the number of pumps in the heart rather than to a selective inhibition of a subset of pumps with different affinities for the drugs.

[Adaptive specific features of energy metabolism in fish ontogenesis].

PubMed

Ozerniuk, N D

2011-01-01

A review of data on the pattern of change of the intensity of oxygen consumption during early ontogenesis of different fish species (rainbow trout, loach, zebrafish, carp, and grass carp) is provided. It has a similar pattern: this index increases in the period of embryonic and larval development and, after passing of larvae to an active feeding, it begins to gradually decline. This dynamics is determined by specific features of an increase in the rate of oxygen uptake and body weight in the course of early stages of fish ontogenesis. For determining optimal temperature conditions of development, a method of total (for a definite stage of development) oxygen uptake was suggested, which makes it possible to determine minimal energy expenditures necessary for the process of a particular stage of embryogenesis to take place. Analysis of temperature dependence of kinetic properties of enzymes with reference to the Michaelis constant (Km) for lactate dehydrogenase demonstrated that minimal Km, corresponding to maximal enzyme-substrate affinity, for embryos of different fish species differs in correspondence with differences in temperature conditions of development of these species in nature. For embryos of one species developing at changing temperature conditions (salmonids), this index changes in accordance with a temperature drift in nature.

Analysis of Protein Interactions with Picomolar Binding Affinity by Fluorescence-Detected Sedimentation Velocity

PubMed Central

2014-01-01

The study of high-affinity protein interactions with equilibrium dissociation constants (KD) in the picomolar range is of significant interest in many fields, but the characterization of stoichiometry and free energy of such high-affinity binding can be far from trivial. Analytical ultracentrifugation has long been considered a gold standard in the study of protein interactions but is typically applied to systems with micromolar KD. Here we present a new approach for the study of high-affinity interactions using fluorescence detected sedimentation velocity analytical ultracentrifugation (FDS-SV). Taking full advantage of the large data sets in FDS-SV by direct boundary modeling with sedimentation coefficient distributions c(s), we demonstrate detection and hydrodynamic resolution of protein complexes at low picomolar concentrations. We show how this permits the characterization of the antibody–antigen interactions with low picomolar binding constants, 2 orders of magnitude lower than previously achieved. The strongly size-dependent separation and quantitation by concentration, size, and shape of free and complex species in free solution by FDS-SV has significant potential for studying high-affinity multistep and multicomponent protein assemblies. PMID:24552356

Diffusion Limitations in Root Uptake of Cadmium and Zinc, But Not Nickel, and Resulting Bias in the Michaelis Constant1[W][OA

PubMed Central

Degryse, Fien; Shahbazi, Afsaneh; Verheyen, Liesbeth; Smolders, Erik

2012-01-01

It has long been recognized that diffusive boundary layers affect the determination of active transport parameters, but this has been largely overlooked in plant physiological research. We studied the short-term uptake of cadmium (Cd), zinc (Zn), and nickel (Ni) by spinach (Spinacia oleracea) and tomato (Lycopersicon esculentum) in solutions with or without metal complexes. At same free ion concentration, the presence of complexes, which enhance the diffusion flux, increased the uptake of Cd and Zn, whereas Ni uptake was unaffected. Competition effects of protons on Cd and Zn uptake were observed only at a very large degree of buffering, while competition of magnesium ions on Ni uptake was observed even in unbuffered solutions. These results strongly suggest that uptake of Cd and Zn is limited by diffusion of the free ion to the roots, except at very high degree of solution buffering, whereas Ni uptake is generally internalization limited. All results could be well described by a model that combined a diffusion equation with a competitive Michaelis-Menten equation. Direct uptake of the complex was estimated to be a major contribution only at millimolar concentrations of the complex or at very large ratios of complex to free ion concentration. The true Km for uptake of Cd2+ and Zn2+ was estimated at <5 nm, three orders of magnitude smaller than the Km measured in unbuffered solutions. Published Michaelis constants for plant uptake of Cd and Zn likely strongly overestimate physiological ones and should not be interpreted as an indicator of transporter affinity. PMID:22864584

Inversion of substrate stereoselectivity of horse liver alcohol dehydrogenase by substitutions of Ser-48 and Phe-93

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Keehyuk; Plapp, Bryce V.

The substrate specificities of alcohol dehydrogenases (ADH) are of continuing interest for understanding the physiological functions of these enzymes. Ser-48 and Phe-93 have been identified as important residues in the substrate binding sites of ADHs, but more comprehensive structural and kinetic studies are required. The S48T substitution in horse ADH1E has small effects on kinetic constants and catalytic efficiency (V/Km) with ethanol, but decreases activity with benzyl alcohol and affinity for 2,2,2-trifluoroethanol (TFE) and 2,3,4,5,6-pentafluorobenzyl alcohol (PFB). Nevertheless, atomic resolution crystal structures of the S48T enzyme complexed with NAD+ and TFE or PFB are very similar to the structures formore » the wild-type enzyme. (The S48A substitution greatly diminishes catalytic activity.) The F93A substitution significantly decreases catalytic efficiency (V/Km) for ethanol and acetaldehyde while increasing activity for larger secondary alcohols and the enantioselectivity for the R-isomer relative to the S-isomer of 2-alcohols. The doubly substituted S48T/F93A enzyme has kinetic constants for primary and secondary alcohols similar to those for the F93A enzyme, but the effect of the S48T substitution is to decrease V/Km for (S)-2-alcohols without changing V/Km for (R)-2-alcohols. Thus, the S48T/F93A substitutions invert the enantioselectivity for alcohol oxidation, increasing the R/S ratio by 10, 590, and 200-fold for 2-butanol, 2-octanol, and sec-phenethyl alcohol, respectively. Transient kinetic studies and simulations of the ordered bi bi mechanism for the oxidation of the 2-butanols by the S48T/F93A ADH show that the rate of hydride transfer is increased about 7-fold for both isomers (relative to wild-type enzyme) and that the inversion of enantioselectivity is due to more productive binding for (R)-2-butanol than for (S)-2-butanol in the ternary complex. Molecular modeling suggests that both of the sec-phenethyl alcohols could bind to the enzyme and that dynamics must affect the rates of catalysis.« less

Hepatic Disposition of Gemfibrozil and Its Major Metabolite Gemfibrozil 1-O-β-Glucuronide.

PubMed

Kimoto, Emi; Li, Rui; Scialis, Renato J; Lai, Yurong; Varma, Manthena V S

2015-11-02

Gemfibrozil (GEM), which decreases serum triglycerides and low density lipoprotein, perpetrates drug-drug interactions (DDIs) with several drugs. These DDIs are primarily attributed to the inhibition of drug transporters and metabolic enzymes, particularly cytochrome P450 (CYP) 2C8 by the major circulating metabolite gemfibrozil 1-O-β-glucuronide (GG). Here, we characterized the transporter-mediated hepatic disposition of GEM and GG using sandwich-cultured human hepatocytes (SCHH) and transporter-transfect systems. Significant active uptake was noted in SCHH for the metabolite. GG, but not GEM, showed substrate affinity to organic anion transporting polypeptide (OATP) 1B1, 1B3, and 2B1. In SCHH, glucuronidation was characterized affinity constants (Km) of 7.9 and 61.4 μM, and biliary excretion of GG was observed. Furthermore, GG showed active basolateral efflux from preloaded SCHH and ATP-dependent uptake into membrane vesicles overexpressing multidrug resistance-associated protein (MRP) 2, MRP3, and MRP4. A mathematical model was developed to estimate hepatic uptake and efflux kinetics of GEM and GG based on SCHH studies. Collectively, the hepatic transporters play a key role in the disposition and thus determine the local concentrations of GEM and more so for GG, which is the predominant inhibitory species against CYP2C8 and OATP1B1.

Lithospheric structure of the Arabian Shield from the joint inversion of receiver functions and surface-wave group velocities

NASA Astrophysics Data System (ADS)

Julià, Jordi; Ammon, Charles J.; Herrmann, Robert B.

2003-08-01

We estimate lithospheric velocity structure for the Arabian Shield by jointly modeling receiver functions and fundamental-mode group velocities from events recorded by the 1995-1997 Saudi Arabian Portable Broadband Deployment. Receiver functions are primarily sensitive to shear-wave velocity contrasts and vertical travel times, and surface-wave dispersion measurements are sensitive to vertical shear-wave velocity averages, so that their combination bridge resolution gaps associated with each individual data set. Our resulting models correlate well with the observed surface geology; the Asir terrane to the West consists of a 10-km-thick upper crust of 3.3 km/s overlying a lower crust of 3.7-3.8 km/s; in the Afif terrane to the East, the upper crust is 20 km thick and has an average velocity of 3.6 km/s, and the lower crust is about 3.8 km/s; separating the terranes, the Nabitah mobile belt is made of a gradational upper crust up to 3.6 km/s at 15 km overlying an also gradational lower crust up to 4.0 km/s. The crust-mantle transition is found to be sharp in terranes of continental affinity (east) and gradual in terranes of oceanic affinity (west). The upper mantle shear velocities range from 4.3 to 4.6 km/s. Temperatures around 1000 °C are obtained from our velocity models for a thin upper mantle lid observed beneath station TAIF, and suggest that the lithosphere could be as thin as 50-60 km under this station.

Ophiolitic basement to the Great Valley forearc basin, California, from seismic and gravity data: Implications for crustal growth at the North American continental margin

USGS Publications Warehouse

Godfrey, N.J.; Beaudoin, B.C.; Klemperer, S.L.; Levander, A.; Luetgert, J.; Meltzer, A.; Mooney, W.; Tréhu, A.

1997-01-01

The nature of the Great Valley basement, whether oceanic or continental, has long been a source of controversy. A velocity model (derived from a 200-km-long east-west reflection-refraction profile collected south of the Mendocino triple junction, northern California, in 1993), further constrained by density and magnetic models, reveals an ophiolite underlying the Great Valley (Great Valley ophiolite), which in turn is underlain by a westward extension of lower-density continental crust (Sierran affinity material). We used an integrated modeling philosophy, first modeling the seismic-refraction data to obtain a final velocity model, and then modeling the long-wavelength features of the gravity data to obtain a final density model that is constrained in the upper crust by our velocity model. The crustal section of Great Valley ophiolite is 7-8 km thick, and the Great Valley ophiolite relict oceanic Moho is at 11-16 km depth. The Great Valley ophiolite does not extend west beneath the Coast Ranges, but only as far as the western margin of the Great Valley, where the 5-7-km-thick Great Valley ophiolite mantle section dips west into the present-day mantle. There are 16-18 km of lower-density Sierran affinity material beneath the Great Valley ophiolite mantle section, such that a second, deeper, "present-day" continental Moho is at about 34 km depth. At mid-crustal depths, the boundary between the eastern extent of the Great Valley ophiolite and the western extent of Sierran affinity material is a near-vertical velocity and density discontinuity about 80 km east of the western margin of the Great Valley. Our model has important implications for crustal growth at the North American continental margin. We suggest that a thick ophiolite sequence was obducted onto continental material, probably during the Jurassic Nevadan orogeny, so that the Great Valley basement is oceanic crust above oceanic mantle vertically stacked above continental crust and continental mantle.

Studies on lectins. XXXII. Application of affinity electrophoresis to the study of the interaction of lectins and their derivatives with sugars.

PubMed

Horejsí, V; Tichá, M; Kocourek, J

1977-09-29

Affinity electrophoresis was used to study the sugar binding heterogeneity of lectins or their derivatives. Commercial and demetallized preparations of concanavalin A could be resolved by affinity electrophoresis into three components with different affinity to immobilized sugar. Similarly the Vicia cracca lectin obtained by affinity chromatography behaved on affinity gels as a mixture of active and inactive molecular species. Affinity electrophoresis has shown that the nonhemagglutinating acetylated lentil lectin and photo-oxidized or sulfenylated pea lectin retain their sugar binding properties; dissociation constants of saccharide complexes of these derivatives are similar to those of native lectins. The presence of specific immobilized sugar in the affinity gel improved the resolution of isolectins from Dolichos biflorus and Ricinus communis seeds.

Insights into ligand binding to a Glutathione S-transferase from mango: structure, thermodynamics and kinetics

PubMed Central

Valenzuela-Chavira, Ignacio; Contreras-Vergara, Carmen A.; Arvizu-Flores, Aldo A.; Serrano-Posada, Hugo; Lopez-Zavala, Alonso A.; García-Orozco, Karina D.; Hernandez-Paredes, Javier; Rudiño-Piñera, Enrique; Stojanoff, Vivian; Sotelo-Mundo, Rogerio R.; Islas-Osuna, Maria A.

2017-01-01

We studied a mango glutathione S-transferase (GST) (Mangifera indica) bound to glutathione (GSH) and S-hexyl glutathione (GSX). This GST Tau class (MiGSTU) had a molecular mass of 25.5 kDa. MiGSTU Michaelis-Menten kinetic constants were determined for their substrates obtaining a Km, Vmax and kcat for CDNB of 0.792 mM, 80.58 mM·min−1 and 68.49 s−1 respectively and 0.693 mM, 105.32 mM·min−1 and 89.57 s−1, for reduced GSH respectively. MiGSTU had a micromolar affinity towards GSH (5.2 μM) or GSX (7.8 μM). The crystal structure of the MiGSTU in apo or bound to GSH or GSX generated a model that explains the thermodynamic signatures of binding and showed the importance of enthalpic-entropic compensation in ligand binding to Tau-class GST enzymes. PMID:28104507

Polyaniline-carboxymethyl cellulose nanocomposite for cholesterol detection.

PubMed

Barik, Abdul; Solanki, Pratima R; Kaushik, Ajeet; Ali, Azahar; Pandey, M K; Kim, C G; Malhotra, B D

2010-10-01

Cholesterol oxidase (ChOx) has been covalently immobilized onto polyaniline-carboxymethyl cellulose (PANI-CMC) nanocomposite film deposited onto indium-tin-oxide (ITO) coated glass plate using glutaraldehyde as a cross-linker. Fourier transform infrared (FTIR) spectroscopic and electrochemical studies have been used to characterize the PANI-CMC/ITO nanocomposite electrode and ChOx/PANI-CMC/ITO bioelectrode. Scanning electron microscopy (SEM) studies reveal the formation of PANI-CMC nanocomposite fibers of size approximately 150 nm in diameter. The ChOx/PANI-CMC/ITO bioelectrode exhibits linearity as 0.5-22 mM, detection limit as 1.31 mM, sensitivity as 0.14 mA/mM cm2, response time as 10 s and shelf-life of about 10 weeks when bioelectrode is stored at 4 degrees C. The low value of Michaelis-Menten constant (K(m)) obtained as 2.71 mM reveals high affinity of immobilized ChOx for PANI-CMC/ITO nanocomposite electrode.

Preparation and characterization of tannase immobilized onto carboxyl-functionalized superparamagnetic ferroferric oxide nanoparticles.

PubMed

Wu, Changzheng; Xu, Caiyun; Ni, Hui; Yang, Qiuming; Cai, Huinong; Xiao, Anfeng

2016-04-01

Tannase from Aspergillus tubingensis was immobilized onto carboxyl-functionalized Fe3O4 nanoparticles (CMNPs), and conditions affecting tannase immobilization were investigated. Successful binding between CMNPs and tannase was confirmed by Fourier transform infrared spectroscopy and thermogravimetric analysis. Vibrating sample magnetometry and X-ray diffraction showed that the CMNPs and immobilized tannase exhibit distinct magnetic responses and superparamagnetic properties. Free and immobilized tannase exhibited identical optimal temperatures of 50°C and differing pH optima at 6 and 7, respectively. The thermal, pH, and storage stabilities of the immobilized tannase were superior to those of free tannase. After six cycles of catalytic hydrolysis of propyl gallate, the immobilized tannase maintained over 60% of its initial activity. The Michaelis constant (Km) of the immobilized enzyme indicated its higher affinity for substrate binding than the free enzyme. Copyright © 2016 Elsevier Ltd. All rights reserved.

Point mutation increases a form of the NK1 receptor with high affinity for neurokinin A and B and septide

PubMed Central

Ciucci, Alessandra; Palma, Carla; Manzini, Stefano; Werge, Thomas M

1998-01-01

The binding modalities of substance P and neurokinin A on the wild type and Gly166 to-Cys mutant NK1 receptors expressed on CHO cells were investigated in homologous and heterologous binding experiments using both radiolabelled substance P and neurokinin A.On the wild type NK1 receptor NKA displaces radiolabelled substance P with very low apparent affinity, despite its high-affinity binding constant (determined in homologous binding experiments). The Gly166 to-Cys substitution in the NK1 tachykinin receptor greatly enhances the apparent affinity of neurokinin A in competition for radiolabelled substance P, but it does not change the binding constant of neurokinin A. The mutation, thereby, eliminates the discrepancy between the low apparent affinity and the high binding constant of neurokinin A.On the wild type receptor the binding capacity of neurokinin A is significantly smaller than that of substance P. In contrast, the two tachykinins bind to approximately the same number of sites on the mutant receptor.Simultaneous mass action law analysis of binding data in which multiple radioligands were employed in parallel demonstrated that a one-site model was unable to accommodate all the experimental data, whereas a two-site model provided a dramatically better description.These two receptor-sites display equally high affinity for substance P, while neurokinin A strongly discriminates between a high and a low affinity component. The binding affinities of neurokinin A are not affected by the mutation, which instead specifically alters the distribution between receptor sites in favour of a high affinity neurokinin A binding form.The low apparent affinity and binding capacity of neurokinin A on the wild type receptor results from neurokinin A binding with high affinity only to a fraction of the sites labelled by substance P. The mutation increases the proportion of this site, and consequently enhances the apparent affinity and binding capacity of neurokinin A.The binding modalities of septide-like ligands (i.e. neurokinin B, SP(6-11), SP-methyl ester) are affected similarly to neurokinin A and are better resolved into two sites. The mutation leaves the affinity of these ligands for the two receptor forms unchanged, but increases the fraction of high-affinity sites. On the other hand, the binding of non-peptide and peptide antagonists (SR140.333 and FK888) behaved similarly to substance P with a single high affinity site that is unaffected by the mutation.These findings may suggest that the NK1 receptor exists in two different forms with similar affinity for substance P and NK1 antagonists, but with a high and a low affinity for neurokinin A and septide-like ligands. Hence, the Gly166 in the NK1 receptor would seem to control the distribution between a pan-reactive form and a substance P-selective form of the receptor. PMID:9786514

Catchment Area Treatment (CAT) Plan and Crop Area Optimization for Integrated Management in a Water Resource Project

NASA Astrophysics Data System (ADS)

Jaiswal, R. K.; Thomas, T.; Galkate, R. V.; Ghosh, N. C.; Singh, S.

2013-09-01

A scientifically developed catchment area treatment (CAT) plan and optimized pattern of crop areas may be the key for sustainable development of water resource, profitability in agriculture and improvement of overall economy in drought affected Bundelkhand region of Madhya Pradesh (India). In this study, an attempt has been made to develop a CAT plan using spatial variation of geology, geomorphology, soil, drainage, land use in geographical information system for selection of soil and water conservation measures and crop area optimization using linear programming for maximization of return considering water availability, area affinity, fertilizers, social and market constraints in Benisagar reservoir project of Chhatarpur district (M.P.). The scientifically developed CAT plan based on overlaying of spatial information consists of 58 mechanical measure (49 boulder bunds, 1 check dam, 7 cully plug and 1 percolation tank), 2.60 km2 land for agro forestry, 2.08 km2 land for afforestation in Benisagar dam and 67 mechanical measures (45 boulder bunds and 22 gully plugs), 7.79 km2 land for agro forestry, 5.24 km2 land for afforestation in Beniganj weir catchment with various agronomic measures for agriculture areas. The linear programming has been used for optimization of crop areas in Benisagar command for sustainable development considering various scenarios of water availability, efficiencies, affinity and fertilizers availability in the command. Considering present supply condition of water, fertilizers, area affinity and making command self sufficient in most of crops, the net benefit can be increase to Rs. 1.93 crores from 41.70 km2 irrigable area in Benisagar command by optimizing cropping pattern and reducing losses during conveyance and application of water.

Biochemical characterization of recombinant mevalonate kinase from Bacopa monniera.

PubMed

Kumari, Uma; Vishwakarma, Rishi K; Sonawane, Prashant; Abbassi, Shakeel; Khan, Bashir M

2015-01-01

Mevalonate kinase (MK; ATP: mevalonate 5-phosphotransferase; EC 2.7.1.36) plays a key role in isoprenoid biosynthetic pathway in plants. MK catalyzes the phosphorylation of mevalonate to form mevalonate-5-phosphate. The recombinant BmMK was cloned and over-expressed in E. coli BL21 (DE3), and purified to homogeneity by affinity chromatography followed by gel filtration. Optimum pH and temperature for forward reaction was found to be 7.0 and 30 °C, respectively. The enzyme was most stable at pH 8 at 25 °C with deactivation rate constant (Kd*) 1.398 × 10(-4) and half life (t1/2) 49 h. pH activity profile of BmMK indicates the involvement of carboxylate ion, histidine, lysine, arginine or aspartic acid at the active site of enzyme. Activity of recombinant BmMK was confirmed by phosphorylation of RS-mevalonate in the presence of Mg(2+), having Km and Vmax 331.9 μM and 719.1 pKat μg(-1), respectively. The values of kcat and kcat/Km for RS-mevalonate were determined to be 143.82 s(-1) and 0.43332 M(-1) s(-1) and kcat and kcat/Km values for ATP were found 150.9 s(-1) and 1.023 M(-1) s(-1). The metal ion studies suggested that BmMK is a metal dependent enzyme and highly active in the presence of MgCl2. Copyright © 2014 Elsevier B.V. All rights reserved.

Affine theory of gravitation

NASA Astrophysics Data System (ADS)

Popławski, Nikodem

2014-01-01

We propose a theory of gravitation, in which the affine connection is the only dynamical variable describing the gravitational field. We construct a simple dynamical Lagrangian density that is entirely composed from the connection, via its curvature and torsion, and is a polynomial function of its derivatives. It is given by the contraction of the Ricci tensor with a tensor which is inverse to the symmetric, contracted square of the torsion tensor, . We vary the total action for the gravitational field and matter with respect to the affine connection, assuming that the matter fields couple to the connection only through . We derive the resulting field equations and show that they are identical with the Einstein equations of general relativity with a nonzero cosmological constant if the tensor is regarded as proportional to the metric tensor. The cosmological constant is simply a constant of proportionality between the two tensors, which together with and provides a natural system of units in gravitational physics. This theory therefore provides a physical construction of the metric as a polynomial function of the connection, and explains dark energy as an intrinsic property of spacetime.

The neonatal Fc receptor (FcRn) binds independently to both sites of the IgG homodimer with identical affinity.

PubMed

Abdiche, Yasmina Noubia; Yeung, Yik Andy; Chaparro-Riggers, Javier; Barman, Ishita; Strop, Pavel; Chin, Sherman Michael; Pham, Amber; Bolton, Gary; McDonough, Dan; Lindquist, Kevin; Pons, Jaume; Rajpal, Arvind

2015-01-01

The neonatal Fc receptor (FcRn) is expressed by cells of epithelial, endothelial and myeloid lineages and performs multiple roles in adaptive immunity. Characterizing the FcRn/IgG interaction is fundamental to designing therapeutic antibodies because IgGs with moderately increased binding affinities for FcRn exhibit superior serum half-lives and efficacy. It has been hypothesized that 2 FcRn molecules bind an IgG homodimer with disparate affinities, yet their affinity constants are inconsistent across the literature. Using surface plasmon resonance biosensor assays that eliminated confounding experimental artifacts, we present data supporting an alternate hypothesis: 2 FcRn molecules saturate an IgG homodimer with identical affinities at independent sites, consistent with the symmetrical arrangement of the FcRn/Fc complex observed in the crystal structure published by Burmeister et al. in 1994. We find that human FcRn binds human IgG1 with an equilibrium dissociation constant (KD) of 760 ± 60 nM (N = 14) at 25°C and pH 5.8, and shows less than 25% variation across the other human subtypes. Human IgG1 binds cynomolgus monkey FcRn with a 2-fold higher affinity than human FcRn, and binds both mouse and rat FcRn with a 10-fold higher affinity than human FcRn. FcRn/IgG interactions from multiple species show less than a 2-fold weaker affinity at 37°C than at 25°C and appear independent of an IgG's variable region. Our in vivo data in mouse and rat models demonstrate that both affinity and avidity influence an IgG's serum half-life, which should be considered when choosing animals, especially transgenic systems, as surrogates.

Identification of two proline transport systems in Staphylococcus aureus and their possible roles in osmoregulation.

PubMed

Bae, J H; Miller, K J

1992-02-01

The food-borne pathogen Staphylococcus aureus is distinguished from other food-borne pathogens by its ability to grow at water activity values below 0.90. Previous studies have indicated that proline accumulation mediated by transport represents a primary osmoregulatory strategy utilized by this bacterium (C. B. Anderson and L. D. Witter, Appl. Environ, Microbiol. 43:1501-1503, 1982; I. Koujima, H. Hayashi, K. Tomochika, A. Okabe, and Y. Kanemasa, Appl. Environ. Microbiol. 35:467-470, 1978; K. J. Miller, S. C. Zelt, and J.-H. Bae, Curr. Microbiol. 23:131-137, 1991). In this study, we demonstrate the presence of two proline transport systems within whole cells of S. aureus, a high-affinity transport system (Km, 7 microM) and a low-affinity transport system (Km, 420 microM). Our results indicate that the low-affinity proline transport system is osmotically activated and is the primary system responsible for the accumulation of proline by this pathogen during growth at low water activity.

The Role of Human Cytochrome P450 Enzymes in the Formation of 2-Hydroxymetronidazole: CYP2A6 is the High Affinity (Low Km) Catalyst

PubMed Central

Cohen-Wolkowiez, Michael; Sampson, Mario R.; Kearns, Gregory L.

2013-01-01

Despite metronidazole’s widespread clinical use since the 1960s, the specific enzymes involved in its biotransformation have not been previously identified. Hence, in vitro studies were conducted to identify and characterize the cytochrome P450 enzymes involved in the formation of the major metabolite, 2-hydroxymetronidazole. Formation of 2-hydroxymetronidazole in human liver microsomes was consistent with biphasic, Michaelis-Menten kinetics. Although several cDNA-expressed P450 enzymes catalyzed 2-hydroxymetronidazole formation at a supratherapeutic concentration of metronidazole (2000 μM), at a “therapeutic concentration” of 100 μM only CYPs 2A6, 3A4, 3A5, and 3A7 catalyzed metronidazole 2-hydroxylation at rates substantially greater than control vector, and CYP2A6 catalyzed 2-hydroxymetronidazole formation at rates 6-fold higher than the next most active enzyme. Kinetic studies with these recombinant enzymes revealed that CYP2A6 has a Km = 289 μM which is comparable to the Km for the high-affinity (low-Km) enzyme in human liver microsomes, whereas the Km values for the CYP3A enzymes corresponded with the low-affinity (high-Km) component. The sample-to-sample variation in 2-hydroxymetronidazole formation correlated significantly with CYP2A6 activity (r ≥ 0.970, P < 0.001) at substrate concentrations of 100 and 300 μM. Selective chemical inhibitors of CYP2A6 inhibited metronidazole 2-hydroxylation in a concentration-dependent manner and inhibitory antibodies against CYP2A6 virtually eliminated metronidazole 2-hydroxylation (>99%). Chemical and antibody inhibitors of other P450 enzymes had little or no effect on metronidazole 2-hydroxylation. These results suggest that CYP2A6 is the primary catalyst responsible for the 2-hydroxylation of metronidazole, a reaction that may function as a marker of CYP2A6 activity both in vitro and in vivo. PMID:23813797

The first armadillo repeat is involved in the recognition and regulation of beta-catenin phosphorylation by protein kinase CK1.

PubMed

Bustos, Victor H; Ferrarese, Anna; Venerando, Andrea; Marin, Oriano; Allende, Jorge E; Pinna, Lorenzo A

2006-12-26

Multiple phosphorylation of beta-catenin by glycogen synthase kinase 3 (GSK3) in the Wnt pathway is primed by CK1 through phosphorylation of Ser-45, which lacks a typical CK1 canonical sequence. Synthetic peptides encompassing amino acids 38-64 of beta-catenin are phosphorylated by CK1 on Ser-45 with low affinity (K(m) approximately 1 mM), whereas intact beta-catenin is phosphorylated at Ser-45 with very high affinity (K(m) approximately 200 nM). Peptides extended to include a putative CK1 docking motif (FXXXF) at 70-74 positions or a F74AA mutation in full-length beta-catenin had no significant effect on CK1 phosphorylation efficiency. beta-Catenin C-terminal deletion mutants up to residue 181 maintained their high affinity, whereas removal of the 131-181 fragment, corresponding to the first armadillo repeat, was deleterious, resulting in a 50-fold increase in K(m) value. Implication of the first armadillo repeat in beta-catenin targeting by CK1 is supported in that the Y142E mutation, which mimics phosphorylation of Tyr-142 by tyrosine kinases and promotes dissociation of beta-catenin from alpha-catenin, further improves CK1 phosphorylation efficiency, lowering the K(m) value to <50 nM, approximating the physiological concentration of beta-catenin. In contrast, alpha-catenin, which interacts with the N-terminal region of beta-catenin, prevents Ser-45 phosphorylation of CK1 in a dose-dependent manner. Our data show that the integrity of the N-terminal region and the first armadillo repeat are necessary and sufficient for high-affinity phosphorylation by CK1 of Ser-45. They also suggest that beta-catenin association with alpha-catenin and beta-catenin phosphorylation by CK1 at Ser-45 are mutually exclusive.

The Intrinsic Reactivity of ATP and the Catalytic Proficiencies of Kinases Acting on Glucose, N-Acetylgalactosamine, and Homoserine

PubMed Central

Stockbridge, Randy B.; Wolfenden, Richard

2009-01-01

To evaluate the rate enhancements produced by representative kinases and their thermodynamic basis, rate constants were determined as a function of changing temperature for 1) the spontaneous methanolysis of ATP and 2) reactions catalyzed by kinases to which different mechanisms of action have been ascribed. For each of these enzymes, the minor effects of changing viscosity indicate that kcat/Km is governed by the central chemical events in the enzyme-substrate complex rather than by enzyme-substrate encounter. Individual Arrhenius plots, obtained at intervals between pH 4.8 and 11.0, yielded ΔH‡ and TΔS‡ for the nonenzymatic methanolysis of ATP2−, ATP3−, and ATP4− in the absence of Mg2+. The addition of Mg2+ led to partly compensating changes in ΔH‡ and TΔS‡, accelerating the nonenzymatic methanolysis of ATP 11-fold at pH 7 and 25 °C. The rate enhancements produced by yeast hexokinase, homoserine kinase, and N-acetylgalactosamine kinase (obtained by comparison of their kcat/Km values in the presence of saturating phosphoryl acceptor with the second order rate constant for methanolysis of MgATP) ranged between 1012- and 1014-fold. Their nominal affinities for the altered substrates in the transition state were 2.1 × 10−16 m for N-acetylgalactosamine kinase, 7.4 × 10−17 m for homoserine kinase, and 6.4 × 10−18 m for hexokinase. Compared with nonenzymatic phosphoryl transfer, all three kinases were found to produce major reductions in the entropy of activation, in accord with the likelihood that substrate juxtaposition and desolvation play prominent roles in their catalytic action. PMID:19531469

A nitrogen-dependent switch in the high affinity ammonium transport in Medicago truncatula.

PubMed

Straub, Daniel; Ludewig, Uwe; Neuhäuser, Benjamin

2014-11-01

Ammonium transporters (AMTs) are crucial for the high affinity primary uptake and translocation of ammonium in plants. In the model legume Medicago truncatula, the genomic set of AMT-type ammonium transporters comprises eight members. Only four genes were abundantly expressed in young seedlings, both in roots and shoots. While the expression of all AMTs in the shoot was not affected by the nitrogen availability, the dominating MtAMT1;1 gene was repressed by nitrogen in roots, despite that cellular nitrogen concentrations were far above deficiency levels. A contrasting de-repression by nitrogen was observed for MtAMT1;4 and MtAMT2;1, which were both expressed at intermediate level. Weak expression was found for MtAMT1;2 and MtAMT2;3, while the other AMTs were not detected in young seedlings. When expressed from their endogenous promoters, translational fusion proteins of MtAMT1;1 and MtAMT2;1 with green fluorescent protein were co-localized in the plasma membrane of rhizodermal cells, but also detected in cortical root layers. Both transporter proteins similarly functionally complemented a yeast strain that is deficient in high affinity ammonium transport, both at acidic and neutral pH. The uptake into yeast mediated by these transporters saturated with Km AMT1;1 = 89 µM and Km AMT2;1 = 123 µM, respectively. When expressed in oocytes, MtAMT1;1 mediated much larger (15)N-ammonium uptake than MtAMT2;1, but NH4 (+) currents were only recorded for MtAMT1;1. These currents saturated with a voltage-dependent Km = 90 µM at -80 mV. The cellular localization and regulation of the AMTs suggests that MtAMT1;1 encodes the major high affinity ammonium transporter gene in low nitrogen grown young M. truncatula roots and despite the similar localization and substrate affinity, MtAMT2;1 appears functionally distinct and more important at higher nitrogen supply.

Biphasic Kinetic Behavior of Nitrate Reductase from Heterocystous, Nitrogen-Fixing Cyanobacteria 1

PubMed Central

Martin-Nieto, José; Flores, Enrique; Herrero, Antonia

1992-01-01

Nitrate reductase activity from filamentous, heterocyst-forming cyanobacteria showed a biphasic kinetic behavior with respect to nitrate as the variable substrate. Two kinetic components were detected, the first showing a higher affinity for nitrate (Km, 0.05-0.25 mm) and a lower catalytic activity and the second showing a lower affinity for nitrate (Km, 5-25 mm) and a higher (3- to 5-fold) catalytic activity. In contrast, among unicellular cyanobacteria, most representatives studied exhibited a monophasic, Michaelis-Menten kinetic pattern for nitrate reductase activity. Biphasic kinetics remained unchanged with the use of different assay conditions (i.e. cell disruption or permeabilization, two different electron donors) or throughout partial purification of the enzyme. PMID:16652939

Improved Accuracy of Low Affinity Protein-Ligand Equilibrium Dissociation Constants Directly Determined by Electrospray Ionization Mass Spectrometry

NASA Astrophysics Data System (ADS)

Jaquillard, Lucie; Saab, Fabienne; Schoentgen, Françoise; Cadene, Martine

2012-05-01

There is continued interest in the determination by ESI-MS of equilibrium dissociation constants (KD) that accurately reflect the affinity of a protein-ligand complex in solution. Issues in the measurement of KD are compounded in the case of low affinity complexes. Here we present a KD measurement method and corresponding mathematical model dealing with both gas-phase dissociation (GPD) and aggregation. To this end, a rational mathematical correction of GPD (fsat) is combined with the development of an experimental protocol to deal with gas-phase aggregation. A guide to apply the method to noncovalent protein-ligand systems according to their kinetic behavior is provided. The approach is validated by comparing the KD values determined by this method with in-solution KD literature values. The influence of the type of molecular interactions and instrumental setup on fsat is examined as a first step towards a fine dissection of factors affecting GPD. The method can be reliably applied to a wide array of low affinity systems without the need for a reference ligand or protein.

Identification and characterization of a novel high affinity metal-binding site in the hammerhead ribozyme.

PubMed Central

Hansen, M R; Simorre, J P; Hanson, P; Mokler, V; Bellon, L; Beigelman, L; Pardi, A

1999-01-01

A novel metal-binding site has been identified in the hammerhead ribozyme by 31P NMR. The metal-binding site is associated with the A13 phosphate in the catalytic core of the hammerhead ribozyme and is distinct from any previously identified metal-binding sites. 31P NMR spectroscopy was used to measure the metal-binding affinity for this site and leads to an apparent dissociation constant of 250-570 microM at 25 degrees C for binding of a single Mg2+ ion. The NMR data also show evidence of a structural change at this site upon metal binding and these results are compared with previous data on metal-induced structural changes in the core of the hammerhead ribozyme. These NMR data were combined with the X-ray structure of the hammerhead ribozyme (Pley HW, Flaherty KM, McKay DB. 1994. Nature 372:68-74) to model RNA ligands involved in binding the metal at this A13 site. In this model, the A13 metal-binding site is structurally similar to the previously identified A(g) metal-binding site and illustrates the symmetrical nature of the tandem G x A base pairs in domain 2 of the hammerhead ribozyme. These results demonstrate that 31P NMR represents an important method for both identification and characterization of metal-binding sites in nucleic acids. PMID:10445883

Low-temperature (10°C) anaerobic digestion of dilute dairy wastewater in an EGSB bioreactor: microbial community structure, population dynamics, and kinetics of methanogenic populations.

PubMed

Bialek, Katarzyna; Cysneiros, Denise; O'Flaherty, Vincent

2013-01-01

The feasibility of anaerobic digestion of dairy wastewater at 10°C was investigated in a high height : diameter ratio EGSB reactor. Stable performance was observed at an applied organic loading rate (OLR) of 0.5-2 kg COD m(-3) d(-1) with chemical oxygen demand (COD) removal efficiencies above 85%. When applied OLR increased to values above 2 kg COD m(-3) d(-1), biotreatment efficiency deteriorated, with methanogenesis being the rate-limiting step. The bioreactor recovered quickly (3 days) after reduction of the OLR. qPCR results showed a reduction in the abundance of hydrogenotrophic methanogenic Methanomicrobiales and Methanobacteriales throughout the steady state period followed by a sharp increase in their numbers (111-fold) after the load shock. Specific methanogenic activity and maximum substrate utilising rate (A(max)) of the biomass at the end of trial indicated increased activity and preference towards hydrogenotrophic methanogenesis, which correlated well with the increased abundance of hydrogenotrophic methanogens. Acetoclastic Methanosaeta spp. remained at stable levels throughout the trial. However, increased apparent half-saturation constant (K(m)) at the end of the trial indicated a decrease in the specific substrate affinity for acetate of the sludge, suggesting that Methanosaeta spp., which have high substrate affinity, started to be outcompeted in the reactor.

[Cell-ELA-based determination of binding affinity of DNA aptamer against U87-EGFRvIII cell].

PubMed

Tan, Yan; Liang, Huiyu; Wu, Xidong; Gao, Yubo; Zhang, Xingmei

2013-05-01

A15, a DNA aptamer with binding specificity for U87 glioma cells stably overexpressing the epidermal growth factor receptor variant III (U87-EGFRvIII), was generated by cell systematic evolution of ligands by exponential enrichment (cell-SELEX) using a random nucleotide library. Subsequently, we established a cell enzyme-linked assay (cell-ELA) to detect the affinity of A15 compared to an EGFR antibody. We used A15 as a detection probe and cultured U87-EGFRvIII cells as targets. Our data indicate that the equilibrium dissociation constants (K(d)) for A15 were below 100 nmol/L and had similar affinity compared to an EGFR antibody for U87-EGFRvIII. We demonstrated that the cell-ELA was a useful method to determine the equilibrium dissociation constants (K(d)) of aptamers generated by cell-SELEX.

Identification of the dominant runoff pathways from data-based mechanistic modelling of nested catchments in temperate UK

NASA Astrophysics Data System (ADS)

Ockenden, M. C.; Chappell, N. A.

2011-05-01

SummaryUnderstanding hydrological flow pathways is important for modelling stream response, in order to address a range of environmental problems such as flood prediction, prediction of chemical loads and identification of contaminant pathways for subsequent remediation. This paper describes the use of parametrically efficient, low order models to identify the dominant modes of stream response for catchments within the Upper Eden, UK. A first order linear model adequately identified the dominant mode in all but one of the sub-catchments. A consistent pattern of time constants and pure time delays between catchments was observed over different periods of data. In the nested catchments, time constants increased as the catchment size increased from 1.1 km 2 at Gais Gill (2-7 h) to 69.4 km 2 at Kirkby Stephen (5-10 h) to 223.4 km 2 at Great Musgrave (7-16 h) to 616.4 km 2 at Temple Sowerby (11-22 h), but Blind Beck (a small catchment 8.8 km 2, time constants 11-21 h) had time constants most similar to Temple Sowerby. This was attributed to a combination of the storage role of permeable rock strata, where present, and the effect of scale on sub-surface and channel routing. A first order model could not be identified for the 1.0 km 2 Low Hall catchment, which comprises permeable sandstone overlain by Quaternary sediments. A second-order model of Low Hall stream showed a higher proportion of water taking a slower pathway (76% via a slow pathway; time constant 252 h) than a model with the same structure for the 8.8 km 2 Blind Beck (46% via slow pathway; time constant 60 h), where only 38% of the basin was underlain by the same permeable sandstone. This highlights the need to quantify the role of deep pathways through permeable rock, where present, in addition to the effect of catchment size on response times.

Single-shot characterization of enzymatic reaction constants Km and kcat by an acoustic-driven, bubble-based fast micromixer.

PubMed

Xie, Yuliang; Ahmed, Daniel; Lapsley, Michael Ian; Lin, Sz-Chin Steven; Nawaz, Ahmad Ahsan; Wang, Lin; Huang, Tony Jun

2012-09-04

In this work we present an acoustofluidic approach for rapid, single-shot characterization of enzymatic reaction constants K(m) and k(cat). The acoustofluidic design involves a bubble anchored in a horseshoe structure which can be stimulated by a piezoelectric transducer to generate vortices in the fluid. The enzyme and substrate can thus be mixed rapidly, within 100 ms, by the vortices to yield the product. Enzymatic reaction constants K(m) and k(cat) can then be obtained from the reaction rate curves for different concentrations of substrate while holding the enzyme concentration constant. We studied the enzymatic reaction for β-galactosidase and its substrate (resorufin-β-D-galactopyranoside) and found K(m) and k(cat) to be 333 ± 130 μM and 64 ± 8 s(-1), respectively, which are in agreement with published data. Our approach is valuable for studying the kinetics of high-speed enzymatic reactions and other chemical reactions.

Investigation into constant envelope orthogonal frequency division multiplexing for polarization-division multiplexing coherent optical communication

NASA Astrophysics Data System (ADS)

Li, Yupeng; Ding, Ding

2017-09-01

Benefiting from the high spectral efficiency and low peak-to-average power ratio, constant envelope orthogonal frequency division multiplexing (OFDM) is a promising technique in coherent optical communication. Polarization-division multiplexing (PDM) has been employed as an effective way to double the transmission capacity in the commercial 100 Gb/s PDM-QPSK system. We investigated constant envelope OFDM together with PDM. Simulation results show that the acceptable maximum launch power into the fiber improves 10 and 6 dB for 80- and 320-km transmission, respectively (compared with the conventional PDM OFDM system). The maximum reachable distance of the constant envelope OFDM system is able to reach 800 km, and even 1200 km is reachable if an ideal erbium doped fiber amplifier is employed.

Functional activity of L-carnitine transporters in human airway epithelial cells.

PubMed

Ingoglia, Filippo; Visigalli, Rossana; Rotoli, Bianca Maria; Barilli, Amelia; Riccardi, Benedetta; Puccini, Paola; Dall'Asta, Valeria

2016-02-01

Carnitine plays a physiologically important role in the β-oxidation of fatty acids, facilitating the transport of long-chain fatty acids across the inner mitochondrial membrane. Distribution of carnitine within the body tissues is mainly performed by novel organic cation transporter (OCTN) family, including the isoforms OCTN1 (SLC22A4) and OCTN2 (SLC22A5) expressed in human. We performed here a characterization of carnitine transport in human airway epithelial cells A549, Calu-3, NCl-H441, and BEAS-2B, by means of an integrated approach combining data of mRNA/protein expression with the kinetic and inhibition analyses of L-[(3)H]carnitine transport. Carnitine uptake was strictly Na(+)-dependent in all cell models. In A549 and BEAS-2B cells, carnitine uptake was mediated by one high-affinity component (Km<2 μM) identifiable with OCTN2. In both these cell models, indeed, carnitine uptake was maximally inhibited by betaine and strongly reduced by SLC22A5/OCTN2 silencing. Conversely, Calu-3 and NCl-H441 exhibited both a high (Km~20 μM) and a low affinity (Km>1 mM) transport component. While the high affinity component is identifiable with OCTN2, the low affinity uptake is mediated by ATB(0,+), a Na(+), and Cl(-)-coupled transport system for neutral and cationic amino acids, as demonstrated by the inhibition by leucine and arginine, as well as by SLC6A14/ATB(0,+) silencing. The presence of this transporter leads to a massive accumulation of carnitine inside the cells and may be of peculiar relevance in pathologic conditions of carnitine deficiency, such as those associated to OCTN2 defects. Copyright © 2015 Elsevier B.V. All rights reserved.

Quantum gravity in the Eddington purely affine picture

NASA Astrophysics Data System (ADS)

Martellini, M.

1984-06-01

It was shown by Kijowski and Tulczjew that pure gravity with a cosmological constant can be obtained by a covariant Legendre transformation of a purely affine Lagrangian "in the manner of Eddington" constructed from a symmetric linear connection. In this paper I prove by explicit calculations that the Eddington Lagrangian is equivalent, in the sense which gives the same field equations, to a polynomial effective Lagrangian which turns out to be power-counting renormalizable. Then a formal proof of the unitarity of this theory is stated in the Kugo-Ojima formalism on the basis of the existence of two local Becchi-Rouet-Stora symmetries. These supertransformations are related to the algebra of the diffeomorphisms of the space-time, as well as to that of the volume-preserving space-time transformations which are not fixed by the gauge fixing used for the diffeomorphism group itself. Furthermore, I find that in the purely affine picture quantum gravity exhibits an infrared freedom. Since now the self-coupling constant is given by the cosmological constant, such a property could explain the observed almost zero value of the cosmological term at very large distances, i.e., to very low energies.

Characterization of deltamethrin metabolism by rat plasma and liver microsomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Anand, Sathanandam S.; Bruckner, James V.; Haines, Wendy T.

2006-04-15

Deltamethrin, a widely used type II pyrethroid insecticide, is a relatively potent neurotoxicant. While the toxicity has been extensively examined, toxicokinetic studies of deltamethrin and most other pyrethroids are very limited. The aims of this study were to identify, characterize, and assess the relative contributions of esterases and cytochrome P450s (CYP450s) responsible for deltamethrin metabolism by measuring deltamethrin disappearance following incubation of various concentrations (2 to 400 {mu}M) in plasma (esterases) and liver microsomes (esterases and CYP450s) prepared from adult male rats. While the carboxylesterase metabolism in plasma and liver was characterized using an inhibitor, tetra isopropyl pyrophosphoramide (isoOMPA), CYP450more » metabolism was characterized using the cofactor, NADPH. Michaelis-Menten rate constants were calculated using linear and nonlinear regression as applicable. The metabolic efficiency of these pathways was estimated by calculating intrinsic clearance (Vmax/Km). In plasma, isoOMPA completely inhibited deltamethrin biotransformation at concentrations (2 and 20 {mu}M of deltamethrin) that are 2- to 10-fold higher than previously reported peak blood levels in deltamethrin-poisoned rats. For carboxylesterase-mediated deltamethrin metabolism in plasma, Vmax = 325.3 {+-} 53.4 nmol/h/ml and Km = 165.4 {+-} 41.9 {mu}M. Calcium chelation by EGTA did not inhibit deltamethrin metabolism in plasma or liver microsomes, indicating that A-esterases do not metabolize deltamethrin. In liver microsomes, esterase-mediated deltamethrin metabolism was completely inhibited by isoOMPA, confirming the role of carboxylesterases. The rate constants for liver carboxylesterases were Vmax = 1981.8 {+-} 132.3 nmol/h/g liver and Km = 172.5 {+-} 22.5 {mu}M. Liver microsomal CYP450-mediated biotransformation of deltamethrin was a higher capacity (Vmax = 2611.3 {+-} 134.1 nmol/h/g liver) and higher affinity (Km = 74.9 {+-} 5.9 {mu}M) process than carboxylesterase (plasma or liver) detoxification. Genetically engineered individual rat CYP450s (Supersomes) were used to identify specific CYP450 isozyme(s) involved in the deltamethrin metabolism. CYP1A2, CYP1A1, and CYP2C11 in decreasing order of importance quantitatively, metabolized deltamethrin. Intrinsic clearance by liver CYP450s (35.5) was more efficient than that by liver (12.0) or plasma carboxylesterases (2.4)« less

Papain hydrolysis of X-phenyl-N-methanesulfonyl glycinates: a quantitative structure-activity relationship and molecular graphics analysis.

PubMed

Carotti, A; Smith, R N; Wong, S; Hansch, C; Blaney, J M; Langridge, R

1984-02-15

The hydrolysis of 32 X-phenyl-N-methanesulfonyl glycinates by papain was investigated. It was found that the variation in the Michaelis constants could be rationalized by the following correlation equation: log 1/Km = 0.61 pi '3 + 0.46 MR4 + 0.55 sigma + 2.00 with a correlation coefficient of 0.945. In this expression, pi '3 is the hydrophobic constant for the more lipophilic of the two possible meta substituents, MR4 is the molar refractivity of 4-substituents, and sigma is the Hammett constant summed for all substituents. Using this equation, we designed, synthesized, and successfully predicted Km for a new congener intended to maximize binding (1/Km). The interactions involved in enzyme-substrate binding, as characterized by the correlation equation, are interpreted using a computer-constructed color three-dimensional-graphics molecular model of the enzyme active site. The nonenzymatic hydrolysis (both acid and basic) of phenyl hippurates yield rate constants which are well correlated by Hammett equations; however, log k for both acid and alkaline hydrolysis are not linearly related to log 1/Km or log kcat/Km.

Semisupervised Gaussian Process for Automated Enzyme Search.

PubMed

Mellor, Joseph; Grigoras, Ioana; Carbonell, Pablo; Faulon, Jean-Loup

2016-06-17

Synthetic biology is today harnessing the design of novel and greener biosynthesis routes for the production of added-value chemicals and natural products. The design of novel pathways often requires a detailed selection of enzyme sequences to import into the chassis at each of the reaction steps. To address such design requirements in an automated way, we present here a tool for exploring the space of enzymatic reactions. Given a reaction and an enzyme the tool provides a probability estimate that the enzyme catalyzes the reaction. Our tool first considers the similarity of a reaction to known biochemical reactions with respect to signatures around their reaction centers. Signatures are defined based on chemical transformation rules by using extended connectivity fingerprint descriptors. A semisupervised Gaussian process model associated with the similar known reactions then provides the probability estimate. The Gaussian process model uses information about both the reaction and the enzyme in providing the estimate. These estimates were validated experimentally by the application of the Gaussian process model to a newly identified metabolite in Escherichia coli in order to search for the enzymes catalyzing its associated reactions. Furthermore, we show with several pathway design examples how such ability to assign probability estimates to enzymatic reactions provides the potential to assist in bioengineering applications, providing experimental validation to our proposed approach. To the best of our knowledge, the proposed approach is the first application of Gaussian processes dealing with biological sequences and chemicals, the use of a semisupervised Gaussian process framework is also novel in the context of machine learning applied to bioinformatics. However, the ability of an enzyme to catalyze a reaction depends on the affinity between the substrates of the reaction and the enzyme. This affinity is generally quantified by the Michaelis constant KM. Therefore, we also demonstrate using Gaussian process regression to predict KM given a substrate-enzyme pair.

Influence of N-ethylmaleimide on cholinoceptors and responses in longitudinal muscles from guinea-pig ileum.

PubMed Central

Aronstam, R. S.; Carrier, G. O.

1982-01-01

1 The binding of carbamylcholine to membranes prepared from the longitudinal muscle of guinea-pig ileum was determined from its inhibition of the binding of [3H]-3-quinuclidinyl benzilate. Carbamylcholine binding was resolved into high and low affinity components with apparent dissociation constants of 0.11 +/- 0.02 and 11 +/- 1 microM; 42% of the receptors displayed high affinity carbamylcholine binding. 2 Alkylation of longitudinal muscle membranes with N-ethylmaleimide increased muscarinic receptor affinity for carbamylcholine in a manner consistent with a conversion of low affinity to high affinity receptors. After exposure the muscle membrane fragments to 1 mM N-ethylmaleimide for 20 min at 35 degrees C, carbamylcholine binding was resolved into two components with apparent dissociation constants of 0.11 +/- 0.01 and 9 +/- 2 microM, with 74% of the receptors displaying the higher affinity. 3 Exposure of longitudinal membranes mounted in an organ chamber to 1 mM N-ethylmaleimide for 30s depressed isometric contractions in response to acetylcholine by 80%, while contractions induced by K+ and Ba2+ were reduced by less than 20% and 10%, respectively. Acetylcholine dose-response curves were shifted to the right while Ba2+ curves were unaffected. 4 It is suggested that N-ethylmaleimide has a selective effect on muscarinic responses in the longitudinal muscle by disrupting processes occurring after receptor occupancy but before the induction of phospholipid turnover or calcium influx in the postsynaptic membrane. PMID:7126999

Acquired Substrate Preference for GAB1 Protein Bestows Transforming Activity to ERBB2 Kinase Lung Cancer Mutants

PubMed Central

Fan, Ying-Xin; Wong, Lily; Marino, Michael P.; Ou, Wu; Shen, Yi; Wu, Wen Jin; Wong, Kwok-Kin; Reiser, Jakob; Johnson, Gibbes R.

2013-01-01

Activating mutations in the αC-β4 loop of the ERBB2 kinase domain, such as ERBB2YVMA and ERBB2G776VC, have been identified in human lung cancers and found to drive tumor formation. Here we observe that the docking protein GAB1 is hyper-phosphorylated in carcinomas from transgenic mice and in cell lines expressing these ERBB2 cancer mutants. Using dominant negative GAB1 mutants lacking canonical tyrosine residues for SHP2 and PI3K interactions or lentiviral shRNA that targets GAB1, we demonstrate that GAB1 phosphorylation is required for ERBB2 mutant-induced cell signaling, cell transformation, and tumorigenesis. An enzyme kinetic analysis comparing ERBB2YVMA to wild type using physiologically relevant peptide substrates reveals that ERBB2YVMA kinase adopts a striking preference for GAB1 phosphorylation sites as evidenced by ∼150-fold increases in the specificity constants (kcat/Km) for several GAB1 peptides, and this change in substrate selectivity was predominantly attributed to the peptide binding affinities as reflected by the apparent Km values. Furthermore, we demonstrate that ERBB2YVMA phosphorylates GAB1 protein ∼70-fold faster than wild type ERBB2 in vitro. Notably, the mutation does not significantly alter the Km for ATP or sensitivity to lapatinib, suggesting that, unlike EGFR lung cancer mutants, the ATP binding cleft of the kinase is not significantly changed. Taken together, our results indicate that the acquired substrate preference for GAB1 is critical for the ERBB2 mutant-induced oncogenesis. PMID:23612964

A laboratory nanoseismological study on deep-focus earthquake micromechanics

PubMed Central

Wang, Yanbin; Zhu, Lupei; Shi, Feng; Schubnel, Alexandre; Hilairet, Nadege; Yu, Tony; Rivers, Mark; Gasc, Julien; Addad, Ahmed; Deldicque, Damien; Li, Ziyu; Brunet, Fabrice

2017-01-01

Global earthquake occurring rate displays an exponential decay down to ~300 km and then peaks around 550 to 600 km before terminating abruptly near 700 km. How fractures initiate, nucleate, and propagate at these depths remains one of the greatest puzzles in earth science, as increasing pressure inhibits fracture propagation. We report nanoseismological analysis on high-resolution acoustic emission (AE) records obtained during ruptures triggered by partial transformation from olivine to spinel in Mg2GeO4, an analog to the dominant mineral (Mg,Fe)2SiO4 olivine in the upper mantle, using state-of-the-art seismological techniques, in the laboratory. AEs’ focal mechanisms, as well as their distribution in both space and time during deformation, are carefully analyzed. Microstructure analysis shows that AEs are produced by the dynamic propagation of shear bands consisting of nanograined spinel. These nanoshear bands have a near constant thickness (~100 nm) but varying lengths and self-organize during deformation. This precursory seismic process leads to ultimate macroscopic failure of the samples. Several source parameters of AE events were extracted from the recorded waveforms, allowing close tracking of event initiation, clustering, and propagation throughout the deformation/transformation process. AEs follow the Gutenberg-Richter statistics with a well-defined b value of 1.5 over three orders of moment magnitudes, suggesting that laboratory failure processes are self-affine. The seismic relation between magnitude and rupture area correctly predicts AE magnitude at millimeter scales. A rupture propagation model based on strain localization theory is proposed. Future numerical analyses may help resolve scaling issues between laboratory AE events and deep-focus earthquakes. PMID:28776024

Revised Model of Calcium and Magnesium Binding to the Bacterial Cell Wall

PubMed Central

Thomas, Kieth J.; Rice, Charles V.

2014-01-01

Metals bind to the bacterial cell wall yet the binding mechanisms and affinity constants are not fully understood. The cell wall of gram positive bacteria is characterized by a thick layer of peptidoglycan and anionic teichoic acids anchored in the cytoplasmic membrane (lipoteichoic acid) or covalently bound to the cell wall (wall teichoic acid). The polyphosphate groups of teichoic acid provide one-half of the metal binding sites for calcium and magnesium, contradicting previous reports that calcium binding is 100% dependent on teichoic acid. The remaining binding sites are formed with the carboxyl units of peptidoglycan. In this work we report equilibrium association constants and total metal binding capacities for the interaction of calcium and magnesium ions with the bacterial cell wall. Metal binding is much stronger and previously reported. Curvature of Scatchard plots from the binding data and the resulting two regions of binding affinity suggest the presence of negative cooperative binding, meaning that the binding affinity decreases as more ions become bound to the sample. For Ca2+, Region I has a KA = (1.0 ± 0.2) × 106 M−1 and Region II has a KA = (0.075 ± 0.058) × 106 M−1. For Mg2+, KA1 = (1.5 ± 0.1) × 106 and KA2 = (0.17 ± 0.10) × 106. A binding capacity (η) is reported for both regions. However, since binding is still occurring in Region II, the total binding capacity is denoted by η2, which are 0.70 ± 0.04 µmol/mg and 0.67 ± 0.03 µmol/mg for Ca2+ and Mg2+ respectively. These data contradict the current paradigm of there being a single metal affinity value that is constant over a range of concentrations. We also find that measurement of equilibrium binding constants is highly sample dependent, suggesting a role for diffusion of metals through heterogeneous cell wall fragments. As a result, we are able to reconcile many contradictory theories that describe binding affinity and the binding mode of divalent metal cations. PMID:25315444

Actomyosin kinetics and in vitro motility of wild-type Drosophila actin and the effects of two mutations in the Act88F gene.

PubMed Central

Anson, M; Drummond, D R; Geeves, M A; Hennessey, E S; Ritchie, M D; Sparrow, J C

1995-01-01

Two missense mutations of the flight muscle-specific actin gene of Drosophila melanogaster, Act88F, assemble into normally structured myofibrils but affect the flight ability of flies and the mechanical kinetics of isolated muscle fibers. We describe the isolation of actin from different homozygous Act88F strains, including wild-type, an Act88F null mutant (KM88), and two Act88F single point mutations (E316K and G368E), their biochemical interactions with rabbit myosin subfragment 1 (S1), and behavior with rabbit myosin and heavy meromyosin in in vitro motility assays. The rabbit and wild-type Drosophila actins have different association rate constants with S1 (2.64 and 1.77 microM-1 s-1, respectively) and in vitro motilities (2.51, 1.60 microns s-1) clearly demonstrating an isoform-specific difference. The G368E mutation shows a reduced affinity for rabbit S1 compared with the wild type (increasing from 0.11 to 0.17 microM) and a reduced velocity in vitro (reduced by 19%). The E316K mutant actin has no change in affinity for myosin S1 or in vitro motility with heavy meromyosin but does have a reduced in vitro motility (15%) with myosin. These results are discussed with respect to the recently published atomic models for the actomyosin structure and our findings that G368E fibers show a reduced rate constant for delayed tension development and increased fiber stiffness. We interpret these results as possibly caused either by effects on A1 myosin light chain binding or conformational changes within the subdomain 1 of actin, which contains the myosin binding site. E316K is discussed with respect to its likely position within the tropomyosin binding site of actin. Images FIGURE 1 FIGURE 9 PMID:7612841

Kinetic Characterisation of a Single Chain Antibody against the Hormone Abscisic Acid: Comparison with Its Parental Monoclonal

PubMed Central

Badescu, George O.; Marsh, Andrew; Smith, Timothy R.; Thompson, Andrew J.; Napier, Richard M.

2016-01-01

A single-chain Fv fragment antibody (scFv) specific for the plant hormone abscisic acid (ABA) has been expressed in the bacterium Escherichia coli as a fusion protein. The kinetics of ABA binding have been measured using surface plasmon resonance spectrometry (BIAcore 2000) using surface and solution assays. Care was taken to calculate the concentration of active protein in each sample using initial rate measurements under conditions of partial mass transport limitation. The fusion product, parental monoclonal antibody and the free scFv all have low nanomolar affinity constants, but there is a lower dissociation rate constant for the parental monoclonal resulting in a three-fold greater affinity. Analogue specificity was tested and structure-activity binding preferences measured. The biologically-active (+)-ABA enantiomer is recognised with an affinity three orders of magnitude higher than the inactive (-)-ABA. Metabolites of ABA including phaseic acid, dihydrophaseic acid and deoxy-ABA have affinities over 100-fold lower than that for (+)-ABA. These properties of the scFv make it suitable as a sensor domain in bioreporters specific for the naturally occurring form of ABA. PMID:27023768

Determination of equilibrium dissociation constants for recombinant antibodies by high-throughput affinity electrophoresis.

PubMed

Pan, Yuchen; Sackmann, Eric K; Wypisniak, Karolina; Hornsby, Michael; Datwani, Sammy S; Herr, Amy E

2016-12-23

High-quality immunoreagents enhance the performance and reproducibility of immunoassays and, in turn, the quality of both biological and clinical measurements. High quality recombinant immunoreagents are generated using antibody-phage display. One metric of antibody quality - the binding affinity - is quantified through the dissociation constant (K D ) of each recombinant antibody and the target antigen. To characterize the K D of recombinant antibodies and target antigen, we introduce affinity electrophoretic mobility shift assays (EMSAs) in a high-throughput format suitable for small volume samples. A microfluidic card comprised of free-standing polyacrylamide gel (fsPAG) separation lanes supports 384 concurrent EMSAs in 30 s using a single power source. Sample is dispensed onto the microfluidic EMSA card by acoustic droplet ejection (ADE), which reduces EMSA variability compared to sample dispensing using manual or pin tools. The K D for each of a six-member fragment antigen-binding fragment library is reported using ~25-fold less sample mass and ~5-fold less time than conventional heterogeneous assays. Given the form factor and performance of this micro- and mesofluidic workflow, we have developed a sample-sparing, high-throughput, solution-phase alternative for biomolecular affinity characterization.

Determination of equilibrium dissociation constants for recombinant antibodies by high-throughput affinity electrophoresis

PubMed Central

Pan, Yuchen; Sackmann, Eric K.; Wypisniak, Karolina; Hornsby, Michael; Datwani, Sammy S.; Herr, Amy E.

2016-01-01

High-quality immunoreagents enhance the performance and reproducibility of immunoassays and, in turn, the quality of both biological and clinical measurements. High quality recombinant immunoreagents are generated using antibody-phage display. One metric of antibody quality – the binding affinity – is quantified through the dissociation constant (KD) of each recombinant antibody and the target antigen. To characterize the KD of recombinant antibodies and target antigen, we introduce affinity electrophoretic mobility shift assays (EMSAs) in a high-throughput format suitable for small volume samples. A microfluidic card comprised of free-standing polyacrylamide gel (fsPAG) separation lanes supports 384 concurrent EMSAs in 30 s using a single power source. Sample is dispensed onto the microfluidic EMSA card by acoustic droplet ejection (ADE), which reduces EMSA variability compared to sample dispensing using manual or pin tools. The KD for each of a six-member fragment antigen-binding fragment library is reported using ~25-fold less sample mass and ~5-fold less time than conventional heterogeneous assays. Given the form factor and performance of this micro- and mesofluidic workflow, we have developed a sample-sparing, high-throughput, solution-phase alternative for biomolecular affinity characterization. PMID:28008969

Selection of staphylococcal enterotoxin B (SEB)-binding peptide using phage display technology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Soykut, Esra Acar; Dudak, Fahriye Ceyda; Boyaci, Ismail Hakki

In this study, peptides were selected to recognize staphylococcal enterotoxin B (SEB) which cause food intoxication and can be used as a biological war agent. By using commercial M13 phage library, single plaque isolation of 38 phages was done and binding affinities were investigated with phage-ELISA. The specificities of the selected phage clones showing high affinity to SEB were checked by using different protein molecules which can be found in food samples. Furthermore, the affinities of three selected phage clones were determined by using surface plasmon resonance (SPR) sensors. Sequence analysis was realized for three peptides showing high binding affinitymore » to SEB and WWRPLTPESPPA, MNLHDYHRLFWY, and QHPQINQTLYRM amino acid sequences were obtained. The peptide sequence with highest affinity to SEB was synthesized with solid phase peptide synthesis technique and thermodynamic constants of the peptide-SEB interaction were determined by using isothermal titration calorimetry (ITC) and compared with those of antibody-SEB interaction. The binding constant of the peptide was determined as 4.2 {+-} 0.7 x 10{sup 5} M{sup -1} which indicates a strong binding close to that of antibody.« less

Binding of resveratrol with sodium caseinate in aqueous solutions.

PubMed

Acharya, Durga P; Sanguansri, Luz; Augustin, Mary Ann

2013-11-15

The interaction between resveratrol (Res) and sodium caseinate (Na-Cas) has been studied by measuring fluorescence quenching of the protein by resveratrol. Quenching constants were determined using Stern-Volmer equation, which suggests that both dynamic and static quenching occur between Na-Cas and Res. Binding constants for the complexation between Na-Cas and Res were determined at different temperatures. The large binding constants (3.7-5.1×10(5)M(-1)) suggest that Res has strong affinity for Na-Cas. This affinity decreases as the temperature is raised from 25 to 37°C. The binding involves both hydrogen bonding and hydrophobic interaction, as suggested by negative enthalpy change and positive entropy change for the binding reaction. The present study indicates that Na-Cas, a common food protein, may be used as a carrier of Res, a bioactive polyphenol which is insoluble in both water and oils. Copyright © 2013 Elsevier Ltd. All rights reserved.

Two classes of cholesterol binding sites for the β2AR revealed by thermostability and NMR.

PubMed

Gater, Deborah L; Saurel, Olivier; Iordanov, Iordan; Liu, Wei; Cherezov, Vadim; Milon, Alain

2014-11-18

Cholesterol binding to G protein-coupled receptors (GPCRs) and modulation of their activities in membranes is a fundamental issue for understanding their function. Despite the identification of cholesterol binding sites in high-resolution x-ray structures of the ?2 adrenergic receptor (β2AR) and other GPCRs, the binding affinity of cholesterol for this receptor and exchange rates between the free and bound cholesterol remain unknown. In this study we report the existence of two classes of cholesterol binding sites in β2AR. By analyzing the β2AR unfolding temperature in lipidic cubic phase (LCP) as a function of cholesterol concentration we observed high-affinity cooperative binding of cholesterol with sub-nM affinity constant. In contrast, saturation transfer difference (STD) NMR experiments revealed the existence of a second class of cholesterol binding sites, in fast exchange on the STD NMR timescale. Titration of the STD signal as a function of cholesterol concentration provided a lower limit of 100 mM for their dissociation constant. However, these binding sites are specific for both cholesterol and β2AR, as shown with control experiments using ergosterol and a control membrane protein (KpOmpA). We postulate that this specificity is mediated by the high-affinity bound cholesterol molecules and propose the formation of transient cholesterol clusters around the high-affinity binding sites.

Fluorescence resonance energy transfer between ZnSe ZnS quantum dots and bovine serum albumin in bioaffinity assays of anticancer drugs

NASA Astrophysics Data System (ADS)

Shu, Chang; Ding, Li; Zhong, Wenying

2014-10-01

In the current work, using ZnSe ZnS quantum dots (QDs) as representative nanoparticles, the affinities of seven anticancer drugs for bovine serum albumin (BSA) were studied using fluorescence resonance energy transfer (FRET). The FRET efficiency of BSA-QD conjugates can reach as high as 24.87% by electrostatic interaction. The higher binding constant (3.63 × 107 L mol-1) and number of binding sites (1.75) between ZnSe ZnS QDs and BSA demonstrated that the QDs could easily associate to plasma proteins and enhance the transport efficacy of drugs. The magnitude of binding constants (103-106 L mol-1), in the presence of QDs, was between drugs-BSA and drugs-QDs in agreement with common affinities of drugs for serum albumins (104-106 L mol-1) in vivo. ZnSe ZnS QDs significantly increased the affinities for BSA of Vorinostat (SAHA), Docetaxel (DOC), Carmustine (BCNU), Doxorubicin (Dox) and 10-Hydroxycamptothecin (HCPT). However, they slightly reduced the affinities of Vincristine (VCR) and Methotrexate (MTX) for BSA. The recent work will not only provide useful information for appropriately understanding the binding affinity and binding mechanism at the molecular level, but also illustrate the ZnSe ZnS QDs are perfect candidates for nanoscal drug delivery system (DDS).

Na⁺-Dependent High-Affinity Nitrate, Phosphate and Amino Acids Transport in Leaf Cells of the Seagrass Posidonia oceanica (L.) Delile.

PubMed

Rubio, Lourdes; García-Pérez, Delia; García-Sánchez, María Jesús; Fernández, José A

2018-05-24

Posidonia oceanica (L.) Delile is a seagrass, the only group of vascular plants to colonize the marine environment. Seawater is an extreme yet stable environment characterized by high salinity, alkaline pH and low availability of essential nutrients, such as nitrate and phosphate. Classical depletion experiments, membrane potential and cytosolic sodium measurements were used to characterize the high-affinity NO₃ - , Pi and amino acids uptake mechanisms in this species. Net uptake rates of both NO₃ - and Pi were reduced by more than 70% in the absence of Na⁺. Micromolar concentrations of NO₃ - depolarized mesophyll leaf cells plasma membrane. Depolarizations showed saturation kinetics ( Km = 8.7 ± 1 μM NO₃ - ), which were not observed in the absence of Na⁺. NO₃ - induced depolarizations at increasing Na⁺ also showed saturation kinetics ( Km = 7.2 ± 2 mM Na⁺). Cytosolic Na⁺ measured in P. oceanica leaf cells (17 ± 2 mM Na⁺) increased by 0.4 ± 0.2 mM Na⁺ upon the addition of 100 μM NO₃ - . Na⁺-dependence was also observed for high-affinity l-ala and l-cys uptake and high-affinity Pi transport. All together, these results strongly suggest that NO₃ - , amino acids and Pi uptake in P. oceanica leaf cells are mediated by high-affinity Na⁺-dependent transport systems. This mechanism seems to be a key step in the process of adaptation of seagrasses to the marine environment.

Effect of magnesium complexation by fluoroquinolones on their antibacterial properties.

PubMed Central

Lecomte, S; Baron, M H; Chenon, M T; Coupry, C; Moreau, N J

1994-01-01

By using infrared and 19F nuclear magnetic resonance spectroscopies, we localized the binding site and measured the affinity of magnesium for six fluoroquinolones. It was proven that magnesium is situated between the ketone and the carboxylate groups. We determined the binding constants for the 1:1 Mg(2+)-drug complex in solution. Sparfloxacin and pefloxacin, with affinity constants (Ka) of (10.1 +/- 0.6) x 10(2) M-1 and (21 +/- 1) x 10(2) M-1, respectively, were the least and the most bound, respectively. The trend of the affinities of the assayed fluoroquinolones for magnesium was correlated with their antimicrobial activities against four bacteria and with their accumulation by these bacteria. The reference strain, Escherichia coli KL16, and two resistant mutants, NalA (gyrase mutation) and NalB (uptake defect), plus Staphylococcus aureus 209P were used. It appeared that, in every case, an impairment of accumulation is responsible for the increase in the MICs observed upon the addition of magnesium. Images PMID:7695267

Use of 2-(/sup 125/I)iodomelatonin to characterize melatonin binding sites in chicken retina

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dubocovich, M.L.; Takahashi, J.S.

2-(/sup 125/I)Iodomelatonin binds with high affinity to a site possessing the pharmacological characteristics of a melatonin receptor in chicken retinal membranes. The specific binding of 2-(/sup 125/I)iodomelatonin is stable, saturable, and reversible. Saturation experiments indicated that 2-(/sup 125/I)iodomelatonin labeled a single class of sites with an affinity constant (Kd) of 434 +/- 56 pM and a total number of binding sites (Bmax) of 74.0 +/- 13.6 fmol/mg of protein. The affinity constant obtained from kinetic analysis was in close agreement with that obtained in saturation experiments. Competition experiments showed a monophasic reduction of 2-(/sup 125/I)iodomelatonin binding with a pharmacological ordermore » of indole amine affinities characteristic of a melatonin receptor: 2-iodomelatonin greater than 6-chloromelatonin greater than or equal to melatonin greater than or equal to 6,7-dichloro-2-methylmelatonin greater than 6-hydroxymelatonin greater than or equal to 6-methoxymelatonin much greater than N-acetyltryptamine greater than N-acetyl-5-hydroxytryptamine greater than 5-methoxytryptamine greater than 5-hydroxytryptamine (inactive). The affinities of these melatonin analogs in competing for 2-(/sup 125/I)iodomelatonin binding sites were correlated closely with their potencies for inhibition of the calcium-dependent release of (3H)dopamine from chicken and rabbit retinas, indicating association of the binding site with a functional response regulated by melatonin. The results indicate that 2-(/sup 125/I)iodomelatonin is a selective, high-affinity radioligand for the identification and characterization of melatonin receptor sites.« less

Into the theory of the partial-filling affinity capillary electrophoresis and the determination of apparent stability constants of analyte-ligand complexes.

PubMed

Ansorge, Martin; Dubský, Pavel; Ušelová, Kateřina

2018-03-01

The partial-filling affinity capillary electrophoresis (pf-ACE) works with a ligand present in a background electrolyte that forms a weak complex with an analyte. In contrast to a more popular mobility-shift affinity capillary electrophoresis, only a short plug of the ligand is introduced into a capillary in the pf-ACE. Both methods can serve for determining apparent stability constants of the formed complexes but this task is hindered in the pf-ACE by the fact that the analyte spends only a part of its migration time in a contact with the ligand. In 1998, Amini and Westerlund published a linearization strategy that allows for extracting an effective mobility of an analyte in the presence of a neutral ligand out of the pf-ACE data. The main purpose of this paper is to show that the original formula is only approximate. We derive a new formula and demonstrate its applicability by means of computer simulations. We further inspect several strategies of data processing in the pf-ACE regarding a risk of an error propagation. This establishes a good practice of determining apparent stability constants of analyte-ligand complexes by means of the pf-ACE. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Interaction between DNA and Drugs Having Protonable Basic Groups: Characterization through Affinity Constants, Drug Release Kinetics, and Conformational Changes

PubMed Central

Alarcón, Liliana P.; Baena, Yolima; Manzo, Rubén H.

2017-01-01

This paper reports the in vitro characterization of the interaction between the phosphate groups of DNA and the protonated species of drugs with basic groups through the determination of the affinity constants, the reversibility of the interaction, and the effect on the secondary structure of the macromolecule. Affinity constants of the counterionic condensation DNA–drug were in the order of 106. The negative electrokinetic potential of DNA decreased with the increase of the proportion of loading drugs. The drugs were slowly released from the DNA–drug complexes and had release kinetics consistent with the high degree of counterionic condensation. The circular dichroism profile of DNA was not modified by complexation with atenolol, lidocaine, or timolol, but was significantly altered by the more lipophilic drugs benzydamine and propranolol, revealing modifications in the secondary structure of the DNA. The in vitro characterization of such interactions provides a physicochemical basis that would contribute to identify the effects of this kind of drugs in cellular cultures, as well as side effects observed under their clinical use. Moreover, this methodology could also be projected to the fields of intracellular DNA transfection and the use of DNA as a carrier of active drugs. PMID:28054999

Meprin metalloproteases inactivate interleukin 6.

PubMed

Keiffer, Timothy R; Bond, Judith S

2014-03-14

Meprins have been implicated in the pathogenesis of several inflammatory diseases, including inflammatory bowel disease, in which the cytokine IL-6 is a prominent effector molecule. Because IL-6 levels are elevated markedly in meprin α and α/β knockout mice in an experimental model of inflammatory bowel disease, the interaction between meprins and IL-6 was studied. The results demonstrate that rodent and human meprin A and B cleave IL-6 to a smaller product and, subsequently, are capable of extensive degradation of the cytokine. Analysis of the limited degradation product formed by meprin A indicated that three to five amino acids are removed from the C terminus of the cytokine. Meprin A and meprin B cleaved IL-6 with micromolar affinities (Km of 4.7 and 12.0 μM, respectively) and with high efficiencies (kcat/Km of 0.2 and 2.5 (M(-1)/s(-1)) × 10(6), respectively). These efficiency constants are among the highest for known meprin substrates. Madin-Darby canine kidney cells transiently transfected with meprin α or meprin β constructs also cleave exogenous IL-6. Both human and murine IL-6 cleaved by meprin A or B are inactivated, as demonstrated by their decreased capability to stimulate proliferation of B9 cells. These results are consistent with the proposition that one function of meprin metalloproteases is to modulate inflammation by inactivating IL-6.

Chitosan-based biocatalytic nanoparticles for pollutant removal from wastewater.

PubMed

Alarcón-Payán, Dulce A; Koyani, Rina D; Vazquez-Duhalt, Rafael

2017-05-01

Chitosan, a renewable biopolymer has the prospective applications in different fields due to its gelation capacity. Nanoconfiguration of chitosan through ionotropic gelation to encapsulate enzymatic activity offers numerous potential applications. In the present study, the preparation and characterization of chitosan nanoparticles loaded with versatile peroxidase are reported. Their performance in bioremediation process and the resistance enhancement against natural microbial biodegradation were studied. The average diameter of enzymatic nanoparticles was 120nm and showed a high enzyme loading capacity. The kinetic parameters of nanoparticles exhibited a slightly lower catalytic activity (k cat ), similar affinity constant (Km) for hydrogen peroxide and higher Km value for the phenolic compound when compared with the free enzyme. The enzymatic nanoparticles showed higher thermostability and the same pH activity profile than those from free enzyme. Ten phenolic compounds, including pesticides, halogenated compounds, endocrine disruptors and antibacterials were transformed by the enzymatic nanoparticles. The transformation rate was lower than those obtained with free enzyme suggesting mass transfer limitations. But very importantly, the enzymatic nanoparticles showed a significant increase of the operational stability in real conditions of wastewater treatment process. Moreover, chemical modification of nanoparticles with different aldehydes still enhanced the operational stability of nanoparticulated enzymes. This enhancement of stability in real conditions and the potential use of biocatalytic nanoparticles in bioremediation processes are discussed. Copyright © 2017 Elsevier Inc. All rights reserved.

Tropine dehydrogenase: purification, some properties and an evaluation of its role in the bacterial metabolism of tropine.

PubMed

Bartholomew, B A; Smith, M J; Long, M T; Darcy, P J; Trudgill, P W; Hopper, D J

1995-04-15

Tropine dehydrogenase was induced by growth of Pseudomonas AT3 on atropine, tropine or tropinone. It was NADP(+)-dependent and gave no activity with NAD+. The enzyme was very unstable but a rapid purification procedure using affinity chromatography that gave highly purified enzyme was developed. The enzyme gave a single band on isoelectric focusing with an isoelectric point at approximately pH 4. The native enzyme had an M(r) of 58,000 by gel filtration and 28,000 by SDS/PAGE and therefore consists of two subunits of equal size. The enzyme displayed a narrow range of specificity and was active with tropine and nortropine but not with pseudotropine, pseudonortropine, or a number of related compounds. The apparent Kms were 6.06 microM for tropine and 73.4 microM for nortropine with the specificity constant (Vmax/Km) for tropine 7.8 times that for pseudotropine. The apparent Km for NADP+ was 48 microM. The deuterium of [3-2H]tropine and [3-2H]pseudotropine was retained when these compounds were converted into 6-hydroxycyclohepta-1,4-dione, an intermediate in tropine catabolism, showing that the tropine dehydrogenase, although induced by growth on tropine, is not involved in the catabolic pathway for this compound. 6-Hydroxycyclohepta-1,4-dione was also implicated as an intermediate in the pathways for pseudotropine and tropinone catabolism.

Pht2;1 encodes a low-affinity phosphate transporter from Arabidopsis.

PubMed Central

Daram, P; Brunner, S; Rausch, C; Steiner, C; Amrhein, N; Bucher, M

1999-01-01

An Arabidopsis genomic sequence was recently shown to share similarity with bacterial and eukaryotic phosphate (Pi) transporters. We have cloned the corresponding cDNA, which we named Pht2;1, and subsequently performed gene expression studies and functional analysis of the protein product. The cDNA encodes a 61-kD protein with a putative topology of 12 transmembrane (TM) domains interrupted by a large hydrophilic loop between TM8 and TM9. Two boxes of eight and nine amino acids, located in the N- and C-terminal domains, respectively, are highly conserved among species across all kingdoms (eubacteria, archea, fungi, plants, and animals). The Pht2;1 gene is predominantly expressed in green tissue, the amount of transcript staying constant in leaves irrespective of the Pi status of the shoot; in roots, however, there is a marginal increase in mRNA amounts in response to Pi deprivation. Although the protein is highly similar to eukaryotic sodium-dependent Pi transporters, functional analysis of the Pht2;1 protein in mutant yeast cells indicates that it is a proton/Pi symporter dependent on the electrochemical gradient across the plasma membrane. Its fairly high apparent K(m) for Pi (0.4 mM) and high mRNA content in the shoot, especially in leaves, suggest a role for shoot organs in Pi loading. Pht2;1 thus differs from members of the recently described plant Pi transporter family in primary structure, affinity for Pi, and presumed function. PMID:10559441

CHL1 is a dual-affinity nitrate transporter of Arabidopsis involved in multiple phases of nitrate uptake.

PubMed Central

Liu, K H; Huang, C Y; Tsay, Y F

1999-01-01

Higher plants have both high- and low-affinity nitrate uptake systems. These systems are generally thought to be genetically distinct. Here, we demonstrate that a well-known low-affinity nitrate uptake mutant of Arabidopsis, chl1, is also defective in high-affinity nitrate uptake. Two to 3 hr after nitrate induction, uptake activities of various chl1 mutants at 250 microM nitrate (a high-affinity concentration) were only 18 to 30% of those of wild-type plants. In these mutants, both the inducible phase and the constitutive phase of high-affinity nitrate uptake activities were reduced, with the inducible phase being severely reduced. Expressing a CHL1 cDNA driven by the cauliflower mosaic virus 35S promoter in a transgenic chl1 plant effectively recovered the defect in high-affinity uptake for the constitutive phase but not for the induced phase, which is consistent with the constitutive level of CHL1 expression in the transgenic plant. Kinetic analysis of nitrate uptake by CHL1-injected Xenopus oocytes displayed a biphasic pattern with a Michaelis-Menten Km value of approximately 50 microM for the high-affinity phase and approximately 4 mM for the low-affinity phase. These results indicate that in addition to being a low-affinity nitrate transporter, as previously recognized, CHL1 is also involved in both the inducible and constitutive phases of high-affinity nitrate uptake in Arabidopsis. PMID:10330471

S-Wave Velocity Models Under the Saudi Arabian Portable Broadband Deployment: Evidence for Lithospheric Erosion Beneath the Arabian Shield

NASA Astrophysics Data System (ADS)

Julià, J.; Ammon, C. J.; Herrmann, R. B.

2002-12-01

Models of crustal evolution strongly rely on our knowledge on the mineralogical composition of subsurface rocks, as well as pressure and temperature conditions. Direct sampling of subsurface rocks is often not possible, so that constraints have to be placed from indirect estimates of rock properties. Detailed seismic imaging of subsurface rocks has the potential for providing such constraints, and probe the extent at depth of surface geologic observations. In this study, we provide detailed S-wave velocity profiles for the crust and uppermost mantle beneath the Saudi Arabian Portable Broadband Deployment stations. Seismic velocities have been estimated from the joint inversion of receiver functions and fundamental mode group velocities. Receiver functions are sensitive to S-wave velocity contrasts and vertical travel times, and surface-wave dispersion is sensitive to vertical S-wave velocity averages, so that their combination bridge resolution gaps associated with each individual data set. Our resulting models correlate well with surface geology observations in the Arabian Shield and characterize its terranes at depth: the Asir terrane consists of a 10-km thick upper crust of 3.3~km/s overlying a lower crust with shear-wave velocities of 3.7-3.8 km/s; the Afif terrane is made of a 20-km thick upper crust with average velocity of 3.6 km/s and a lower crust with a shear-velocity of about 3.8~km/s; the Nabitah mobile belt has a gradational, 15-km thick upper crust up to 3.6 km/s overlying a gradational lower crust with velocities up to 4.0 km/s. The crust-mantle transition is sharper in terranes of continental affinity and more gradational beneath terranes of oceanic affinity. In the uppermost mantle, our models suggest a thin lid between up to 50-60 km depth overlying a low velocity zone beneath station TAIF, located close to a region of upwelling mantle material. Temperatures in the lid are estimated to be about 1000 C, which are in good agreement with independent xenolith data, and suggest that the lithosphere could be eroded to a thickness as little as 50~km under this station.

A laboratory nanoseismological study on deep-focus earthquake micromechanics

DOE PAGES

Wang, Yanbin; Zhu, Lupei; Shi, Feng; ...

2017-07-21

Global earthquake occurring rate displays an exponential decay down to ~300 km and then peaks around 550 to 600 km before terminating abruptly near 700 km. How fractures initiate, nucleate, and propagate at these depths remains one of the greatest puzzles in earth science, as increasing pressure inhibits fracture propagation. We report nanoseismological analysis on high-resolution acoustic emission (AE) records obtained during ruptures triggered by partial transformation from olivine to spinel in Mg 2GeO 4, an analog to the dominant mineral (Mg,Fe) 2SiO 4 olivine in the upper mantle, using state-of-the-art seismological techniques, in the laboratory. AEs’ focal mechanisms, asmore » well as their distribution in both space and time during deformation, are carefully analyzed. Microstructure analysis shows that AEs are produced by the dynamic propagation of shear bands consisting of nanograined spinel. These nanoshear bands have a near constant thickness (~100 nm) but varying lengths and self-organize during deformation. This precursory seismic process leads to ultimate macroscopic failure of the samples. Several source parameters of AE events were extracted from the recorded waveforms, allowing close tracking of event initiation, clustering, and propagation throughout the deformation/transformation process. AEs follow the Gutenberg-Richter statistics with a well-defined b value of 1.5 over three orders of moment magnitudes, suggesting that laboratory failure processes are self-affine. The seismic relation between magnitude and rupture area correctly predicts AE magnitude at millimeter scales. A rupture propagation model based on strain localization theory is proposed. Future numerical analyses may help resolve scaling issues between laboratory AE events and deep-focus earthquakes.« less

A laboratory nanoseismological study on deep-focus earthquake micromechanics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Yanbin; Zhu, Lupei; Shi, Feng

Global earthquake occurring rate displays an exponential decay down to ~300 km and then peaks around 550 to 600 km before terminating abruptly near 700 km. How fractures initiate, nucleate, and propagate at these depths remains one of the greatest puzzles in earth science, as increasing pressure inhibits fracture propagation. We report nanoseismological analysis on high-resolution acoustic emission (AE) records obtained during ruptures triggered by partial transformation from olivine to spinel in Mg2GeO4, an analog to the dominant mineral (Mg,Fe)2SiO4 olivine in the upper mantle, using state-of-the-art seismological techniques, in the laboratory. AEs’ focal mechanisms, as well as their distributionmore » in both space and time during deformation, are carefully analyzed. Microstructure analysis shows that AEs are produced by the dynamic propagation of shear bands consisting of nanograined spinel. These nanoshear bands have a near constant thickness (~100 nm) but varying lengths and self-organize during deformation. This precursory seismic process leads to ultimate macroscopic failure of the samples. Several source parameters of AE events were extracted from the recorded waveforms, allowing close tracking of event initiation, clustering, and propagation throughout the deformation/transformation process. AEs follow the Gutenberg-Richter statistics with a well-defined b value of 1.5 over three orders of moment magnitudes, suggesting that laboratory failure processes are self-affine. The seismic relation between magnitude and rupture area correctly predicts AE magnitude at millimeter scales. A rupture propagation model based on strain localization theory is proposed. Future numerical analyses may help resolve scaling issues between laboratory AE events and deep-focus earthquakes.« less

A laboratory nanoseismological study on deep-focus earthquake micromechanics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Yanbin; Zhu, Lupei; Shi, Feng

Global earthquake occurring rate displays an exponential decay down to ~300 km and then peaks around 550 to 600 km before terminating abruptly near 700 km. How fractures initiate, nucleate, and propagate at these depths remains one of the greatest puzzles in earth science, as increasing pressure inhibits fracture propagation. We report nanoseismological analysis on high-resolution acoustic emission (AE) records obtained during ruptures triggered by partial transformation from olivine to spinel in Mg 2GeO 4, an analog to the dominant mineral (Mg,Fe) 2SiO 4 olivine in the upper mantle, using state-of-the-art seismological techniques, in the laboratory. AEs’ focal mechanisms, asmore » well as their distribution in both space and time during deformation, are carefully analyzed. Microstructure analysis shows that AEs are produced by the dynamic propagation of shear bands consisting of nanograined spinel. These nanoshear bands have a near constant thickness (~100 nm) but varying lengths and self-organize during deformation. This precursory seismic process leads to ultimate macroscopic failure of the samples. Several source parameters of AE events were extracted from the recorded waveforms, allowing close tracking of event initiation, clustering, and propagation throughout the deformation/transformation process. AEs follow the Gutenberg-Richter statistics with a well-defined b value of 1.5 over three orders of moment magnitudes, suggesting that laboratory failure processes are self-affine. The seismic relation between magnitude and rupture area correctly predicts AE magnitude at millimeter scales. A rupture propagation model based on strain localization theory is proposed. Future numerical analyses may help resolve scaling issues between laboratory AE events and deep-focus earthquakes.« less

Use of Mechanistic Modeling to Assess Interindividual Variability and Interspecies Differences in Active Uptake in Human and Rat Hepatocytes

PubMed Central

Ménochet, Karelle; Kenworthy, Kathryn E.; Houston, J. Brian

2012-01-01

Interindividual variability in activity of uptake transporters is evident in vivo, yet limited data exist in vitro, confounding in vitro-in vivo extrapolation. The uptake kinetics of seven organic anion-transporting polypeptide substrates was investigated over a concentration range in plated cryopreserved human hepatocytes. Active uptake clearance (CLactive, u), bidirectional passive diffusion (Pdiff), intracellular binding, and metabolism were estimated for bosentan, pitavastatin, pravastatin, repaglinide, rosuvastatin, telmisartan, and valsartan in HU4122 donor using a mechanistic two-compartment model in Matlab. Full uptake kinetics of rosuvastatin and repaglinide were also characterized in two additional donors, whereas for the remaining drugs CLactive, u was estimated at a single concentration. The unbound affinity constant (Km, u) and Pdiff values were consistent across donors, whereas Vmax was on average up to 2.8-fold greater in donor HU4122. Consistency in Km, u values allowed extrapolation of single concentration uptake activity data and assessment of interindividual variability in CLactive across donors. The maximal contribution of active transport to total uptake differed among donors, for example, 85 to 96% and 68 to 87% for rosuvastatin and repaglinide, respectively; however, in all cases the active process was the major contributor. In vitro-in vivo extrapolation indicated a general underprediction of hepatic intrinsic clearance, an average empirical scaling factor of 17.1 was estimated on the basis of seven drugs investigated in three hepatocyte donors, and donor-specific differences in empirical factors are discussed. Uptake Km, u and CLactive, u were on average 4.3- and 7.1-fold lower in human hepatocytes compared with our previously published rat data. A strategy for the use of rat uptake data to facilitate the experimental design in human hepatocytes is discussed. PMID:22665271

Chromatographic studies of drug interactions with alpha1-acid glycoprotein by ultrafast affinity extraction and peak profiling.

PubMed

Beeram, Sandya; Bi, Cong; Zheng, Xiwei; Hage, David S

2017-05-12

Interactions with serum proteins such as alpha 1 -acid glycoprotein (AGP) can have a significant effect on the behavior and pharmacokinetics of drugs. Ultrafast affinity extraction and peak profiling were used with AGP microcolumns to examine these processes for several model drugs (i.e., chlorpromazine, disopyramide, imipramine, lidocaine, propranolol and verapamil). The association equilibrium constants measured for these drugs with soluble AGP by ultrafast affinity extraction were in the general range of 10 4 -10 6 M -1 at pH 7.4 and 37°C and gave good agreement with literature values. Some of these values were dependent on the relative drug and protein concentrations that were present when using a single-site binding model; these results suggested a more complex mixed-mode interaction was actually present, which was also then used to analyze the data. The apparent dissociation rate constants that were obtained by ultrafast affinity extraction when using a single-site model varied from 0.14 to 7.0s -1 and were dependent on the relative drug and protein concentrations. Lower apparent dissociation rate constants were obtained by this approach as the relative amount of drug versus protein was decreased, with the results approaching those measured by peak profiling at low drug concentrations. This information should be useful in better understanding how these and other drugs interact with AGP in the circulation. In addition, the chromatographic approaches that were optimized and used in this report to examine these systems can be adapted for the analysis of other solute-protein interactions of biomedical interest. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification of a β-lactamase inhibitory protein variant that is a potent inhibitor of Staphylococcus PC1 β-lactamase

PubMed Central

Yuan, Ji; Chow, Dar-Chone; Huang, Wanzhi; Palzkill, Timothy

2011-01-01

The β-lactamase inhibitory protein (BLIP) binds and inhibits a diverse collection of class A β-lactamases. Widespread resistance to β-lactam antibiotics currently limits treatment strategies for Staphylococcus infections. The goal of this study was to determine the binding affinity of BLIP for S. aureus PC1 β-lactamase and to identify mutants that alter binding affinity. The BLIP inhibition constant (Ki) for the PC1 β-lactamase was measured at 350 nM and isothermal titration calorimetry (ITC) experiments indicated a binding constant (Kd) of 380 nM. A total of 23 residue positions in BLIP that contact β-lactamase were randomized and phage display was used to sort the libraries for tight binders to immobilized PC1 β-lactamase. The BLIP K74G mutant was the dominant clone selected and it was found to inhibit the PC1 β-lactamase with a Ki of 42 nM while calorimetry indicated a Kd of 26 nM. Molecular modeling studies suggested BLIP binds weakly to the PC1 β-lactamase due to the presence of alanine at position 104 of PC1. This position is occupied by glutamate in the TEM-1 enzyme where it forms a salt bridge with BLIP residue Lys74 that is important for the stability of the complex. This hypothesis was confirmed by showing that the A104E PC1 enzyme binds BLIP with 15-fold greater affinity than wild type PC1 β-lactamase. Kinetic measurements indicated similar association rates for all complexes with the variation in affinity due to altered dissociation rate constants suggesting changes in short-range interactions are responsible for the altered binding properties of the mutants. PMID:21238457

Characterization of the functional role of Asp141, Asp194, and Asp464 residues in the Mn2+-L-malate binding of pigeon liver malic enzyme.

PubMed

Chou, W Y; Chang, H P; Huang, C H; Kuo, C C; Tong, L; Chang, G G

2000-02-01

Pigeon liver malic enzyme was inactivated and cleaved at Asp141, Asp194, and Asp464 by the Cu2+-ascorbate system in acidic environment. Site-specific mutagenesis was performed at these putative metal-binding sites. Three point mutants, D141N, D194N, and D464N; three double mutants, D(141,194)N, D(194,464)N, and D(141,464)N; and a triple mutant, D(141,194,464)N; as well as the wild-type malic enzyme (WT) were successfully cloned and expressed in Escherichia coli cells. All recombinant enzymes, except the triple mutant, were purified to apparent homogeneity by successive Q-Sepharose and adenosine-2',5'-bisphosphate-agarose columns. The mutants showed similar apparent Km,NADP values to that of the WT. The Km,Mal value was increased in the D141N and D194N mutants. The Km,Mn value, on the other hand, was increased only in the D141N mutant by 14-fold, corresponding to approximately 1.6 kcal/mol for the Asp141-Mn2+ binding energy. Substrate inhibition by L-malate was only observed in WT, D464N, and D(141,464)N. Initial velocity experiments were performed to derive the various kinetic parameters. The possible interactions between Asp141, Asp194, and Asp464 were analyzed by the double-mutation cycles and triple-mutation box. There are synergistic weakening interactions between Asp141 and Asp194 in the metal binding that impel the D(141,194)N double mutant to an overall specificity constant [k(cat)/(Kd,Mn Km,Mal Km,NADP)] at least four orders of magnitude smaller than the WT value. This difference corresponds to an increase of 6.38 kcal/mol energy barrier for the catalytic efficiency. Mutation at Asp464, on the other hand, has partial additivity on the mutations at Asp141 and Asp194. The overall specificity constants for the double mutants D(194,464)N and D(141,464)N or the triple mutant D(141,194,464)N were decreased by only 10- to 100-fold compared to the WT. These results strongly suggest the involvement of Asp141 in the Mn2+-L-malate binding for the pigeon liver malic enzyme. The Asp194 and Asp464, which may be oxidized by nonspecific binding of Cu2+, are involved in the Mn2+-L-malate binding or catalysis indirectly by modulating the binding affinity of Asp141 with the Mn2+.

Size and molecular flexibility affect the binding of ellagitannins to bovine serum albumin.

PubMed

Dobreva, Marina A; Green, Rebecca J; Mueller-Harvey, Irene; Salminen, Juha-Pekka; Howlin, Brendan J; Frazier, Richard A

2014-09-17

Binding to bovine serum albumin of monomeric (vescalagin and pedunculagin) and dimeric ellagitannins (roburin A, oenothein B, and gemin A) was investigated by isothermal titration calorimetry and fluorescence spectroscopy, which indicated two types of binding sites. Stronger and more specific sites exhibited affinity constants, K1, of 10(4)-10(6) M(-1) and stoichiometries, n1, of 2-13 and dominated at low tannin concentrations. Weaker and less-specific binding sites had K2 constants of 10(3)-10(5) M(-1) and stoichiometries, n2, of 16-30 and dominated at higher tannin concentrations. Binding to stronger sites appeared to be dependent on tannin flexibility and the presence of free galloyl groups. Positive entropies for all but gemin A indicated that hydrophobic interactions dominated during complexation. This was supported by an exponential relationship between the affinity, K1, and the modeled hydrophobic accessible surface area and by a linear relationship between K1 and the Stern-Volmer quenching constant, K(SV).

Rate constants of agonist binding to muscarinic receptors in rat brain medulla. Evaluation by competition kinetics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schreiber, G.; Henis, Y.I.; Sokolovsky, M.

The method of competition kinetics, which measures the binding kinetics of an unlabeled ligand through its effect on the binding kinetics of a labeled ligand, was employed to investigate the kinetics of muscarinic agonist binding to rat brain medulla pons homogenates. The agonists studied were acetylcholine, carbamylcholine, and oxotremorine, with N-methyl-4-(TH)piperidyl benzilate employed as the radiolabeled ligand. Our results suggested that the binding of muscarinic agonists to the high affinity sites is characterized by dissociation rate constants higher by 2 orders of magnitude than those of antagonists, with rather similar association rate constants. Our findings also suggest that isomerization ofmore » the muscarinic receptors following ligand binding is significant in the case of antagonists, but not of agonists. Moreover, it is demonstrated that in the medulla pons preparation, agonist-induced interconversion between high and low affinity bindings sites does not occur to an appreciable extent.« less

Nitrous oxide production kinetics during nitrate reduction in river sediments.

PubMed

Laverman, Anniet M; Garnier, Josette A; Mounier, Emmanuelle M; Roose-Amsaleg, Céline L

2010-03-01

A significant amount of nitrogen entering river basins is denitrified in riparian zones. The aim of this study was to evaluate the influence of nitrate and carbon concentrations on the kinetic parameters of nitrate reduction as well as nitrous oxide emissions in river sediments in a tributary of the Marne (the Seine basin, France). In order to determine these rates, we used flow-through reactors (FTRs) and slurry incubations; flow-through reactors allow determination of rates on intact sediment slices under controlled conditions compared to sediment homogenization in the often used slurry technique. Maximum nitrate reduction rates (R(m)) ranged between 3.0 and 7.1microg Ng(-1)h(-1), and affinity constant (K(m)) ranged from 7.4 to 30.7mg N-NO(3)(-)L(-1). These values were higher in slurry incubations with an R(m) of 37.9microg Ng(-1)h(-1) and a K(m) of 104mg N-NO(3)(-)L(-1). Nitrous oxide production rates did not follow Michaelis-Menten kinetics, and we deduced a rate constant with an average of 0.7 and 5.4ng Ng(-1)h(-1) for FTR and slurry experiments respectively. The addition of carbon (as acetate) showed that carbon was not limiting nitrate reduction rates in these sediments. Similar rates were obtained for FTR and slurries with carbon addition, confirming the hypothesis that homogenization increases rates due to release of and increasing access to carbon in slurries. Nitrous oxide production rates in FTR with carbon additions were low and represented less than 0.01% of the nitrate reduction rates and were even negligible in slurries. Maximum nitrate reduction rates revealed seasonality with high potential rates in fall and winter and low rates in late spring and summer. Under optimal conditions (anoxia, non-limiting nitrate and carbon), nitrous oxide emission rates were low, but significant (0.01% of the nitrate reduction rates). Copyright 2009 Elsevier Ltd. All rights reserved.

The Cutting Edge of Affinity Electrophoresis Technology

PubMed Central

Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Koike, Tohru

2015-01-01

Affinity electrophoresis is an important technique that is widely used to separate and analyze biomolecules in the fields of biology and medicine. Both quantitative and qualitative information can be gained through affinity electrophoresis. Affinity electrophoresis can be applied through a variety of strategies, such as mobility shift electrophoresis, charge shift electrophoresis or capillary affinity electrophoresis. These strategies are based on changes in the electrophoretic patterns of biological macromolecules that result from interactions or complex-formation processes that induce changes in the size or total charge of the molecules. Nucleic acid fragments can be characterized through their affinity to other molecules, for example transcriptional factor proteins. Hydrophobic membrane proteins can be identified by means of a shift in the mobility induced by a charged detergent. The various strategies have also been used in the estimation of association/disassociation constants. Some of these strategies have similarities to affinity chromatography, in that they use a probe or ligand immobilized on a supported matrix for electrophoresis. Such methods have recently contributed to profiling of major posttranslational modifications of proteins, such as glycosylation or phosphorylation. Here, we describe advances in analytical techniques involving affinity electrophoresis that have appeared during the last five years. PMID:28248262

Role of Reversible Histidine Coordination in Hydroxylamine Reduction by Plant Hemoglobins (Phytoglobins).

PubMed

Athwal, Navjot Singh; Alagurajan, Jagannathan; Andreotti, Amy H; Hargrove, Mark S

2016-10-18

Reduction of hydroxylamine to ammonium by phytoglobin, a plant hexacoordinate hemoglobin, is much faster than that of other hexacoordinate hemoglobins or pentacoordinate hemoglobins such as myoglobin, leghemoglobin, and red blood cell hemoglobin. The reason for differences in reactivity is not known but could be intermolecular electron transfer between protein molecules in support of the required two-electron reduction, hydroxylamine binding, or active site architecture favoring the reaction. Experiments were conducted with phytoglobins from rice, tomato, and soybean along with human neuroglobin and soybean leghemoglobin that reveal hydroxylamine binding as the rate-limiting step. For hexacoordinate hemoglobins, binding is limited by the dissociation rate constant for the distal histidine, while leghemoglobin is limited by an intrinsically low affinity for hydroxylamine. When the distal histidine is removed from rice phytoglobin, a hydroxylamine-bound intermediate is formed and the reaction rate is diminished, indicating that the distal histidine imidazole side chain is critical for the reaction, albeit not for electron transfer but rather for direct interaction with the substrate. Together, these results demonstrate that phytoglobins are superior at hydroxylamine reduction because they have distal histidine coordination affinity constants near 1, and facile rate constants for binding and dissociation of the histidine side chain. Hexacoordinate hemoglobins such as neuroglobin are limited by tighter histidine coordination that blocks hydroxylamine binding, and pentacoordinate hemoglobins have intrinsically lower hydroxylamine affinities.

Measurement of the affinity and phosphorylation constants governing irreversible inhibition of cholinesterases by di-isopropyl phosphorofluoridate

PubMed Central

Main, A. R.; Iverson, F.

1966-01-01

1. A procedure is described for determining the affinity constant Ka and the phosphorylation constant kp for the inhibition by di-isopropyl phosphorofluoridate of erythrocyte acetylcholinesterase and serum cholinesterase. The procedure depends on the use of a specially designed reaction vessel with which incubation times as short as 1·2sec. could be obtained at any convenient temperature. 2. The Ka of acetylcholinesterase decreased from 1·58 (±0·22)×10−3m at 5° to 1·17 (±0·10)×10−3m at 25° and the associated change in enthalpy was 2980 cal. 3. The kp of acetylcholinesterase increased from 11·9 (±0·7)min.−1 at 5° to 40·7 (±1·4)min.−1 at 25°, indicating an activational energy of 9600 cal. The change in entropy associated with Ka was 23·5 cal. degree−1 at 25°. 4. At 5°, the Ka and kp of serum cholinesterase were 9·95 (±1·10)×10−6m and 11·2 (±0·63)min.−1 respectively. 5. The 150-fold difference in the inhibitory power of di-isopropyl phosphorofluoridate for the two cholinesterases was attributed entirely to differences in affinity. PMID:5968549

Photolabeling of Tonoplast from Sugar Beet Cell Suspensions by [3H]5-(N-Methyl-N-Isobutyl)-Amiloride, an Inhibitor of the Vacuolar Na+/H+ Antiport 1

PubMed Central

Barkla, Bronwyn J.; Charuk, Jeffrey H. M.; Cragoe, Edward J.; Blumwald, Eduardo

1990-01-01

The effects of 5-(N-methyl-N-isobutyl)-amiloride (MIA), an amiloride analog, was tested on the Na+/H+ antiport activity of intact vacuoles and tonoplast vesicles isolated from sugar beet (Beta vulgaris L.) cell suspension cultures. MIA inhibited Na+/H+ exchange in a competitive manner with a Ki of 2.5 and 5.9 micromolar for ΔpH-dependent 22Na+ influx in tonoplast vesicles and Na+-dependent H+ efflux in intact vacuoles, respectively. Scatchard analysis of the binding of [3H]MIA to tonoplast membranes revealed a high affinity binding component with a Kd of 1.3 micromolar. The close relationship between the dissociation constant value obtained and the constants of inhibition for MIA obtained by fluorescence quenching and isotope exchange suggests that the high affinity component represents a class of sites associated with the tonoplast Na+/H+ antiport. Photolabeling of the tonoplast with [3H]MIA revealed two sets of polypeptides with a different affinity to amiloride and its analog. Images Figure 7 PMID:16667602

Photolabeling of tonoplast from sugar beet cell suspensions by [h]5-(N-methyl-N-isobutyl)-amiloride, an inhibitor of the vacuolar na/h antiport.

PubMed

Barkla, B J; Charuk, J H; Cragoe, E J; Blumwald, E

1990-07-01

The effects of 5-(N-methyl-N-isobutyl)-amiloride (MIA), an amiloride analog, was tested on the Na(+)/H(+) antiport activity of intact vacuoles and tonoplast vesicles isolated from sugar beet (Beta vulgaris L.) cell suspension cultures. MIA inhibited Na(+)/H(+) exchange in a competitive manner with a K(i) of 2.5 and 5.9 micromolar for DeltapH-dependent (22)Na(+) influx in tonoplast vesicles and Na(+)-dependent H(+) efflux in intact vacuoles, respectively. Scatchard analysis of the binding of [(3)H]MIA to tonoplast membranes revealed a high affinity binding component with a K(d) of 1.3 micromolar. The close relationship between the dissociation constant value obtained and the constants of inhibition for MIA obtained by fluorescence quenching and isotope exchange suggests that the high affinity component represents a class of sites associated with the tonoplast Na(+)/H(+) antiport. Photolabeling of the tonoplast with [(3)H]MIA revealed two sets of polypeptides with a different affinity to amiloride and its analog.

A global benchmark study using affinity-based biosensors

PubMed Central

Rich, Rebecca L.; Papalia, Giuseppe A.; Flynn, Peter J.; Furneisen, Jamie; Quinn, John; Klein, Joshua S.; Katsamba, Phini S.; Waddell, M. Brent; Scott, Michael; Thompson, Joshua; Berlier, Judie; Corry, Schuyler; Baltzinger, Mireille; Zeder-Lutz, Gabrielle; Schoenemann, Andreas; Clabbers, Anca; Wieckowski, Sebastien; Murphy, Mary M.; Page, Phillip; Ryan, Thomas E.; Duffner, Jay; Ganguly, Tanmoy; Corbin, John; Gautam, Satyen; Anderluh, Gregor; Bavdek, Andrej; Reichmann, Dana; Yadav, Satya P.; Hommema, Eric; Pol, Ewa; Drake, Andrew; Klakamp, Scott; Chapman, Trevor; Kernaghan, Dawn; Miller, Ken; Schuman, Jason; Lindquist, Kevin; Herlihy, Kara; Murphy, Michael B.; Bohnsack, Richard; Andrien, Bruce; Brandani, Pietro; Terwey, Danny; Millican, Rohn; Darling, Ryan J.; Wang, Liann; Carter, Quincy; Dotzlaf, Joe; Lopez-Sagaseta, Jacinto; Campbell, Islay; Torreri, Paola; Hoos, Sylviane; England, Patrick; Liu, Yang; Abdiche, Yasmina; Malashock, Daniel; Pinkerton, Alanna; Wong, Melanie; Lafer, Eileen; Hinck, Cynthia; Thompson, Kevin; Primo, Carmelo Di; Joyce, Alison; Brooks, Jonathan; Torta, Federico; Bagge Hagel, Anne Birgitte; Krarup, Janus; Pass, Jesper; Ferreira, Monica; Shikov, Sergei; Mikolajczyk, Malgorzata; Abe, Yuki; Barbato, Gaetano; Giannetti, Anthony M.; Krishnamoorthy, Ganeshram; Beusink, Bianca; Satpaev, Daulet; Tsang, Tiffany; Fang, Eric; Partridge, James; Brohawn, Stephen; Horn, James; Pritsch, Otto; Obal, Gonzalo; Nilapwar, Sanjay; Busby, Ben; Gutierrez-Sanchez, Gerardo; Gupta, Ruchira Das; Canepa, Sylvie; Witte, Krista; Nikolovska-Coleska, Zaneta; Cho, Yun Hee; D’Agata, Roberta; Schlick, Kristian; Calvert, Rosy; Munoz, Eva M.; Hernaiz, Maria Jose; Bravman, Tsafir; Dines, Monica; Yang, Min-Hsiang; Puskas, Agnes; Boni, Erica; Li, Jiejin; Wear, Martin; Grinberg, Asya; Baardsnes, Jason; Dolezal, Olan; Gainey, Melicia; Anderson, Henrik; Peng, Jinlin; Lewis, Mark; Spies, Peter; Trinh, Quyhn; Bibikov, Sergei; Raymond, Jill; Yousef, Mohammed; Chandrasekaran, Vidya; Feng, Yuguo; Emerick, Anne; Mundodo, Suparna; Guimaraes, Rejane; McGirr, Katy; Li, Yue-Ji; Hughes, Heather; Mantz, Hubert; Skrabana, Rostislav; Witmer, Mark; Ballard, Joshua; Martin, Loic; Skladal, Petr; Korza, George; Laird-Offringa, Ite; Lee, Charlene S.; Khadir, Abdelkrim; Podlaski, Frank; Neuner, Phillippe; Rothacker, Julie; Rafique, Ashique; Dankbar, Nico; Kainz, Peter; Gedig, Erk; Vuyisich, Momchilo; Boozer, Christina; Ly, Nguyen; Toews, Mark; Uren, Aykut; Kalyuzhniy, Oleksandr; Lewis, Kenneth; Chomey, Eugene; Pak, Brian J.; Myszka, David G.

2013-01-01

To explore the variability in biosensor studies, 150 participants from 20 countries were given the same protein samples and asked to determine kinetic rate constants for the interaction. We chose a protein system that was amenable to analysis using different biosensor platforms as well as by users of different expertise levels. The two proteins (a 50-kDa Fab and a 60-kDa glutathione S-transferase [GST] antigen) form a relatively high-affinity complex, so participants needed to optimize several experimental parameters, including ligand immobilization and regeneration conditions as well as analyte concentrations and injection/dissociation times. Although most participants collected binding responses that could be fit to yield kinetic parameters, the quality of a few data sets could have been improved by optimizing the assay design. Once these outliers were removed, the average reported affinity across the remaining panel of participants was 620 pM with a standard deviation of 980 pM. These results demonstrate that when this biosensor assay was designed and executed appropriately, the reported rate constants were consistent, and independent of which protein was immobilized and which biosensor was used. PMID:19133223

Measurement of radon and xenon binding to a cryptophane molecular host

PubMed Central

Jacobson, David R.; Khan, Najat S.; Collé, Ronald; Fitzgerald, Ryan; Laureano-Pérez, Lizbeth; Bai, Yubin; Dmochowski, Ivan J.

2011-01-01

Xenon and radon have many similar properties, a difference being that all 35 isotopes of radon (195Rn–229Rn) are radioactive. Radon is a pervasive indoor air pollutant believed to cause significant incidence of lung cancer in many geographic regions, yet radon affinity for a discrete molecular species has never been determined. By comparison, the chemistry of xenon has been widely studied and applied in science and technology. Here, both noble gases were found to bind with exceptional affinity to tris-(triazole ethylamine) cryptophane, a previously unsynthesized water-soluble organic host molecule. The cryptophane–xenon association constant, Ka = 42,000 ± 2,000 M-1 at 293 K, was determined by isothermal titration calorimetry. This value represents the highest measured xenon affinity for a host molecule. The partitioning of radon between air and aqueous cryptophane solutions of varying concentration was determined radiometrically to give the cryptophane–radon association constant Ka = 49,000 ± 12,000 M-1 at 293 K. PMID:21690357

Eruptive history of Mount Katmai, Alaska

USGS Publications Warehouse

Hildreth, Edward; Fierstein, Judith

2012-01-01

Compositionally, products of Mount Katmai represent an ordinary medium-K arc array, both tholeiitic and calcalkaline, that extends from 51.6% to 72.3% SiO2. Values of 87Sr/86Sr range from 0.70335 to 0.70372, correlating loosely with fractionation indices. The 5–6 km3 of continuously zoned andesite-dacite magma (58%–68% SiO2) that erupted at Novarupta in 1912 was withdrawn from beneath Mount Katmai and bears close compositional affinity with products of that edifice, not with pre-1912 products of the adjacent Trident cluster. Evidence is presented that the 7–8 km3 of high-silica rhyolite (77% SiO2) released in 1912 is unlikely to have been stored under Novarupta or Trident. Pre-eruptive contiguity with the andesite-dacite reservoir is suggested by (1) eruption of ∼3 km3 of rhyolite magma first, followed by mutual mingling in fluctuating proportions; (2) thermal and redox continuity of the whole zoned sequence despite the wide compositional gap; (3) Nd, Sr, O isotopic, and rare earth element (REE) affinities of the whole array; (4) compositional continuity of the nearly aphyric rhyolite with the glass (melt) phase of the phenocryst-rich dacite; and (5) phase-equilibrium experiments that indicate similar shallow pre-eruptive storage depths (3–6 km) for rhyolite, dacite, and andesite.

Affine generalization of the Komar complex of general relativity

NASA Astrophysics Data System (ADS)

Mielke, Eckehard W.

2001-02-01

On the basis of the ``on shell'' Noether identities of the metric-affine gauge approach of gravity, an affine superpotential is derived which comprises the energy- and angular-momentum content of exact solutions. In the special case of general relativity (GR) or its teleparallel equivalent, the Komar or Freud complex, respectively, are recovered. Applying this to the spontaneously broken anti-de Sitter gauge model of McDowell and Mansouri with an induced Euler term automatically yields the correct mass and spin of the Kerr-AdS solution of GR with a (induced) cosmological constant without the factor two discrepancy of the Komar formula.

Differential Mobility Spectrometry: Preliminary Findings on Determination of Fundamental Constants

NASA Technical Reports Server (NTRS)

Limero, Thomas; Cheng, Patti; Boyd, John

2007-01-01

The electron capture detector (ECD) has been used for 40+ years (1) to derive fundamental constants such as a compound's electron affinity. Given this historical perspective, it is not surprising that differential mobility spectrometry (DMS) might be used in a like manner. This paper will present data from a gas chromatography (GC)-DMS instrument that illustrates the potential capability of this device to derive fundamental constants for electron-capturing compounds. Potential energy curves will be used to provide possible explanation of the data.

Higher Nucleoporin-Importinβ Affinity at the Nuclear Basket Increases Nucleocytoplasmic Import

PubMed Central

Azimi, Mohammad; Mofrad, Mohammad R. K.

2013-01-01

Several in vitro studies have shown the presence of an affinity gradient in nuclear pore complex proteins for the import receptor Importinβ, at least partially contributing to nucleocytoplasmic transport, while others have historically argued against the presence of such a gradient. Nonetheless, the existence of an affinity gradient has remained an uncharacterized contributing factor. To shed light on the affinity gradient theory and better characterize how the existence of such an affinity gradient between the nuclear pore and the import receptor may influence the nucleocytoplasmic traffic, we have developed a general-purpose agent based modeling (ABM) framework that features a new method for relating rate constants to molecular binding and unbinding probabilities, and used our ABM approach to quantify the effects of a wide range of forward and reverse nucleoporin-Importinβ affinity gradients. Our results indicate that transport through the nuclear pore complex is maximized with an effective macroscopic affinity gradient of 2000 µM, 200 µM and 10 µM in the cytoplasmic, central channel and nuclear basket respectively. The transport rate at this gradient is approximately 10% higher than the transport rate for a comparable pore lacking any affinity gradient, which has a peak transport rate when all nucleoporins have an affinity of 200 µM for Importinβ. Furthermore, this optimal ratio of affinity gradients is representative of the ratio of affinities reported for the yeast nuclear pore complex – suggesting that the affinity gradient seen in vitro is highly optimized. PMID:24282617

Binding site and affinity prediction of general anesthetics to protein targets using docking.

PubMed

Liu, Renyu; Perez-Aguilar, Jose Manuel; Liang, David; Saven, Jeffery G

2012-05-01

The protein targets for general anesthetics remain unclear. A tool to predict anesthetic binding for potential binding targets is needed. In this study, we explored whether a computational method, AutoDock, could serve as such a tool. High-resolution crystal data of water-soluble proteins (cytochrome C, apoferritin, and human serum albumin), and a membrane protein (a pentameric ligand-gated ion channel from Gloeobacter violaceus [GLIC]) were used. Isothermal titration calorimetry (ITC) experiments were performed to determine anesthetic affinity in solution conditions for apoferritin. Docking calculations were performed using DockingServer with the Lamarckian genetic algorithm and the Solis and Wets local search method (http://www.dockingserver.com/web). Twenty general anesthetics were docked into apoferritin. The predicted binding constants were compared with those obtained from ITC experiments for potential correlations. In the case of apoferritin, details of the binding site and their interactions were compared with recent cocrystallization data. Docking calculations for 6 general anesthetics currently used in clinical settings (isoflurane, sevoflurane, desflurane, halothane, propofol, and etomidate) with known 50% effective concentration (EC(50)) values were also performed in all tested proteins. The binding constants derived from docking experiments were compared with known EC(50) values and octanol/water partition coefficients for the 6 general anesthetics. All 20 general anesthetics docked unambiguously into the anesthetic binding site identified in the crystal structure of apoferritin. The binding constants for 20 anesthetics obtained from the docking calculations correlate significantly with those obtained from ITC experiments (P = 0.04). In the case of GLIC, the identified anesthetic binding sites in the crystal structure are among the docking predicted binding sites, but not the top ranked site. Docking calculations suggest a most probable binding site located in the extracellular domain of GLIC. The predicted affinities correlated significantly with the known EC(50) values for the 6 frequently used anesthetics in GLIC for the site identified in the experimental crystal data (P = 0.006). However, predicted affinities in apoferritin, human serum albumin, and cytochrome C did not correlate with these 6 anesthetics' known experimental EC(50) values. A weak correlation between the predicted affinities and the octanol/water partition coefficients was observed for the sites in GLIC. We demonstrated that anesthetic binding sites and relative affinities can be predicted using docking calculations in an automatic docking server (AutoDock) for both water-soluble and membrane proteins. Correlation of predicted affinity and EC(50) for 6 frequently used general anesthetics was only observed in GLIC, a member of a protein family relevant to anesthetic mechanism.

Binding Site and Affinity Prediction of General Anesthetics to Protein Targets Using Docking

PubMed Central

Liu, Renyu; Perez-Aguilar, Jose Manuel; Liang, David; Saven, Jeffery G.

2012-01-01

Background The protein targets for general anesthetics remain unclear. A tool to predict anesthetic binding for potential binding targets is needed. In this study, we explore whether a computational method, AutoDock, could serve as such a tool. Methods High-resolution crystal data of water soluble proteins (cytochrome C, apoferritin and human serum albumin), and a membrane protein (a pentameric ligand-gated ion channel from Gloeobacter violaceus, GLIC) were used. Isothermal titration calorimetry (ITC) experiments were performed to determine anesthetic affinity in solution conditions for apoferritin. Docking calculations were performed using DockingServer with the Lamarckian genetic algorithm and the Solis and Wets local search method (https://www.dockingserver.com/web). Twenty general anesthetics were docked into apoferritin. The predicted binding constants are compared with those obtained from ITC experiments for potential correlations. In the case of apoferritin, details of the binding site and their interactions were compared with recent co-crystallization data. Docking calculations for six general anesthetics currently used in clinical settings (isoflurane, sevoflurane, desflurane, halothane, propofol, and etomidate) with known EC50 were also performed in all tested proteins. The binding constants derived from docking experiments were compared with known EC50s and octanol/water partition coefficients for the six general anesthetics. Results All 20 general anesthetics docked unambiguously into the anesthetic binding site identified in the crystal structure of apoferritin. The binding constants for 20 anesthetics obtained from the docking calculations correlate significantly with those obtained from ITC experiments (p=0.04). In the case of GLIC, the identified anesthetic binding sites in the crystal structure are among the docking predicted binding sites, but not the top ranked site. Docking calculations suggest a most probable binding site located in the extracellular domain of GLIC. The predicted affinities correlated significantly with the known EC50s for the six commonly used anesthetics in GLIC for the site identified in the experimental crystal data (p=0.006). However, predicted affinities in apoferritin, human serum albumin, and cytochrome C did not correlate with these six anesthetics’ known experimental EC50s. A weak correlation between the predicted affinities and the octanol/water partition coefficients was observed for the sites in GLIC. Conclusion We demonstrated that anesthetic binding sites and relative affinities can be predicted using docking calculations in an automatic docking server (Autodock) for both water soluble and membrane proteins. Correlation of predicted affinity and EC50 for six commonly used general anesthetics was only observed in GLIC, a member of a protein family relevant to anesthetic mechanism. PMID:22392968

Kinetics of sulfate and hydrogen uptake by the thermophilic sulfate-reducing bacteria thermodesulfobacterium sp. Strain JSP and thermodesulfovibrio sp. Strain R1Ha3

PubMed

Sonne-Hansen; Westermann; Ahring

1999-03-01

Half-saturation constants (Km), maximum uptake rates (Vmax), and threshold concentrations for sulfate and hydrogen were determined for two thermophilic sulfate-reducing bacteria (SRB) in an incubation system without headspace. Km values determined for the thermophilic SRB were similar to the constants described for mesophilic SRB isolated from environments with low sulfate concentrations.

Kinetics of Sulfate and Hydrogen Uptake by the Thermophilic Sulfate-Reducing Bacteria Thermodesulfobacterium sp. Strain JSP and Thermodesulfovibrio sp. Strain R1Ha3

PubMed Central

Sonne-Hansen, Jacob; Westermann, Peter; Ahring, Birgitte K.

1999-01-01

Half-saturation constants (Km), maximum uptake rates (Vmax), and threshold concentrations for sulfate and hydrogen were determined for two thermophilic sulfate-reducing bacteria (SRB) in an incubation system without headspace. Km values determined for the thermophilic SRB were similar to the constants described for mesophilic SRB isolated from environments with low sulfate concentrations. PMID:10049897

MOFzyme: Intrinsic protease-like activity of Cu-MOF.

PubMed

Li, Bin; Chen, Daomei; Wang, Jiaqiang; Yan, Zhiying; Jiang, Liang; Deliang Duan; He, Jiao; Luo, Zhongrui; Zhang, Jinping; Yuan, Fagui

2014-10-24

The construction of efficient enzyme mimetics for the hydrolysis of peptide bonds in proteins is challenging due to the high stability of peptide bonds and the importance of proteases in biology and industry. Metal-organic frameworks (MOFs) consisting of infinite crystalline lattices with metal clusters and organic linkers may provide opportunities for protease mimic which has remained unknown. Herein, we report that Cu₂(C₉H₃O₆)₄/₃ MOF (which is well known as HKUST-1 and denoted as Cu-MOF here), possesses an intrinsic enzyme mimicking activity similar to that found in natural trypsin to bovine serum albumin (BSA) and casein. The Michaelis constant (Km) of Cu-MOF is about 26,000-fold smaller than that of free trypsin indicating a much higher affinity of BSA for Cu-MOF surface. Cu-MOF also exhibited significantly higher catalytic efficiency than homogeneous artificial metalloprotease Cu(II) complexes and could be reused for ten times without losing in its activity. Moreover, Cu-MOF was successfully used to simulate trypsinization in cell culture since it dissociated cells in culture even without EDTA.

MOFzyme: Intrinsic protease-like activity of Cu-MOF

NASA Astrophysics Data System (ADS)

Li, Bin; Chen, Daomei; Wang, Jiaqiang; Yan, Zhiying; Jiang, Liang; Deliang Duan; He, Jiao; Luo, Zhongrui; Zhang, Jinping; Yuan, Fagui

2014-10-01

The construction of efficient enzyme mimetics for the hydrolysis of peptide bonds in proteins is challenging due to the high stability of peptide bonds and the importance of proteases in biology and industry. Metal-organic frameworks (MOFs) consisting of infinite crystalline lattices with metal clusters and organic linkers may provide opportunities for protease mimic which has remained unknown. Herein, we report that Cu2(C9H3O6)4/3 MOF (which is well known as HKUST-1 and denoted as Cu-MOF here), possesses an intrinsic enzyme mimicking activity similar to that found in natural trypsin to bovine serum albumin (BSA) and casein. The Michaelis constant (Km) of Cu-MOF is about 26,000-fold smaller than that of free trypsin indicating a much higher affinity of BSA for Cu-MOF surface. Cu-MOF also exhibited significantly higher catalytic efficiency than homogeneous artificial metalloprotease Cu(II) complexes and could be reused for ten times without losing in its activity. Moreover, Cu-MOF was successfully used to simulate trypsinization in cell culture since it dissociated cells in culture even without EDTA.

MOFzyme: Intrinsic protease-like activity of Cu-MOF

PubMed Central

Li, Bin; Chen, Daomei; Wang, Jiaqiang; Yan, Zhiying; Jiang, Liang; Deliang Duan; He, Jiao; Luo, Zhongrui; Zhang, Jinping; Yuan, Fagui

2014-01-01

The construction of efficient enzyme mimetics for the hydrolysis of peptide bonds in proteins is challenging due to the high stability of peptide bonds and the importance of proteases in biology and industry. Metal-organic frameworks (MOFs) consisting of infinite crystalline lattices with metal clusters and organic linkers may provide opportunities for protease mimic which has remained unknown. Herein, we report that Cu2(C9H3O6)4/3 MOF (which is well known as HKUST-1 and denoted as Cu-MOF here), possesses an intrinsic enzyme mimicking activity similar to that found in natural trypsin to bovine serum albumin (BSA) and casein. The Michaelis constant (Km) of Cu-MOF is about 26,000-fold smaller than that of free trypsin indicating a much higher affinity of BSA for Cu-MOF surface. Cu-MOF also exhibited significantly higher catalytic efficiency than homogeneous artificial metalloprotease Cu(II) complexes and could be reused for ten times without losing in its activity. Moreover, Cu-MOF was successfully used to simulate trypsinization in cell culture since it dissociated cells in culture even without EDTA. PMID:25342169

Ammonia oxidation kinetics determine niche separation of nitrifying Archaea and Bacteria.

PubMed

Martens-Habbena, Willm; Berube, Paul M; Urakawa, Hidetoshi; de la Torre, José R; Stahl, David A

2009-10-15

The discovery of ammonia oxidation by mesophilic and thermophilic Crenarchaeota and the widespread distribution of these organisms in marine and terrestrial environments indicated an important role for them in the global nitrogen cycle. However, very little is known about their physiology or their contribution to nitrification. Here we report oligotrophic ammonia oxidation kinetics and cellular characteristics of the mesophilic crenarchaeon 'Candidatus Nitrosopumilus maritimus' strain SCM1. Unlike characterized ammonia-oxidizing bacteria, SCM1 is adapted to life under extreme nutrient limitation, sustaining high specific oxidation rates at ammonium concentrations found in open oceans. Its half-saturation constant (K(m) = 133 nM total ammonium) and substrate threshold (

Familial secondary erythrocytosis due to increased oxygen affinity is caused by destabilization of the T state of hemoglobin Brigham (α2β2Pro100Leu)

PubMed Central

Mollan, Todd L; Abraham, Bindu; Strader, Michael Brad; Jia, Yiping; Lozier, Jay N; Olson, John S; Alayash, Abdu I

2012-01-01

Hemoglobin Brigham (β Pro100 to Leu) was first reported in a patient with familial erythrocytosis. Erythrocytes of an affected individual from the same family contain both HbA and Hb Brigham and exhibit elevated O2 affinity compared with normal cells (P50 = 23 mm Hg vs. 31 mmHg at pH 7.4 at 37°C). O2 affinities measured for hemolysates were sensitive to changes in pH or chloride concentrations, indicating little change in the Bohr and Chloride effects. Hb Brigham was separated from normal HbA by nondenaturing cation exchange liquid chromatography, and the amino acid substitution was verified by mass spectrometry. The properties of Hb Brigham isolated from the patient's blood were then compared with those of recombinant Hb Brigham expressed in Escherichia coli. Kinetic experiments suggest that the rate constants for ligand binding and release in the high (R) and low (T) affinity quaternary states of Hb Brigham are similar to those of native hemoglobin. However, the Brigham mutation decreases the T to R equilibrium constant (L) which accelerates the switch to the R state during ligand binding to deoxy-Hb, increasing the rate of association by approximately twofold, and decelerates the switch during ligand dissociation from HbO2, decreasing the rate approximately twofold. These kinetic data help explain the high O2 affinity characteristics of Hb Brigham and provide further evidence for the importance of the contribution of Pro100 to intersubunit contacts and stabilization of the T quaternary structure. PMID:22821886

Quantifying Protein-Ligand Binding Constants using Electrospray Ionization Mass Spectrometry: A Systematic Binding Affinity Study of a Series of Hydrophobically Modified Trypsin Inhibitors

NASA Astrophysics Data System (ADS)

Cubrilovic, Dragana; Biela, Adam; Sielaff, Frank; Steinmetzer, Torsten; Klebe, Gerhard; Zenobi, Renato

2012-10-01

NanoESI-MS is used for determining binding strengths of trypsin in complex with two different series of five congeneric inhibitors, whose binding affinity in solution depends on the size of the P3 substituent. The ligands of the first series contain a 4-amidinobenzylamide as P1 residue, and form a tight complex with trypsin. The inhibitors of the second series have a 2-aminomethyl-5-chloro-benzylamide as P1 group, and represent a model system for weak binders. The five different inhibitors of each group are based on the same scaffold and differ only in the length of the hydrophobic side chain of their P3 residue, which modulates the interactions in the S3/4 binding pocket of trypsin. The dissociation constants (KD) for high affinity ligands investigated by nanoESI-MS ranges from 15 nM to 450 nM and decreases with larger hydrophobic P3 side chains. Collision-induced dissociation (CID) experiments of five trypsin and benzamidine-based complexes show a correlation between trends in KD and gas-phase stability. For the second inhibitor series we could show that the effect of imidazole, a small stabilizing additive, can avoid the dissociation of the complex ions and as a result increases the relative abundance of weakly bound complexes. Here the KD values ranging from 2.9 to 17.6 μM, some 1-2 orders of magnitude lower than the first series. For both ligand series, the dissociation constants (KD) measured via nanoESI-MS were compared with kinetic inhibition constants (Ki) in solution.

Tropine dehydrogenase: purification, some properties and an evaluation of its role in the bacterial metabolism of tropine.

PubMed Central

Bartholomew, B A; Smith, M J; Long, M T; Darcy, P J; Trudgill, P W; Hopper, D J

1995-01-01

Tropine dehydrogenase was induced by growth of Pseudomonas AT3 on atropine, tropine or tropinone. It was NADP(+)-dependent and gave no activity with NAD+. The enzyme was very unstable but a rapid purification procedure using affinity chromatography that gave highly purified enzyme was developed. The enzyme gave a single band on isoelectric focusing with an isoelectric point at approximately pH 4. The native enzyme had an M(r) of 58,000 by gel filtration and 28,000 by SDS/PAGE and therefore consists of two subunits of equal size. The enzyme displayed a narrow range of specificity and was active with tropine and nortropine but not with pseudotropine, pseudonortropine, or a number of related compounds. The apparent Kms were 6.06 microM for tropine and 73.4 microM for nortropine with the specificity constant (Vmax/Km) for tropine 7.8 times that for pseudotropine. The apparent Km for NADP+ was 48 microM. The deuterium of [3-2H]tropine and [3-2H]pseudotropine was retained when these compounds were converted into 6-hydroxycyclohepta-1,4-dione, an intermediate in tropine catabolism, showing that the tropine dehydrogenase, although induced by growth on tropine, is not involved in the catabolic pathway for this compound. 6-Hydroxycyclohepta-1,4-dione was also implicated as an intermediate in the pathways for pseudotropine and tropinone catabolism. Images Figure 1 PMID:7733902

Novel transporters from Kluyveromyces marxianus and Pichia guilliermondii expressed in Saccharomyces cerevisiae enable growth on L-arabinose and D-xylose.

PubMed

Knoshaug, Eric P; Vidgren, Virve; Magalhães, Frederico; Jarvis, Eric E; Franden, Mary Ann; Zhang, Min; Singh, Arjun

2015-10-01

Genes encoding L-arabinose transporters in Kluyveromyces marxianus and Pichia guilliermondii were identified by functional complementation of Saccharomyces cerevisiae whose growth on L-arabinose was dependent on a functioning L-arabinose transporter, or by screening a differential display library, respectively. These transporters also transport D-xylose and were designated KmAXT1 (arabinose-xylose transporter) and PgAXT1, respectively. Transport assays using L-arabinose showed that KmAxt1p has K(m) 263 mM and V(max) 57 nM/mg/min, and PgAxt1p has K(m) 0.13 mM and V(max) 18 nM/mg/min. Glucose, galactose and xylose significantly inhibit L-arabinose transport by both transporters. Transport assays using D-xylose showed that KmAxt1p has K(m) 27 mM and V(max) 3.8 nM/mg/min, and PgAxt1p has K(m) 65 mM and V(max) 8.7 nM/mg/min. Neither transporter is capable of recovering growth on glucose or galactose in a S. cerevisiae strain deleted for hexose and galactose transporters. Transport kinetics of S. cerevisiae Gal2p showed K(m) 371 mM and V(max) 341 nM/mg/min for L-arabinose, and K(m) 25 mM and V(max) 76 nM/mg/min for galactose. Due to the ability of Gal2p and these two newly characterized transporters to transport both L-arabinose and D-xylose, one scenario for the complete usage of biomass-derived pentose sugars would require only the low-affinity, high-throughput transporter Gal2p and one additional high-affinity general pentose transporter, rather than dedicated D-xylose or L-arabinose transporters. Additionally, alignment of these transporters with other characterized pentose transporters provides potential targets for substrate recognition engineering. Copyright © 2015 John Wiley & Sons, Ltd.

Solubilization and purification of melatonin receptors from lizard brain.

PubMed

Rivkees, S A; Conron, R W; Reppert, S M

1990-09-01

Melatonin receptors in lizard brain were identified and characterized using 125I-labeled melatonin ([125I]MEL) after solubilization with the detergent digitonin. Saturation studies of solubilized material revealed a high affinity binding site, with an apparent equilibrium dissociation constant of 181 +/- 45 pM. Binding was reversible and inhibited by melatonin and closely related analogs, but not by serotonin or norepinephrine. Treatment of solubilized material with the non-hydrolyzable GTP analog, guanosine 5'-(3-O-thiotriphosphate) (GTP-gamma-S), significantly reduced receptor affinity. Gel filtration chromatography of solubilized melatonin receptors revealed a high affinity, large (Mr 400,000) peak of specific binding. Pretreatment with GTP-gamma-S before solubilization resulted in elution of a lower affinity, smaller (Mr 150,000) peak of specific binding. To purify solubilized receptors, a novel affinity chromatography resin was developed by coupling 6-hydroxymelatonin with Epoxy-activated Sepharose 6B. Using this resin, melatonin receptors were purified approximately 10,000-fold. Purified material retained the pharmacologic specificity of melatonin receptors. These results show that melatonin receptors that bind ligand after detergent treatment can be solubilized and substantially purified by affinity chromatography.

Kinetics of phloretin binding to phosphatidylcholine vesicle membranes

PubMed Central

1980-01-01

The submillisecond kinetics for phloretin binding to unilamellar phosphatidylcholine (PC) vesicles was investigated using the temperature-jump technique. Spectrophotometric studies of the equilibrium binding performed at 328 nm demonstrated that phloretin binds to a single set of independent, equivalent sites on the vesicle with a dissociation constant of 8.0 microM and a lipid/site ratio of 4.0. The temperature of the phloretin-vesicle solution was jumped by 4 degrees C within 4 microseconds producing a monoexponential, concentration-dependent relaxation process with time constants in the 30--200-microseconds time range. An analysis of the concentration dependence of relaxation time constants at pH 7.30 and 24 degrees C yielded a binding rate constant of 2.7 X 10(8) M-1 s-1 and an unbinding constant of 2,900 s-1; approximately 66 percent of total binding sites are exposed at the outer vesicle surface. The value of the binding rate constant and three additional observations suggest that the binding kinetics are diffusion limited. The phloretin analogue, naringenin, which has a diffusion coefficient similar to phloretin yet a dissociation constant equal to 24 microM, bound to PC vesicle with the same rate constant as phloretin did. In addition, the phloretin-PC system was studied in buffers made one to six times more viscous than water by addition of sucrose or glycerol to the differ. The equilibrium affinity for phloretin binding to PC vesicles is independent of viscosity, yet the binding rate constant decreases with the expected dependence (kappa binding alpha 1/viscosity) for diffusion-limited processes. Thus, the binding rate constant is not altered by differences in binding affinity, yet depends upon the diffusion coefficient in buffer. Finally, studies of the pH dependence of the binding rate constant showed a dependence (kappa binding alpha [1 + 10pH-pK]) consistent with the diffusion-limited binding of a weak acid. PMID:7391812

Toxic metals (Ni2+, Pb2+, Hg2+) binding affinity of dissolved organic matter (DOM) derived from different ages municipal landfill leachate

NASA Astrophysics Data System (ADS)

Rikta, S. Y.; Tareq, Shafi M.; Uddin, M. Khabir

2018-03-01

Solid waste production is rapidly increasing in Bangladesh and landfill leachate is the consequence of the decomposition of this waste. These leachates contain heavy metals and significant amount of dissolved organic matter (DOM). DOM is known to have considerable role in heavy metals speciation. Hence, it is important to characterize DOM/leachate and evaluate toxic metals binding affinity of DOM. The objectives of this study were to characterize the DOM in landfill leachate through physico-chemical and optical analyses and to investigate the toxic metals (Ni2+, Pb2+ and Hg2+) binding affinity of three different ages (fresh sample L-1, young sample L-2 and mature sample L-3) DOM samples. Results suggested that leachate is a potential pollutant which contained very high organic pollutant load. Conditional stability constant (Log K) and percentages of fluorophores that correspond to metal binding (% f) values indicated that young DOM sample (L-2) had the highest binding affinity to all the three metals ions. In general, DOM samples showed the following order affinity to the metal ions; Ni2+ binding affinity: L-2 > L-3 > L-1, Pb2+ binding affinity: L-2 > L-3 > L-1 and Hg2+ binding affinity: L-2 > L-1 > L-3.

Active uptake of substance P carboxy-terminal heptapeptide (5-11) into rat brain and rabbit spinal cord slices.

PubMed

Nakata, Y; Kusaka, Y; Yajima, H; Segawa, T

1981-12-01

We previously reported that nerve terminals and glial cells lack an active uptake system capable of terminating transmitter action of substance P (SP). In the present study, we demonstrated the existence of an active uptake system for SP carboxy-terminal heptapeptide, (5-11)SP. When the slices from either rat brain or rabbit spinal cord were incubated with [3H](5-11)SP, the uptake of (5-11)SP into slices was observed. The uptake system has the properties of an active transport mechanism: it is dependent on temperature and sensitive to hypoosmotic treatment and is inhibited by ouabain and dinitrophenol (DNP). In the brain, (5-11)SP was accumulated by means of a high-affinity and a low-affinity uptake system. The Km and the Vmax values for the high-affinity system were 4.20 x 10(-8) M and 7.59 fmol/10 mg wet weight/min, respectively, whereas these values for the low-affinity system were 1.00 x 10(-6) M and 100 fmol/10 mg wet weight/min, respectively. In the spinal cord, there was only one uptake system, with a Km value of 2.16 x 10(-7) M and Vmax value of 26.2 fmol/10 mg wet weight/min. These results suggest that when SP is released from nerve terminals, it is hydrolysed into (5-11)SP before or after acting as a neurotransmitter, which is in turn accumulated into nerve terminals. Therefore, the uptake system may represent a possible mechanism for the inactivation of SP.

Time parameterizations and spin supplementary conditions of the Mathisson-Papapetrou-Dixon equations

NASA Astrophysics Data System (ADS)

Lukes-Gerakopoulos, Georgios

2017-11-01

The implications of two different time constraints on the Mathisson-Papapetrou-Dixon (MPD) equations are discussed under three spin supplementary conditions (SSCs). For this reason the MPD equations are revisited without specifying the affine parameter and several relations are reintroduced in their general form. The latter allows one to investigate the consequences of combining the Mathisson-Pirani (MP) SSC, the Tulczyjew-Dixon (TD) SSC and the Ohashi-Kyrian-Semerák (OKS) SSC with two affine parameter types: the proper time on one hand and the parameterizations introduced in [Gen. Relativ. Gravit. 8, 197 (1977), 10.1007/BF00763547] on the other. For the MP SSC and the TD SSC it is shown that quantities that are constant of motion for the one affine parameter are not for the other, while for the OKS SSC it is shown that the two affine parameters are the same. To clarify the relation between the two affine parameters in the case of the TD SSC the MPD equations are evolved and discussed.

Label-Free Determination of Protein Binding in Aqueous Solution using Overlayer Enhanced Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (OE-ATR-FTIR)

NASA Astrophysics Data System (ADS)

Ruthenburg, Travis; Aweda, Tolulope; Park, Simon; Meares, Claude; Land, Donald

2009-03-01

Protein binding/affinity studies are often performed using Surface Plasmon Resonance techniques that don't produce much spectral information. Measurement of protein binding affinity using FTIR is traditionally performed using high protein concentration or deuterated solvent. By immobilizing a protein near the surface of a gold-coated germanium internal reflection element interactions can be measured between an immobilized protein and free proteins or small molecules in aqueous solution. By monitoring the on and off rates of these interactions, the dissociation constant for the system can be determined. The dissociation constant for the molecule Yttrium-DOTA binding to the antibody 2D12.5 system was determined to be 100nM. Results will also be presented from our measurements of Bovine Serum Albumin (BSA) binding to anti-BSA.

Peroxidative oxidation of halides catalysed by myeloperoxidase. Effect of fluoride on halide oxidation.

PubMed

Zgliczyński, J M; Stelmaszyńska, T; Olszowska, E; Krawczyk, A; Kwasnowska, E; Wróbel, J T

1983-01-01

It was found that all halides can compete with cyanide for binding with myeloperoxidase. The lower is the pH, the higher is the affinity of halides. The apparent dissociation constants (Kd) of myeloperoxidase-cyanide complex were determined in the presence of F-, Cl-, Br- and I- in the pH range of 4 to 7. In slightly acidic pH (4 - 6) fluoride and chloride exhibit a higher affinity towards the enzyme than bromide and iodide. Taking into account competition between cyanide and halides for binding with myeloperoxidase the dissociation constants of halide-myeloperoxidase complexes were calculated. All halides except fluoride can be oxidized by H2O2 in the presence of myeloperoxidase. However, since fluoride can bind with myeloperoxidase, it can competitively inhibit the oxidation of other halides. Fluoride was a competitive inhibitor with respect to other halides as well as to H2O2. Inhibition constants (Ki) for fluoride as a competitive inhibitor with respect to H2O2 increased from iodide oxidation through bromide to chloride oxidation.

Effect of Red Blood Cell Storage on Cardiac Performance. Improved Myocardial Oxygen Delivery and Function during Constant Flow Coronary Perfusion with Low Oxy-Hemoglobin Affinity Human Red Blood Cells in Normothermic and Hypothermic Rabbit Hearts.

DTIC Science & Technology

1983-02-01

with an isovolumic left ven- tricular balloon. Coronary flow was held constant to simulate the physiolog of coronary atherosclerosis and other...erythrocyte DPG content can potentially benefit patients with coronary atherosclerosis , or other states with a limited coronary vasodilator reserve, who...Coronary flow was held constant to simulate the physiology of coronary atherosclerosis and other conditions of limited coronary vasodilator reserve

Kinetics of the monomer-dimer reaction of yeast hexokinase PI.

PubMed

Hoggett, J G; Kellett, G L

1992-10-15

Kinetic studies of the glucose-dependent monomer-dimer reaction of yeast hexokinase PI at pH 8.0 in the presence of 0.1 M-KCl have been carried out using the fluorescence temperature-jump technique. A slow-relaxation effect was observed which was attributed from its dependence on enzyme concentration to the monomer-dimer reaction; the reciprocal relaxation times tau-1 varied from 3 s-1 at low concentrations of glucose to 42 s-1 at saturating concentrations. Rate constants for association (kass.) and dissociation (kdiss.) were determined as a function of glucose concentration using values of the equilibrium association constant of the monomer-dimer reaction derived from sedimentation ultracentrifugation studies under similar conditions, and also from the dependence of tau-2 on enzyme concentration. kass. was almost independent of glucose concentration and its value (2 x 10(5) M-1.s-1) was close to that expected for a diffusion-controlled process. The influence of glucose on the monomer-dimer reaction is entirely due to effects on kdiss., which increases from 0.21 s-1 in the absence of glucose to 25 s-1 at saturating concentrations. The monomer and dimer forms of hexokinase have different affinities and Km values for glucose, and the results reported here imply that there may be a significant lag in the response of the monomer-dimer reaction to changes in glucose concentrations in vivo with consequent hysteretic effects on the hexokinase activity.

Expression, purification and enzymatic characterization of Brugia malayi dihydrofolate reductase.

PubMed

Perez-Abraham, Romy; Sanchez, Karla Garabiles; Alfonso, Melany; Gubler, Ueli; Siekierka, John J; Goodey, Nina M

2016-12-01

Brugia malayi (B. malayi) is one of the three causative agents of lymphatic filariasis, a neglected parasitic disease. Current literature suggests that dihydrofolate reductase is a potential drug target for the elimination of B. malayi. Here we report the recombinant expression and purification of a ∼20 kDa B. malayi dihydrofolate reductase (BmDHFR). A His6-tagged construct was expressed in E. coli and purified by affinity chromatography to yield active and homogeneous enzyme for steady-state kinetic characterization and inhibition studies. The catalytic activity kcat was found to be 1.4 ± 0.1 s(-1), the Michaelis Menten constant KM for dihydrofolate 14.7 ± 3.6 μM, and the equilibrium dissociation constant KD for NADPH 25 ± 24 nM. For BmDHFR, IC50 values for a six DHFR inhibitors were determined to be 3.1 ± 0.2 nM for methotrexate, 32 ± 22 μM for trimethoprim, 109 ± 34 μM for pyrimethamine, 154 ± 46 μM for 2,4-diaminoquinazoline, 771 ± 44 μM for cycloguanil, and >20,000 μM for 2,4-diaminopyrimidine. Our findings suggest that antifolate compounds can serve as inhibitors of BmDHFR. Copyright © 2016 Elsevier Inc. All rights reserved.

Kinetics of the monomer-dimer reaction of yeast hexokinase PI.

PubMed Central

Hoggett, J G; Kellett, G L

1992-01-01

Kinetic studies of the glucose-dependent monomer-dimer reaction of yeast hexokinase PI at pH 8.0 in the presence of 0.1 M-KCl have been carried out using the fluorescence temperature-jump technique. A slow-relaxation effect was observed which was attributed from its dependence on enzyme concentration to the monomer-dimer reaction; the reciprocal relaxation times tau-1 varied from 3 s-1 at low concentrations of glucose to 42 s-1 at saturating concentrations. Rate constants for association (kass.) and dissociation (kdiss.) were determined as a function of glucose concentration using values of the equilibrium association constant of the monomer-dimer reaction derived from sedimentation ultracentrifugation studies under similar conditions, and also from the dependence of tau-2 on enzyme concentration. kass. was almost independent of glucose concentration and its value (2 x 10(5) M-1.s-1) was close to that expected for a diffusion-controlled process. The influence of glucose on the monomer-dimer reaction is entirely due to effects on kdiss., which increases from 0.21 s-1 in the absence of glucose to 25 s-1 at saturating concentrations. The monomer and dimer forms of hexokinase have different affinities and Km values for glucose, and the results reported here imply that there may be a significant lag in the response of the monomer-dimer reaction to changes in glucose concentrations in vivo with consequent hysteretic effects on the hexokinase activity. Images Fig. 1. PMID:1445216

The ATP-binding cassette transporter Cbc (choline/betaine/carnitine) recruits multiple substrate-binding proteins with strong specificity for distinct quaternary ammonium compounds

PubMed Central

Chen, Chiliang; Malek, Adel A.; Wargo, Matthew J.; Hogan, Deborah A.; Beattie, Gwyn A.

2017-01-01

Summary We identified a choline, betaine and carnitine transporter, designated Cbc, from Pseudomonas syringae and Pseudomonas aeruginosa that is unusual among members of the ATP-binding cassette (ABC) transporter family in its use of multiple periplasmic substrate-binding proteins (SBPs) that are highly specific for their substrates. The SBP encoded by the cbcXWV operon, CbcX, binds choline with a high affinity (Km, 2.6 μM) and, although it also binds betaine (Km, 24.2 μM), CbcXWV-mediated betaine uptake did not occur in the presence of choline. The CbcX orthologue ChoX from Sinorhizobium meliloti was similar to CbcX in these binding properties. The core transporter CbcWV also interacts with the carnitine-specific SBP CaiX (Km, 24 μM) and the betaine-specific SBP BetX (Km, 0.6 μM). Unlike most ABC transporter loci, caiX, betX and cbcXWV are separated in the genome. CaiX-mediated carnitine uptake was reduced by CbcX and BetX only when they were bound by their individual ligands, providing the first in vivo evidence for a higher affinity for ligand-bound than ligand-free SBPs by an ABC transporter. These studies demonstrate not only that the Cbc transporter serves as a useful model for exploring ABC transporter component interactions, but also that the orphan SBP genes common to bacterial genomes can encode functional SBPs. PMID:19919675

The ATP-binding cassette transporter Cbc (choline/betaine/carnitine) recruits multiple substrate-binding proteins with strong specificity for distinct quaternary ammonium compounds.

PubMed

Chen, Chiliang; Malek, Adel A; Wargo, Matthew J; Hogan, Deborah A; Beattie, Gwyn A

2010-01-01

We identified a choline, betaine and carnitine transporter, designated Cbc, from Pseudomonas syringae and Pseudomonas aeruginosa that is unusual among members of the ATP-binding cassette (ABC) transporter family in its use of multiple periplasmic substrate-binding proteins (SBPs) that are highly specific for their substrates. The SBP encoded by the cbcXWV operon, CbcX, binds choline with a high affinity (K(m), 2.6 microM) and, although it also binds betaine (K(m), 24.2 microM), CbcXWV-mediated betaine uptake did not occur in the presence of choline. The CbcX orthologue ChoX from Sinorhizobium meliloti was similar to CbcX in these binding properties. The core transporter CbcWV also interacts with the carnitine-specific SBP CaiX (K(m), 24 microM) and the betaine-specific SBP BetX (K(m), 0.6 microM). Unlike most ABC transporter loci, caiX, betX and cbcXWV are separated in the genome. CaiX-mediated carnitine uptake was reduced by CbcX and BetX only when they were bound by their individual ligands, providing the first in vivo evidence for a higher affinity for ligand-bound than ligand-free SBPs by an ABC transporter. These studies demonstrate not only that the Cbc transporter serves as a useful model for exploring ABC transporter component interactions, but also that the orphan SBP genes common to bacterial genomes can encode functional SBPs.

Determination of Acid Dissociation Constants (pKa) of Bicyclic Thiohydantoin-Pyrrolidine Compounds in 20% Ethanol-Water Hydroorganic Solvent

PubMed Central

Nural, Yahya; Döndaş, H. Ali; Sarı, Hayati; Atabey, Hasan; Belveren, Samet; Gemili, Müge

2014-01-01

The acid dissociation constants of potential bioactive fused ring thiohydantoin-pyrrolidine compounds were determined by potentiometric titration in 20% (v/v) ethanol-water mixed at 25 ± 0.1°C, at an ionic background of 0.1 mol/L of NaCl using the HYPERQUAD computer program. Proton affinities of potential donor atoms of the ligands were calculated by AM1 and PM3 semiempiric methods. We found, potentiometrically, three different acid dissociation constants for 1a–f. We suggest that these acid dissociation constants are related to the carboxyl, enol, and amino groups. PMID:24799905

Engineering an antibody with picomolar affinity to DOTA chelates of multiple radionuclides for pretargeted radioimmunotherapy and imaging

PubMed Central

Orcutt, Kelly Davis; Slusarczyk, Adrian L; Cieslewicz, Maryelise; Ruiz-Yi, Benjamin; Bhushan, Kumar R; Frangioni, John V; Wittrup, K Dane

2014-01-01

Introduction In pretargeted radioimmunotherapy (PRIT), a bifunctional antibody is administered and allowed to pre-localize to tumor cells. Subsequently, a chelated radionuclide is administered and captured by cell-bound antibody while unbound hapten clears rapidly from the body. We aim to engineer high-affinity binders to DOTA chelates for use in PRIT applications. Methods We mathematically modeled antibody and hapten pharmacokinetics to analyze hapten tumor retention as a function of hapten binding affinity. Motivated by model predictions, we used directed evolution and yeast surface display to affinity mature the 2D12.5 antibody to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), reformatted as a single chain variable fragment (scFv). Results Modeling predicts that for high antigen density and saturating bsAb dose, a hapten binding affinity of 100 picomolar (pM) is needed for near-maximal hapten retention. We affinity matured 2D12.5 with an initial binding constant of about 10 nanomolar (nM) to DOTA-yttrium chelates. Affinity maturation resulted in a 1000-fold affinity improvement to biotinylated DOTA-yttrium, yielding an 8.2 ± 1.9 picomolar binder. The high-affinity scFv binds DOTA complexes of lutetium and gadolinium with similar picomolar affinity and indium chelates with low nanomolar affinity. When engineered into a bispecific antibody construct targeting carcinoembryonic antigen (CEA), pretargeted high-affinity scFv results in significantly higher tumor retention of a 111In-DOTA hapten compared to pretargeted wild-type scFv in a xenograft mouse model. Conclusions We have engineered a versatile, high-affinity DOTA-chelate-binding scFv. We anticipate it will prove useful in developing pretargeted imaging and therapy protocols to exploit the potential of a variety of radiometals. PMID:21315278

Characterizing low affinity epibatidine binding to α4β2 nicotinic acetylcholine receptors with ligand depletion and nonspecific binding

PubMed Central

2011-01-01

Background Along with high affinity binding of epibatidine (Kd1≈10 pM) to α4β2 nicotinic acetylcholine receptor (nAChR), low affinity binding of epibatidine (Kd2≈1-10 nM) to an independent binding site has been reported. Studying this low affinity binding is important because it might contribute understanding about the structure and synthesis of α4β2 nAChR. The binding behavior of epibatidine and α4β2 AChR raises a question about interpreting binding data from two independent sites with ligand depletion and nonspecific binding, both of which can affect equilibrium binding of [3H]epibatidine and α4β2 nAChR. If modeled incorrectly, ligand depletion and nonspecific binding lead to inaccurate estimates of binding constants. Fitting total equilibrium binding as a function of total ligand accurately characterizes a single site with ligand depletion and nonspecific binding. The goal of this study was to determine whether this approach is sufficient with two independent high and low affinity sites. Results Computer simulations of binding revealed complexities beyond fitting total binding for characterizing the second, low affinity site of α4β2 nAChR. First, distinguishing low-affinity specific binding from nonspecific binding was a potential problem with saturation data. Varying the maximum concentration of [3H]epibatidine, simultaneously fitting independently measured nonspecific binding, and varying α4β2 nAChR concentration were effective remedies. Second, ligand depletion helped identify the low affinity site when nonspecific binding was significant in saturation or competition data, contrary to a common belief that ligand depletion always is detrimental. Third, measuring nonspecific binding without α4β2 nAChR distinguished better between nonspecific binding and low-affinity specific binding under some circumstances of competitive binding than did presuming nonspecific binding to be residual [3H]epibatidine binding after adding a large concentration of cold competitor. Fourth, nonspecific binding of a heterologous competitor changed estimates of high and low inhibition constants but did not change the ratio of those estimates. Conclusions Investigating the low affinity site of α4β2 nAChR with equilibrium binding when ligand depletion and nonspecific binding are present likely needs special attention to experimental design and data interpretation beyond fitting total binding data. Manipulation of maximum ligand and receptor concentrations and intentionally increasing ligand depletion are potentially helpful approaches. PMID:22112852

Evaluation of interactions between RAW264.7 macrophages and small molecules by capillary electrophoresis.

PubMed

Wang, Feng-Qin; Li, Qiao-Qiao; Zhang, Qian; Wang, Yin-Zhen; Hu, Yuan-Jia; Li, Peng; Wan, Jian-Bo; Yang, Feng-Qing; Xia, Zhi-Ning

2017-03-01

In this study, the affinity interactions between RAW 264.7 macrophages and three small molecules including naringin, oleuropein and paeoniflorin were evaluated by affinity capillary electrophoresis (ACE), partial filling affinity capillary electrophoresis (PFACE) and frontal analysis capillary electrophoresis (FACE), respectively. The result indicated that ACE (varying concentrations of cell suspension were filled in the capillary as receptor) may not be suitable for the evaluation of interactions between cell and small molecules due to the high viscosity of cell suspension; PFACE can qualitatively evaluate the interaction, but the difference in viscosity between RAW264.7 suspension and buffer effects on the liner relationship between filling length and injection time, which makes the calculation of binding constant difficult. Furthermore, based on the PFACE results, naringin showed stronger interaction with macrophages than the other two molecules; taking advantage of the aggregation phenomenon of cell induced by electric field, FACE was successfully used to determine the stoichiometry (n = 5×10 9 ) and binding constant (K b = 1×10 4 L/mol) of the interaction between RAW264.7 and naringin. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Prostaglandin E and F2 alpha receptors in human myometrium during the menstrual cycle and in pregnancy and labor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Giannopoulos, G.; Jackson, K.; Kredentser, J.

The binding of prostaglandins E1 and F2 alpha has been studied in the human myometrium and cervix during the menstrual cycle and in the myometrium of pregnant patients at term before and during labor. Tritium-labeled prostaglandin E1 and F2 alpha binding was saturable and reversible. Scatchard analysis of tritium-labeled prostaglandin E1 binding was linear, which suggests a single class of high-affinity binding sites with an estimated apparent equilibrium dissociation constant of 2.5 to 5.4 nmol/L and inhibitor affinities of 0.9, 273, 273, and 217 nmol/L for prostaglandins E2, A1, B1, and F2 alpha, respectively. Scatchard analysis of tritium-labeled prostaglandin F2more » alpha, binding was also linear, but the affinity of these binding sites was much lower, with an average dissociation constant of 50 nmol/L and inhibitor affinities of 1.6, 2.2, and 11.2 nmol/L for prostaglandins E1, E2, and A1, respectively. In nonpregnant patients, the concentrations and affinities of tritium-labeled prostaglandin E1 binding sites were similar in the myometrium during the proliferative and secretory phases of the menstrual cycle, but the concentration of these sites was much lower in the cervix. The concentration of the tritium-labeled prostaglandin E1 binding sites was significantly lower in the myometrium of pregnant patients at term than in the myometrium of nonpregnant patients. The concentrations and affinities of tritium-labeled prostaglandin E1 binding sites were not significantly different in the upper and lower myometrium of pregnant patients at term or in the myometrium of such patients before and during labor. The concentrations of the tritium-labeled prostaglandin F2 alpha binding sites during the menstrual cycle and in pregnancy at term were similar to those of tritium-labeled prostaglandin E1 binding sites.« less

NEUTRALIZATION OF THE ASPARTIC ACID RESIDUE D367, BUT NOT D454, INHIBITS BINDING OF NA+ TO THE GLUTAMATE-FREE FORM AND CYCLING OF THE GLUTAMATE TRANSPORTER EAAC1

PubMed Central

Tao, Zhen; Zhang, Zhou; Grewer, Christof

2008-01-01

Substrate transport by the plasma membrane glutamate transporter EAAC1 is coupled to cotransport of three sodium ions. One of these Na+ ions binds to the transporter already in the absence of glutamate. Here, we have investigated the possible involvement of two conserved aspartic acid residues in transmembrane segments 7 and 8 of EAAC1, D367 and D454, in Na+ cotransport. In order to test the effect of charge neutralization mutations in these positions on Na+ binding to the glutamate-free transporter, we recorded the Na+-induced anion leak current to determine the Km of EAAC1 for Na+. For EAAC1WT, this Km was determined as 120 mM. When the negative charge of D367 was neutralized by mutagenesis to asparagine, Na+ activated the anion leak current with a Km of about 2 M, indicating dramatically impaired Na+ binding to the mutant transporter. In contrast, the Na+ affinity of EAAC1D454N was virtually unchanged compared to the wild type transporter (Km = 90 mM). The reduced occupancy of the Na+ binding site of EAAC1D367N resulted in a dramatic reduction in glutamate affinity (Km = 3.6 mM, 140 mM [Na+]), which could be partially overcome by increasing extracellular [Na+]. In addition to impairing Na+ binding, the D367N mutation slowed glutamate transport, as shown by pre-steady-state kinetic analysis of transport currents, by strongly decreasing the rate of a reaction step associated with glutamate translocation. Our data are consistent with a model in which D367, but not D454 is involved in coordinating the bound Na+ in the glutamate-free transporter form. PMID:16478724

Third system for neutral amino acid transport in a marine pseudomonad.

PubMed Central

Pearce, S M; Hildebrandt, V A; Lee, T

1977-01-01

Uptake of leucine by the marine pseudomonad B-16 is an energy-dependent, concentrative process. Respiratory inhibitors, uncouplers, and sulfhydryl reagents block transport. The uptake of leucine is Na+ dependent, although the relationship between the rate of leucine uptake and Na+ concentration depends, to some extent, on the ionic strength of the suspending assay medium and the manner in which cells are washed prior to assay. Leucine transport can be separated into at least two systems: a low-affinity system with an apparent Km of 1.3 X 10(-5) M, and a high-affinity system with an apparent Km of 1.9 X 10(-7) M. The high-affinity system shows a specificity unusual for bacterial systems in that both aromatic and aliphatic amino acids inhibit leucine transport, provided that they have hydrophobic side chains of a length greater than that of two carbon atoms. The system exhibits strict stereospecificity for the L form. Phenylalanine inhibition was investigated in more detail. The Ki for inhibition of leucine transport by phenylalanine is about 1.4 X 10(-7) M. Phenylalanine itself is transported by an energy-dependent process whose specificity is the same as the high-affinity leucine transport system, as is expected if both amino acids share the same transport system. Studies with protoplasts indicate that a periplasmic binding protein is not an essential part of this transport system. Fein and MacLeod (J. Bacteriol. 124:1177-1190, 1975) reported two neutral amino acid transport systems in strain B-16: the DAG system, serving glycine, D-alanine, D-serine, and alpha-aminoisobutyric acid; and the LIV system, serving L-leucine, L-isoleucine, L-valine, and L-alanine. The high-affinity system reported here is a third neutral amino acid transport system in this marine pseudomonad. We propose the name "LIV-II" system. PMID:856786

Selection and identification of a DNA aptamer targeted to Vibrio parahemolyticus.

PubMed

Duan, Nuo; Wu, Shijia; Chen, Xiujuan; Huang, Yukun; Wang, Zhouping

2012-04-25

A whole-bacterium systemic evolution of ligands by exponential enrichment (SELEX) method was applied to a combinatorial library of FAM-labeled single-stranded DNA molecules to identify DNA aptamers demonstrating specific binding to Vibrio parahemolyticus . FAM-labeled aptamer sequences with high binding affinity to V. parahemolyticus were identified by flow cytometric analysis. Aptamer A3P, which showed a particularly high binding affinity in preliminary studies, was chosen for further characterization. This aptamer displayed a dissociation constant (K(d)) of 16.88 ± 1.92 nM. Binding assays to assess the specificity of aptamer A3P showed a high binding affinity (76%) for V. parahemolyticus and a low apparent binding affinity (4%) for other bacteria. Whole-bacterium SELEX is a promising technique for the design of aptamer-based molecular probes for microbial pathogens that does not require the labor-intensive steps of isolating and purifying complex markers or targets.

Muscarinic receptor occupation and receptor activation in the guinea-pig ileum by some acetamides related to oxotremorine.

PubMed Central

Ringdahl, B.

1984-01-01

The dissociation constants (KD values) and relative efficacies of seven acetamide analogues of oxotremorine, including two enantiomeric pairs, at muscarinic receptors in the guinea-pig isolated ileum were determined. The method used involved analysis of dose-response data before and after fractional inactivation of receptors with propylbenzilylcholine mustard. All of the compounds studied had lower affinities than oxotremorine, but some had substantially higher relative efficacies. Replacement of the pyrrolidine ring of N-methyl-N-(4- pyrrolidino -2- butynyl )acetamide(I), the parent compound in the series, by a dimethylamino or a trimethylammonium group decreased the affinity 32 and 4.5 fold, respectively, whereas the relative efficacy increased 5.7-8.3 times. There was no correlation between relative efficacies and affinities of the compounds. The structural requirements for high affinity and high efficacy appeared to be quite different. PMID:6733356

Cloning, heterologous expression, and in situ characterization of the first high affinity nucleobase transporter from a protozoan.

PubMed

Burchmore, Richard J S; Wallace, Lynsey J M; Candlish, Denise; Al-Salabi, Mohammed I; Beal, Paul R; Barrett, Michael P; Baldwin, Stephen A; de Koning, Harry P

2003-06-27

While multiple nucleoside transporters, some of which can also transport nucleobases, have been cloned in recent years from many different organisms, no sequence information is available for the high affinity, nucleobase-selective transporters of metazoa, parazoa, or protozoa. We have identified a gene, TbNBT1, from Trypanosoma brucei brucei that encodes a 435-residue protein of the equilibrative nucleoside transporter superfamily. The gene was expressed in both the procyclic and bloodstream forms of the organism. Expression of TbNBT1 in a Saccharomyces cerevisiae strain lacking an endogenous purine transporter allowed growth on adenine as sole purine source and introduced a high affinity transport activity for adenine and hypoxanthine, with Km values of 2.1 +/- 0.6 and 0.66 +/- 0.22 microm, respectively, as well as high affinity for xanthine, guanine, guanosine, and allopurinol and moderate affinity for inosine. A transporter with an indistinguishable kinetic profile was identified in T. b. brucei procyclics and designated H4. RNA interference of TbNBT1 in procyclics reduced cognate mRNA levels by approximately 80% and H4 transport activity by approximately 90%. Expression of TbNBT1 in Xenopus oocytes further confirmed that this gene encodes the first high affinity nucleobase transporter from protozoa or animals to be identified at the molecular level.

U.S. Standard Atmosphere, 1976

NASA Technical Reports Server (NTRS)

1976-01-01

Part 1 gives the basis for computation of the main tables of atmospheric properties, including values of physical constants, conversion factors, and definitions of derived properties, including values of physical constants, conversion factors, and definitions of derived properties. Part 2 describes the model and data used up to 85 km, in the first section; and the model and data used above 85 km in the second section. The theoretical basis of the high altitude model is given in an appendix. Part 3 contains information on minor constituents in the troposphere, stratosphere, and mesosphere. The main tables of atmospheric properties to 1000 km are given in Part 4. The international system of metric units is used.

Modulating Uranium Binding Affinity in Engineered Calmodulin EF-Hand Peptides: Effect of Phosphorylation

PubMed Central

Pardoux, Romain; Sauge-Merle, Sandrine; Lemaire, David; Delangle, Pascale; Guilloreau, Luc; Adriano, Jean-Marc; Berthomieu, Catherine

2012-01-01

To improve our understanding of uranium toxicity, the determinants of uranyl affinity in proteins must be better characterized. In this work, we analyzed the contribution of a phosphoryl group on uranium binding affinity in a protein binding site, using the site 1 EF-hand motif of calmodulin. The recombinant domain 1 of calmodulin from A. thaliana was engineered to impair metal binding at site 2 and was used as a structured template. Threonine at position 9 of the loop was phosphorylated in vitro, using the recombinant catalytic subunit of protein kinase CK2. Hence, the T9TKE12 sequence was substituted by the CK2 recognition sequence TAAE. A tyrosine was introduced at position 7, so that uranyl and calcium binding affinities could be determined by following tyrosine fluorescence. Phosphorylation was characterized by ESI-MS spectrometry, and the phosphorylated peptide was purified to homogeneity using ion-exchange chromatography. The binding constants for uranyl were determined by competition experiments with iminodiacetate. At pH 6, phosphorylation increased the affinity for uranyl by a factor of ∼5, from Kd = 25±6 nM to Kd = 5±1 nM. The phosphorylated peptide exhibited a much larger affinity at pH 7, with a dissociation constant in the subnanomolar range (Kd = 0.25±0.06 nM). FTIR analyses showed that the phosphothreonine side chain is partly protonated at pH 6, while it is fully deprotonated at pH 7. Moreover, formation of the uranyl-peptide complex at pH 7 resulted in significant frequency shifts of the νas(P-O) and νs(P-O) IR modes of phosphothreonine, supporting its direct interaction with uranyl. Accordingly, a bathochromic shift in νas(UO2)2+ vibration (from 923 cm−1 to 908 cm−1) was observed upon uranyl coordination to the phosphorylated peptide. Together, our data demonstrate that the phosphoryl group plays a determining role in uranyl binding affinity to proteins at physiological pH. PMID:22870263

Diurnal variation of nitric oxide in the upper stratosphere

NASA Technical Reports Server (NTRS)

Kondo, Y.; Aimedieu, P.; Pirre, M.; Ramaroson, R.; Matthews, W. A.

1990-01-01

Two recent measurements of the temporal variation of nitric oxide at constant altitude near 40 km are reported. The observations were made at float altitude with a balloon-borne chemiluminescence detector together with in situ ozone measurements. The first measurement was made at 44 N on September 17, 1987, at an altitude of 40 km from before sunrise until 1000 LT. The second observation was made at the same latitude on June 18, 1988, at 39 km from 0800 to 1230 LT. At an altitude of 40 km, nitric oxide was observed to start increasing very rapidly at sunrise when the solar zenith angle reached about 95 deg. After the rapid initial buildup, the rate of NO increase stabilized for 3 hours at about 1.2 ppbv/hour. Near 1100 LT at 39 km in summer, the NO mixing ratio was observed to become nearly constant. These features of the diurnal variation of NO are in accord with the temporal variation expected from a time-dependent zero-dimensional photochemical model.

Rate Constants and Mechanisms of Protein–Ligand Binding

PubMed Central

Pang, Xiaodong; Zhou, Huan-Xiang

2017-01-01

Whereas protein–ligand binding affinities have long-established prominence, binding rate constants and binding mechanisms have gained increasing attention in recent years. Both new computational methods and new experimental techniques have been developed to characterize the latter properties. It is now realized that binding mechanisms, like binding rate constants, can and should be quantitatively determined. In this review, we summarize studies and synthesize ideas on several topics in the hope of providing a coherent picture of and physical insight into binding kinetics. The topics include microscopic formulation of the kinetic problem and its reduction to simple rate equations; computation of binding rate constants; quantitative determination of binding mechanisms; and elucidation of physical factors that control binding rate constants and mechanisms. PMID:28375732

Determination of thermodynamic values of acidic dissociation constants and complexation constants of profens and their utilization for optimization of separation conditions by Simul 5 Complex.

PubMed

Riesová, Martina; Svobodová, Jana; Ušelová, Kateřina; Tošner, Zdeněk; Zusková, Iva; Gaš, Bohuslav

2014-10-17

In this paper we determine acid dissociation constants, limiting ionic mobilities, complexation constants with β-cyclodextrin or heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin, and mobilities of resulting complexes of profens, using capillary zone electrophoresis and affinity capillary electrophoresis. Complexation parameters are determined for both neutral and fully charged forms of profens and further corrected for actual ionic strength and variable viscosity in order to obtain thermodynamic values of complexation constants. The accuracy of obtained complexation parameters is verified by multidimensional nonlinear regression of affinity capillary electrophoretic data, which provides the acid dissociation and complexation parameters within one set of measurements, and by NMR technique. A good agreement among all discussed methods was obtained. Determined complexation parameters were used as input parameters for simulations of electrophoretic separation of profens by Simul 5 Complex. An excellent agreement of experimental and simulated results was achieved in terms of positions, shapes, and amplitudes of analyte peaks, confirming the applicability of Simul 5 Complex to complex systems, and accuracy of obtained physical-chemical constants. Simultaneously, we were able to demonstrate the influence of electromigration dispersion on the separation efficiency, which is not possible using the common theoretical approaches, and predict the electromigration order reversals of profen peaks. We have shown that determined acid dissociation and complexation parameters in combination with tool Simul 5 Complex software can be used for optimization of separation conditions in capillary electrophoresis. Copyright © 2014 Elsevier B.V. All rights reserved.

Thiacarbocyanine as ligand in dye-affinity chromatography for protein purification. II. Dynamic binding capacity using lysozyme as a model.

PubMed

Boto, R E F; Anyanwu, U; Sousa, F; Almeida, P; Queiroz, J A

2009-09-01

A constant development of dye-affinity chromatography to replace more traditional techniques is verified, with the aim of increasing specificity in the purification of biomolecules. The establishment of a new dye-affinity chromatographic support imposes their complete characterization, namely with relation to the binding capacity for proteins, in order to evaluate its applicability on global purification processes. Following previous studies, the adsorption of lysozyme onto a thiacarbocyanine dye immobilized on beaded cellulose was investigated. The effect of different parameters, such as temperature, ionic strength, pH, protein concentration and flow rate, on the dynamic binding capacity of the support to retain lysozyme was also studied. Increasing the temperature and the lysozyme concentration had a positive effect on the dynamic binding capacity (DBC), whereas increasing the ionic strength and the flow rate resulted in the opposite. It was also discovered that the pH used had an important impact on the lysozyme binding onto the immobilized dye. The maximum DBC value obtained for lysozyme was 8.6 mg/mL, which was achieved at 30 degrees C and pH 9 with a protein concentration of 0.5 mg/mL and a flow rate of 0.05 mL/min. The dissociation constant (K(d)) obtained was 2.61 +/- 0.36 x 10(-5 )m, proving the affinity interaction between the thiacarbocyanine dye ligand and the lysozyme. Copyright (c) 2009 John Wiley & Sons, Ltd.

Influence of heme environment structure on dioxygen affinity for the dual function Amphitrite ornata hemoglobin/dehaloperoxidase. Insights into the evolutional structure-function adaptations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Shengfang; Sono, Masanori; Wang, Chunxue

Sea worm, Amphitrite ornata, has evolved its globin (an O 2 carrier) also to serves as a dehaloperoxidase (DHP) to detoxify haloaromatic pollutants generated by competing species. A previous mutagenesis study by our groups on both DHP and sperm whale myoglobin (SW Mb) revealed some structural factors that influence the dehaloperoxidase activities (significantly lower for Mb) of both proteins. Using an isocyanide/O 2 partition constant measurement method in this study, we have examined the effects of these structural factors on the O 2 equilibrium constants (K O2) of DHP, SW Mb, and their mutants. A clear trend of decreasing Omore » 2 affinity and increasing catalytic activity along with the increase in the distal His N ε–heme iron distance is observed. An H93K/T95H Mb double mutant mimicking the DHP proximal His positioning exhibited markedly enhanced O 2 affinity, confirming the essential effect of proximal His rotation on the globin function of DHP. For DHP, the L100F, T56G and M86E variants showed the effects of distal volume, distal His flexibility and proximal electronic push, respectively, on the O 2 affinity. This study provides insights into how DHP has evolved its heme environment to gain significantly enhanced peroxidase capability without compromising its primary function as an O 2 carrier.« less

Seasonal changes in plasma androgens, glucocorticoids and glucocorticoid-binding proteins in the marsupial sugar glider Petaurus breviceps.

PubMed

Bradley, A J; Stoddart, D M

1992-01-01

An investigation spanning two breeding seasons was carried out to examine endocrine changes associated with reproduction in a wild population of the marsupial sugar glider Petaurus breviceps, a small arboreal gliding possum. Using techniques of equilibrium dialysis and polyacrylamide gel electrophoresis at steady-state conditions, a high-affinity, low-capacity glucocorticoid-binding protein was demonstrated in the plasma of Petaurus breviceps. Equilibrium dialysis at 36 degrees C using cortisol gave a high-affinity binding constant of 95 +/- 5.2 litres/mumol for a presumed corticosteroid-binding globulin (CBG) while the binding constant for the cortisol-albumin interaction was 3.5 +/- 0.4 litres/mmol. There was no difference between the sexes in the affinity of binding of cortisol to CBG; however, the cortisol-binding capacity underwent seasonal variation in both sexes. Progesterone was bound strongly to the presumed CBG while neither oestradiol nor aldosterone appeared to be bound with high affinity to P. breviceps plasma. In the males, peaks in the plasma concentration of testosterone coincided with the July-September breeding season in both years. A significant inverse relationship was shown to exist between the plasma testosterone concentration and the CBG-binding capacity. In both sexes an increase occurred in the plasma concentration of free cortisol during the first breeding season, a pattern which was not repeated in the subsequent breeding season, possibly due to a lower population density in that year.

Single transporter for sulfate, selenate, and selenite in Escherichia coli K-12.

PubMed Central

Lindblow-Kull, C; Kull, F J; Shrift, A

1985-01-01

A Michaelis-Menten kinetic analysis of the transport of sulfate, selenate, and selenite into Escherichia coli K-12 showed that the three dianions were transported by the same carrier. Km values, used as a measure of the affinity of each ligand for the carrier, showed that sulfate was bound 5 times more tightly than selenate and 37 times more tightly than selenite. The specificity ratio, Vmax/Km, also indicated that sulfate was the preferred ligand. There was little difference in the ratios for selenate and selenite. PMID:3897189

The size of the hydroxyl group and its contribution to the affinity of atropine for muscarine-sensitive acetylcholine receptors.

PubMed Central

Barlow, R. B.; Ramtoola, S.

1980-01-01

1 From measurements of the affinity constants of hydratropyltropine and its methiodide for muscarine-sensitive acetylcholine receptors in the guinea-pig ileum, the increment in log K for the hydroxyl group in atropine is 2.06 and in the methiodide it is 2.16. These effects are slightly bigger than any so far recorded with these receptors. 2 The estimate of the increment in apparent molal volume for the hydroxyl group is 1.1 cm3/mol in atropine and 1.0 cm3/mol in the methobromide. 3 The large effect of the group on affinity may be linked to its small apparent size in water as suggested in the previous paper. PMID:7470742

Solubilization and purification of melatonin receptors from lizard brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rivkees, S.A.; Conron, R.W. Jr.; Reppert, S.M.

Melatonin receptors in lizard brain were identified and characterized using {sup 125}I-labeled melatonin (({sup 125}I)MEL) after solubilization with the detergent digitonin. Saturation studies of solubilized material revealed a high affinity binding site, with an apparent equilibrium dissociation constant of 181 +/- 45 pM. Binding was reversible and inhibited by melatonin and closely related analogs, but not by serotonin or norepinephrine. Treatment of solubilized material with the non-hydrolyzable GTP analog, guanosine 5'-(3-O-thiotriphosphate) (GTP-gamma-S), significantly reduced receptor affinity. Gel filtration chromatography of solubilized melatonin receptors revealed a high affinity, large (Mr 400,000) peak of specific binding. Pretreatment with GTP-gamma-S before solubilization resultedmore » in elution of a lower affinity, smaller (Mr 150,000) peak of specific binding. To purify solubilized receptors, a novel affinity chromatography resin was developed by coupling 6-hydroxymelatonin with Epoxy-activated Sepharose 6B. Using this resin, melatonin receptors were purified approximately 10,000-fold. Purified material retained the pharmacologic specificity of melatonin receptors. These results show that melatonin receptors that bind ligand after detergent treatment can be solubilized and substantially purified by affinity chromatography.« less

Fluorogen-Activating-Proteins as Universal Affinity Biosensors for Immunodetection

PubMed Central

Gallo, Eugenio; Vasilev, Kalin V.; Jarvik, Jonathan

2014-01-01

Fluorogen-activating-proteins (FAPs) are a novel platform of fluorescence biosensors utilized for protein discovery. The technology currently demands molecular manipulation methods that limit its application and adaptability. Here, we highlight an alternative approach based on universal affinity reagents for protein detection. The affinity reagents were engineered as bi-partite fusion proteins, where the specificity moiety is derived from IgG-binding proteins –Protein-A or Protein-G – and the signaling element is a FAP. In this manner, primary antibodies provide the antigenic selectivity against a desired protein in biological samples, while FAP affinity reagents target the constant region (Fc) of antibodies and provide the biosensor component of detection. Fluorescence results using various techniques indicate minimal background and high target specificity for exogenous and endogenous proteins in mammalian cells. Additionally, FAP-based affinity reagents provide enhanced properties of detection previously absent using conventional affinity systems. Distinct features explored in this report include: (1) unfixed signal wavelengths (excitation and emission) determined by the particular fluorogen chosen, (2) real-time user controlled fluorescence on-set and off-set, (3) signal wavelength substitution while performing live analysis, and (4) enhanced resistance to photobleaching. PMID:24122476

Application of ADM1 for modeling of biogas production from anaerobic digestion of Hydrilla verticillata.

PubMed

Chen, Xiaojuan; Chen, Zhihua; Wang, Xun; Huo, Chan; Hu, Zhiquan; Xiao, Bo; Hu, Mian

2016-07-01

The present study focused on the application of anaerobic digestion model no. 1 (ADM1) to simulate biogas production from Hydrilla verticillata. Model simulation was carried out by implementing ADM1 in AQUASIM 2.0 software. Sensitivity analysis was used to select the most sensitive parameters for estimation using the absolute-relative sensitivity function. Among all the kinetic parameters, disintegration constant (kdis), hydrolysis constant of protein (khyd_pr), Monod maximum specific substrate uptake rate (km_aa, km_ac, km_h2) and half-saturation constants (Ks_aa, Ks_ac) affect biogas production significantly, which were optimized by fitting of the model equations to the data obtained from batch experiments. The ADM1 model after parameter estimation was able to well predict the experimental results of daily biogas production and biogas composition. The simulation results of evolution of organic acids, bacteria concentrations and inhibition effects also helped to get insight into the reaction mechanisms. Copyright © 2016. Published by Elsevier Ltd.

A possible molecular mechanism of the action of digitalis: ouabain action on calcium binding to sites associated with a purified sodium-potassium-activated adenosine triphosphatase from kidney.

PubMed

Gervais, A; Lane, L K; Anner, B M; Lindenmayer, G E; Schwartz, A

1977-01-01

Calcium binding at 0 degrees C to a purified sheep kidney Na+,K+-ATPase was described by linear Scatchard plots. Binding at saturating free calcium was 65-80 nmol/mg of protein, or 30-40 mol of calcium/mol of enzyme. Aqueous emulsions of lipids extracted from Na+,K+-ATPase yielded dissociation constants and maximum calcium-binding values that were similar to those for native Na+,K+-ATPase. Phospholipase A treatment markedly reduced calcium binding. Pretreatment of native Na+,K+-ATPase with ouabain increased the dissociation constant for calcium binding from 131 +/- 7 to 192 +/- 7 muM without altering maximum calcium binding. Ouabain pretreatment did not affect calcium binding to extracted phospholipids, ouabain-insensitive ATPases, or heat denatured Na+,K+-ATPase, Na+ and K+ (5-20 mM) increased the dissociation constants for calcium, which suggests competition between the monovalent cations and calcium for the binding sites. At higher concentrations of monovalent cations, ouabain increased the apparent affinity of binding sites for calcium. Extrapolation to physiological cation concentrations revealed that the ouabain-induced increase in apparent affinity for calcium may be as much as 2- to 3-fold. These results suggest: (1) calcium binds to phospholipids associated with Na+,K+-ATPase; (2) ouabain interaction with Na+,K+-ATPase induces a perturbation that is transmitted to adjacent phospholipids, altering their affinity for calcium; and (3) at physiological concentrations of Na+ or K+, or both, ouabain interaction with Na+,K+-ATPase may lead to an increased pool of membrane-bound calcium.

The 5-HT1A Receptor PET Radioligand 11C-CUMI-101 Has Significant Binding to α1-Adrenoceptors in Human Cerebellum, Limiting Its Use as a Reference Region.

PubMed

Shrestha, Stal S; Liow, Jeih-San; Jenko, Kimberly; Ikawa, Masamichi; Zoghbi, Sami S; Innis, Robert B

2016-12-01

Prazosin, a potent and selective α 1 -adrenoceptor antagonist, displaces 25% of 11 C-CUMI-101 ([O-methyl- 11 C]2-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-4-methyl-1,2,4-triazine-3,5(2H,4H)dione) binding in monkey cerebellum. We sought to estimate the percentage contamination of 11 C-CUMI-101 binding to α 1 -adrenoceptors in human cerebellum under in vivo conditions. In vitro receptor-binding techniques were used to measure α 1 -adrenoceptor density and the affinity of CUMI-101 for these receptors in human, monkey, and rat cerebellum. Binding potential (maximum number of binding sites × affinity [(1/dissociation constant]) was determined using in vitro homogenate binding assays in human, monkey, and rat cerebellum. 3 H-prazosin was used to determine the maximum number of binding sites, as well as the dissociation constant of 3 H-prazosin and the inhibition constant of CUMI-101. α 1 -adrenoceptor density and the affinity of CUMI-101 for these receptors were similar across species. Cerebellar binding potentials were 3.7 for humans, 2.3 for monkeys, and 3.4 for rats. Reasoning by analogy, 25% of 11 C-CUMI-101 uptake in human cerebellum reflects binding to α 1 -adrenoceptors, suggesting that the cerebellum is of limited usefulness as a reference tissue for quantification in human studies. © 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

The Universal Statistical Distributions of the Affinity, Equilibrium Constants, Kinetics and Specificity in Biomolecular Recognition

PubMed Central

Zheng, Xiliang; Wang, Jin

2015-01-01

We uncovered the universal statistical laws for the biomolecular recognition/binding process. We quantified the statistical energy landscapes for binding, from which we can characterize the distributions of the binding free energy (affinity), the equilibrium constants, the kinetics and the specificity by exploring the different ligands binding with a particular receptor. The results of the analytical studies are confirmed by the microscopic flexible docking simulations. The distribution of binding affinity is Gaussian around the mean and becomes exponential near the tail. The equilibrium constants of the binding follow a log-normal distribution around the mean and a power law distribution in the tail. The intrinsic specificity for biomolecular recognition measures the degree of discrimination of native versus non-native binding and the optimization of which becomes the maximization of the ratio of the free energy gap between the native state and the average of non-native states versus the roughness measured by the variance of the free energy landscape around its mean. The intrinsic specificity obeys a Gaussian distribution near the mean and an exponential distribution near the tail. Furthermore, the kinetics of binding follows a log-normal distribution near the mean and a power law distribution at the tail. Our study provides new insights into the statistical nature of thermodynamics, kinetics and function from different ligands binding with a specific receptor or equivalently specific ligand binding with different receptors. The elucidation of distributions of the kinetics and free energy has guiding roles in studying biomolecular recognition and function through small-molecule evolution and chemical genetics. PMID:25885453

The computational analysis and modelling of substitution effects on hydrolysis of formanilides in acidic aqueous solutions

NASA Astrophysics Data System (ADS)

Lukeš, Vladimír; Škorňa, Peter; Michalík, Martin; Klein, Erik

2017-11-01

Various para, meta and ortho substituted formanilides have been theoretically studied. For trans and cis-isomers of non-substituted formanilide, the calculated B3LYP vibration normal modes were analyzed. Substituent effect on the selected normal modes was described and the comparison with the available experimental data is presented. The calculated B3LYP proton affinities were correlated with Hammett constants, Fujita-Nishioka equation and the rate constants of the hydrolysis in 1 M HCl. Found linear dependences allow predictions of dissociation constants (pKBH+) and hydrolysis rate constants. Obtained results indicate that protonation of amide group may represent the rate determining step of acid catalyzed hydrolysis.

The Hubble Constant.

PubMed

Jackson, Neal

2015-01-01

I review the current state of determinations of the Hubble constant, which gives the length scale of the Universe by relating the expansion velocity of objects to their distance. There are two broad categories of measurements. The first uses individual astrophysical objects which have some property that allows their intrinsic luminosity or size to be determined, or allows the determination of their distance by geometric means. The second category comprises the use of all-sky cosmic microwave background, or correlations between large samples of galaxies, to determine information about the geometry of the Universe and hence the Hubble constant, typically in a combination with other cosmological parameters. Many, but not all, object-based measurements give H 0 values of around 72-74 km s -1 Mpc -1 , with typical errors of 2-3 km s -1 Mpc -1 . This is in mild discrepancy with CMB-based measurements, in particular those from the Planck satellite, which give values of 67-68 km s -1 Mpc -1 and typical errors of 1-2 km s -1 Mpc -1 . The size of the remaining systematics indicate that accuracy rather than precision is the remaining problem in a good determination of the Hubble constant. Whether a discrepancy exists, and whether new physics is needed to resolve it, depends on details of the systematics of the object-based methods, and also on the assumptions about other cosmological parameters and which datasets are combined in the case of the all-sky methods.

Expression of drug transporters and drug metabolizing enzymes in the bladder urothelium in man and affinity of the bladder spasmolytic trospium chloride to transporters likely involved in its pharmacokinetics.

PubMed

Bexten, Maria; Oswald, Stefan; Grube, Markus; Jia, Jia; Graf, Tanja; Zimmermann, Uwe; Rodewald, Kathrin; Zolk, Oliver; Schwantes, Ulrich; Siegmund, Werner; Keiser, Markus

2015-01-05

The cationic, water-soluble quaternary trospium chloride (TC) is incompletely absorbed from the gut and undergoes wide distribution but does not pass the blood-brain barrier. It is secreted by the kidneys, liver, and intestine. To evaluate potential transport mechanisms for TC, we measured affinity of the drug to the human uptake and efflux transporters known to be of pharmacokinetic relevance. Affinity of TC to the uptake transporters OATP1A2, -1B1, -1B3, -2B1, OCT1, -2, -3, OCTN2, NTCP, and ASBT and the efflux carriers P-gp, MRP2 and MRP3 transfected in HEK293 and MDCK2 cells was measured. To identify relevant pharmacokinetic mechanisms in the bladder urothelium, mRNA expression of multidrug transporters, drug metabolizing enzymes, and nuclear receptors, and the uptake of TC into primary human bladder urothelium (HBU) cells were measured. TC was shown to be a substrate of OATP1A2 (Km = 6.9 ± 1.3 μmol/L; Vmax = 41.6 ± 1.8 pmol/mg·min), OCT1 (Km = 106 ± 16 μmol/L; Vmax = 269 ± 18 pmol/mg·min), and P-gp (Km = 34.9 ± 7.5 μmol/L; Vmax = 105 ± 9.1 pmol/mg·min, lipovesicle assay). The genetic OATP1A2 variants *2 and *3 were loss-of-function transporters for TC. The mRNA expression analysis identified the following transporter proteins in the human urothelium: ABCB1 (P-gp), ABCC1-5 (MRP1-5), ABCG2 (BCRP), SLCO2B1 (OATP2B1), SLCO4A1 (OATP4A1), SLC22A1 (OCT1), SLC22A3 (OCT3), SLC22A4 (OCTN1), SLC22A5 (OCTN2), and SLC47A1 (MATE1). Immuno-reactive P-gp and OATP1A2 were localized to the apical cell layers. Drug metabolizing enzymes CYP3A5, -2B6, -2B7 -2E1, SULT1A1-4, UGT1A1-10, and UGT2B15, and nuclear receptors NR1H3 and NR1H4 were also expressed on mRNA level. TC was taken up into HBU cells (Km = 18.5 ± 4.8 μmol/L; Vmax = 106 ± 11.3 pmol/mg·min) by mechanisms that could be synergistically inhibited by naringin (IC50 = 10.8 (8.4; 13.8) μmol/L) and verapamil (IC50 = 4.6 (2.8; 7.5) μmol/L), inhibitors of OATP1A2 and OCT1, respectively. Affinity of TC to OCT1 and P-glycoprotein may be the reason for incomplete oral absorption, wide distribution into liver and kidneys, and substantial intestinal and renal secretions. Absence of brain distribution may result from affinity to P-gp and a low affinity to OATP1A2. The human urothelium expresses many drug transporters and drug metabolizing enzymes that may interact with TC and other drugs eliminated into the urine.

High-affinity RNA aptamers to C-reactive protein (CRP): newly developed pre-elution methods for aptamer selection

NASA Astrophysics Data System (ADS)

Orito, N.; Umekage, S.; Sato, K.; Kawauchi, S.; Tanaka, H.; Sakai, E.; Tanaka, T.; Kikuchi, Y.

2012-03-01

We have developed a modified SELEX (systematic evolution of ligands by exponential enrichment) method to obtain RNA aptamers with high affinity to C-reactive protein (CRP). CRP is a clinical biomarker present in plasma, the level of which increases in response to infections and noninfectious inflammation. The CRP level is also an important prognostic indicator in patients with several syndromes. At present, CRP content in blood is measured immunochemically using antibodies. To develop a more sensitive method using RNA aptamers, we have attempted to obtain high-affinity RNA aptamers to CRP. We succeeded in obtaining an RNA aptamer with high affinity to CRP using a CRP-immobilized Sepharose column and pre-elution procedure. Pre-elution is a method that removes the weak binding portion from a selected RNA population by washing for a short time with buffer containing CRP. By surface plasmon-resonance (SPR) analysis, the affinity constant of this aptamer for CRP was calculated to be KD = 2.25×10-9 (M). The secondary structure, contact sites with CRP protein, and application of this aptamer will be described.

Comparative study of substrate and product binding to the human ABO(H) blood group glycosyltransferases.

PubMed

Soya, Naoto; Shoemaker, Glen K; Palcic, Monica M; Klassen, John S

2009-11-01

The first comparative thermodynamic study of the human blood group glycosyltransferases, alpha-(1-->3)-N-acetylgalactosaminyltransferase (GTA) and alpha-(1-->3)-galactosyltransferase (GTB), interacting with donor substrates, donor and acceptor analogs, and trisaccharide products in vitro is reported. The binding constants, measured at 24 degrees C with the direct electrospray ionization mass spectrometry (ES-MS) assay, provide new insights into these model GTs and their interactions with substrate and product. Notably, the recombinant forms of GTA and GTB used in this study are shown to exist as homodimers, stabilized by noncovalent interactions at neutral pH. In the absence of divalent metal ion, neither GTA nor GTB exhibits any appreciable affinity for its native donors (UDP-GalNAc, UDP-Gal). Upon introduction of Mn(2+), both donors undergo enzyme-catalyzed hydrolysis in the presence of either GTA or GTB. Hydrolysis of UDP-GalNAc in the presence of GTA proceeds very rapidly under the solution conditions investigated and a binding constant could not be directly measured. In contrast, the rate of hydrolysis of UDP-Gal in the presence of GTB is significantly slower and, utilizing a modified approach to analyze the ES-MS data, a binding constant of 2 x 10(4) M(-1) was established. GTA and GTB bind the donor analogs UDP-GlcNAc, UDP-Glc with affinities similar to those measured for UDP-Gal and UDP-GalNAc (GTB only), suggesting that the native donors and donor analogs bind to the GTA and GTB through similar interactions. The binding constant determined for GTA and UDP-GlcNAc (approximately 1 x 10(4) M(-1)), therefore, provides an estimate for the binding constant for GTA and UDP-GalNAc. Binding of GTA and GTB with the A and B trisaccharide products was also investigated for the first time. In the absence of UDP and Mn(2+), both GTA and GTB recognize their respective trisaccharide products but with a low affinity approximately 10(3) M(-1); the presence of UDP and Mn(2+) has no effect on A trisaccharide binding but precludes B-trisaccharide binding.

Affinity of microbial transglutaminase to αs1-, β-, and acid casein under atmospheric and high pressure conditions.

PubMed

Menéndez, Orquídea; Schwarzenbolz, Uwe; Partschefeld, Claudia; Henle, Thomas

2009-05-27

Kinetics for the reaction of microbial transglutaminase (MTG) with individual caseins in a TRIS-acetate buffer at pH 6.0 was evaluated under atmospheric pressure (0.1 MPa) and high pressure (400 MPa) at 40 °C. The reaction was monitored under the following limitations: The kinetics from the initial velocities was obtained from nonprogressive enzymatic reactions assuming that the individual catalytic constants of reactive glutamine residues are represented by the reaction between MTG and casein monomers. Enzyme reaction kinetics carried out at 0.1 MPa at 40 °C showed Henri-Michaelis-Menten behavior with maximal velocities of 2.7 ± 0.02 × 10(-3), 0.8 ± 0.01 × 10(-3), and 1.3 ± 0.30 × 10(-3) mmol/L · min and K(m) values of 59 ± 2 × 10(-3), 64 ± 3 × 10(-3), and 50 ± 2 × 10(-3) mmol/L for β-, α(s1)-, and acid casein, respectively. Enzyme reaction kinetics of β-casein carried out at 400 MPa and 40 °C also showed a Henri-Michaelis-Menten behavior with a similar maximal velocity of 2.5 ± 0.33 × 10(-3) mmol/L · min, but, comparable to a competitive inhibition, the K(m) value increased to 144 ± 34 × 10(-3) mmol/L. The reaction of MTG with α(s1)-casein under high pressure did not fit in to Henri-Michaelis-Menten kinetics, indicating the complex influence of pressure on protein-enzyme interactions.

Ammonium and nitrite oxidation at nanomolar oxygen concentrations in oxygen minimum zone waters

PubMed Central

Bristow, Laura A.; Dalsgaard, Tage; Tiano, Laura; Mills, Daniel B.; Bertagnolli, Anthony D.; Wright, Jody J.; Hallam, Steven J.; Ulloa, Osvaldo; Canfield, Donald E.; Revsbech, Niels Peter; Thamdrup, Bo

2016-01-01

A major percentage of fixed nitrogen (N) loss in the oceans occurs within nitrite-rich oxygen minimum zones (OMZs) via denitrification and anammox. It remains unclear to what extent ammonium and nitrite oxidation co-occur, either supplying or competing for substrates involved in nitrogen loss in the OMZ core. Assessment of the oxygen (O2) sensitivity of these processes down to the O2 concentrations present in the OMZ core (<10 nmol⋅L−1) is therefore essential for understanding and modeling nitrogen loss in OMZs. We determined rates of ammonium and nitrite oxidation in the seasonal OMZ off Concepcion, Chile at manipulated O2 levels between 5 nmol⋅L−1 and 20 μmol⋅L−1. Rates of both processes were detectable in the low nanomolar range (5–33 nmol⋅L−1 O2), but demonstrated a strong dependence on O2 concentrations with apparent half-saturation constants (Kms) of 333 ± 130 nmol⋅L−1 O2 for ammonium oxidation and 778 ± 168 nmol⋅L−1 O2 for nitrite oxidation assuming one-component Michaelis–Menten kinetics. Nitrite oxidation rates, however, were better described with a two-component Michaelis–Menten model, indicating a high-affinity component with a Km of just a few nanomolar. As the communities of ammonium and nitrite oxidizers were similar to other OMZs, these kinetics should apply across OMZ systems. The high O2 affinities imply that ammonium and nitrite oxidation can occur within the OMZ core whenever O2 is supplied, for example, by episodic intrusions. These processes therefore compete with anammox and denitrification for ammonium and nitrite, thereby exerting an important control over nitrogen loss. PMID:27601665

Ligand-receptor binding affinities from saturation transfer difference (STD) NMR spectroscopy: the binding isotherm of STD initial growth rates.

PubMed

Angulo, Jesús; Enríquez-Navas, Pedro M; Nieto, Pedro M

2010-07-12

The direct evaluation of dissociation constants (K(D)) from the variation of saturation transfer difference (STD) NMR spectroscopy values with the receptor-ligand ratio is not feasible due to the complex dependence of STD intensities on the spectral properties of the observed signals. Indirect evaluation, by competition experiments, allows the determination of K(D), as long as a ligand of known affinity is available for the protein under study. Herein, we present a novel protocol based on STD NMR spectroscopy for the direct measurements of receptor-ligand dissociation constants (K(D)) from single-ligand titration experiments. The influence of several experimental factors on STD values has been studied in detail, confirming the marked impact on standard determinations of protein-ligand affinities by STD NMR spectroscopy. These factors, namely, STD saturation time, ligand residence time in the complex, and the intensity of the signal, affect the accumulation of saturation in the free ligand by processes closely related to fast protein-ligand rebinding and longitudinal relaxation of the ligand signals. The proposed method avoids the dependence of the magnitudes of ligand STD signals at a given saturation time on spurious factors by constructing the binding isotherms using the initial growth rates of the STD amplification factors, in a similar way to the use of NOE growing rates to estimate cross relaxation rates for distance evaluations. Herein, it is demonstrated that the effects of these factors are cancelled out by analyzing the protein-ligand association curve using STD values at the limit of zero saturation time, when virtually no ligand rebinding or relaxation takes place. The approach is validated for two well-studied protein-ligand systems: the binding of the saccharides GlcNAc and GlcNAcbeta1,4GlcNAc (chitobiose) to the wheat germ agglutinin (WGA) lectin, and the interaction of the amino acid L-tryptophan to bovine serum albumin (BSA). In all cases, the experimental K(D) measured under different experimental conditions converged to the thermodynamic values. The proposed protocol allows accurate determinations of protein-ligand dissociation constants, extending the applicability of the STD NMR spectroscopy for affinity measurements, which is of particular relevance for those proteins for which a ligand of known affinity is not available.

Development of a Gd(III)-based receptor-induced magnetization enhancement (RIME) contrast agent for β-glucuronidase activity profiling.

PubMed

Chen, Shih-Hsien; Kuo, Yu-Ting; Singh, Gyan; Cheng, Tian-Lu; Su, Yu-Zheng; Wang, Tzu-Pin; Chiu, Yen-Yu; Lai, Jui-Jen; Chang, Chih-Ching; Jaw, Twei-Shiun; Tzou, Shey-Cherng; Liu, Gin-Chung; Wang, Yun-Ming

2012-11-19

β-Glucuronidase is a key lysosomal enzyme and is often overexpressed in necrotic tumor masses. We report here the synthesis of a pro receptor-induced magnetization enhancement (pro-RIME) magnetic resonance imaging (MRI) contrast agent ([Gd(DOTA-FPβGu)]) for molecular imaging of β-glucuronidase activity in tumor tissues. The contrast agent consists of two parts, a gadolinium complex and a β-glucuronidase substrate (β-d-glucopyranuronic acid). The binding association constant (KA) of [Gd(DOTA-FPβGu)] is 7.42 × 10(2), which is significantly lower than that of a commercially available MS-325 (KA = 3.0 × 10(4)) RIME contrast agent. The low KA value of [Gd(DOTA-FPβGu)] is due to the pendant β-d-glucopyranuronic acid moiety. Therefore, [Gd(DOTA-FPβGu)] can be used for detection of β-glucuronidase through RIME modulation. The detail mechanism of enzymatic activation of [Gd(DOTA-FPβGu)] was elucidated by LC-MS. The kinetics of β-glucuronidase catalyzed hydrolysis of [Eu(DOTA-FPβGu)] at pH 7.4 best fit the Miechalis-Menten kinetic mode with Km = 1.38 mM, kcat = 3.76 × 10(3), and kcat/Km = 2.72 × 10(3) M(-1) s(-1). The low Km value indicates high affinity of β-glucuronidase for [Gd(DOTA-FPβGu)] at physiological pH. Relaxometric studies revealed that T1 relaxivity of [Gd(DOTA-FPβGu)] changes in response to the concentration of β-glucuronidase. Consistent with the relaxometric studies, [Gd(DOTA-FPβGu)] showed significant change in MR image signal in the presence of β-glucuronidase and HSA. In vitro and in vivo MR images demonstrated appreciable differences in signal enhancement in the cell lines and tumor xenografts in accordance to their expression levels of β-glucuronidase.

Critical evaluation of methods to incorporate entropy loss upon binding in high-throughput docking.

PubMed

Salaniwal, Sumeet; Manas, Eric S; Alvarez, Juan C; Unwalla, Rayomand J

2007-02-01

Proper accounting of the positional/orientational/conformational entropy loss associated with protein-ligand binding is important to obtain reliable predictions of binding affinity. Herein, we critically examine two simplified statistical mechanics-based approaches, namely a constant penalty per rotor method, and a more rigorous method, referred to here as the partition function-based scoring (PFS) method, to account for such entropy losses in high-throughput docking calculations. Our results on the estrogen receptor beta and dihydrofolate reductase proteins demonstrate that, while the constant penalty method over-penalizes molecules for their conformational flexibility, the PFS method behaves in a more "DeltaG-like" manner by penalizing different rotors differently depending on their residual entropy in the bound state. Furthermore, in contrast to no entropic penalty or the constant penalty approximation, the PFS method does not exhibit any bias towards either rigid or flexible molecules in the hit list. Preliminary enrichment studies using a lead-like random molecular database suggest that an accurate representation of the "true" energy landscape of the protein-ligand complex is critical for reliable predictions of relative binding affinities by the PFS method. Copyright 2006 Wiley-Liss, Inc.

Mobility-based correction for accurate determination of binding constants by capillary electrophoresis-frontal analysis.

PubMed

Qian, Cheng; Kovalchik, Kevin A; MacLennan, Matthew S; Huang, Xiaohua; Chen, David D Y

2017-06-01

Capillary electrophoresis frontal analysis (CE-FA) can be used to determine binding affinity of molecular interactions. However, its current data processing method mandate specific requirement on the mobilities of the binding pair in order to obtain accurate binding constants. This work shows that significant errors are resulted when the mobilities of the interacting species do not meet these requirements. Therefore, the applicability of CE-FA in many real word applications becomes questionable. An electrophoretic mobility-based correction method is developed in this work based on the flux of each species. A simulation program and a pair of model compounds are used to verify the new equations and evaluate the effectiveness of this method. Ibuprofen and hydroxypropyl-β-cyclodextrinare used to demonstrate the differences in the obtained binding constant by CE-FA when different calculation methods are used, and the results are compared with those obtained by affinity capillary electrophoresis (ACE). The results suggest that CE-FA, with the mobility-based correction method, can be a generally applicable method for a much wider range of applications. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A high-resolution photoelectron imaging and theoretical study of CP- and C2P-

NASA Astrophysics Data System (ADS)

Czekner, Joseph; Cheung, Ling Fung; Johnson, Eric L.; Fortenberry, Ryan C.; Wang, Lai-Sheng

2018-01-01

The discovery of interstellar anions has been a milestone in astrochemistry. In the search for new interstellar anions, CP- and C2P- are viable candidates since their corresponding neutrals have already been detected astronomically. However, scarce data exist for these negatively charged species. Here we report the electron affinities of CP and C2P along with the vibrational frequencies of their anions using high-resolution photoelectron imaging. These results along with previous spectroscopic data of the neutral species are used further to benchmark very accurate quartic force field quantum chemical methods that are applied to CP, CP-, C2P, and two electronic states of C2P-. The predicted electron affinities, vibrational frequencies, and rotational constants are in excellent agreement with the experimental data. The electron affinities of CP (2.8508 ± 0.0007 eV) and C2P (2.6328 ± 0.0006 eV) are measured accurately and found to be quite high, suggesting that the CP- and C2P- anions are thermodynamically stable and possibly observable. The current study suggests that the combination of high-resolution photoelectron imaging and quantum chemistry can be used to determine accurate molecular constants for exotic radical species of astronomical interest.

Interaction of flavonols with human serum albumin: a biophysical study showing structure-activity relationship and enhancement when coated on silver nanoparticles.

PubMed

Das, Pratyusa; Chaudhari, Sunil Kumar; Das, Asmita; Kundu, Somashree; Saha, Chabita

2018-04-24

Binding affinities of flavonols namely quercetin, myricetin, and kaempferol to human serum albumin (HSA) were determined fluorimetrically and the order was observed to be myricetin > quercetin > kaempferol demonstrating structure-activity relationship. Quercetin-coated silver nanoparticles (AgNPs) show higher binding affinity to HSA compared to free quercetin with binding constants 6.04 × 10 7  M -1 and 4.2 × 10 6  M -1 , respectively. Using site-specific markers it is concluded that free quercetin and that coated on AgNPs bind at different sites. Significant structural changes in circular dichroism (CD) spectra of HSA were recorded with quercetin-coated AgNPs compared to free quercetin. These results were further substantiated by time-resolved fluorescence spectroscopy where fluorescence life time of the tryptophan residue in HSA-quercetin-coated AgNPs complex decreased to 3.63 ns from 4.22 ns in HSA-quercetin complex. Isothermal calorimetric studies reveal two binding modes for quercetin-coated AgNPs and also higher binding constants compared to free quercetin. These higher binding affinities are attributed to altered properties of quercetin when coated on AgNPs enabling it to reach the binding sites other than site II where free quercetin mainly binds.

A high-resolution photoelectron imaging and theoretical study of CP- and C2P.

PubMed

Czekner, Joseph; Cheung, Ling Fung; Johnson, Eric L; Fortenberry, Ryan C; Wang, Lai-Sheng

2018-01-28

The discovery of interstellar anions has been a milestone in astrochemistry. In the search for new interstellar anions, CP - and C 2 P - are viable candidates since their corresponding neutrals have already been detected astronomically. However, scarce data exist for these negatively charged species. Here we report the electron affinities of CP and C 2 P along with the vibrational frequencies of their anions using high-resolution photoelectron imaging. These results along with previous spectroscopic data of the neutral species are used further to benchmark very accurate quartic force field quantum chemical methods that are applied to CP, CP - , C 2 P, and two electronic states of C 2 P - . The predicted electron affinities, vibrational frequencies, and rotational constants are in excellent agreement with the experimental data. The electron affinities of CP (2.8508 ± 0.0007 eV) and C 2 P (2.6328 ± 0.0006 eV) are measured accurately and found to be quite high, suggesting that the CP - and C 2 P - anions are thermodynamically stable and possibly observable. The current study suggests that the combination of high-resolution photoelectron imaging and quantum chemistry can be used to determine accurate molecular constants for exotic radical species of astronomical interest.

Modelling of the acid-base properties of natural and synthetic adsorbent materials used for heavy metal removal from aqueous solutions.

PubMed

Pagnanelli, Francesca; Vegliò, Francesco; Toro, Luigi

2004-02-01

In this paper a comparison about kinetic behaviour, acid-base properties and copper removal capacities was carried out between two different adsorbent materials used for heavy metal removal from aqueous solutions: an aminodiacetic chelating resin as commercial product (Lewatit TP207) and a lyophilised bacterial biomass of Sphaerotilus natans. The acid-base properties of a S. natans cell suspension were well described by simplified mechanistic models without electrostatic corrections considering two kinds of weakly acidic active sites. In particular the introduction of two-peak distribution function for the proton affinity constants allows a better representation of the experimental data reproducing the site heterogeneity. A priori knowledge about resin functional groups (aminodiacetic groups) is the base for preliminary simulations of titration curve assuming a Donnan gel structure for the resin phase considered as a concentrated aqueous solution of aminodiacetic acid (ADA). Departures from experimental and simulated data can be interpreted by considering the heterogeneity of the functional groups and the effect of ionic concentration in the resin phase. Two-site continuous model describes adequately the experimental data. Moreover the values of apparent protonation constants (as adjustable parameters found by non-linear regression) are very near to the apparent constants evaluated by a Donnan model assuming the intrinsic constants in resin phase equal to the equilibrium constants in aqueous solution of ADA and considering the amphoteric nature of active sites for the evaluation of counter-ion concentration in the resin phase. Copper removal outlined the strong affinity of the active groups of the resin for this ion in solution compared to the S. natans biomass according to the complexation constants between aminodiacetic and mono-carboxylic groups and copper ions.

Three new defined proton affinities for polybasic molecules in the gas-phase: Proton microaffinity, proton macroaffinity and proton overallaffinity

NASA Astrophysics Data System (ADS)

Salehzadeh, Sadegh; Bayat, Mehdi

2006-08-01

A theoretical study on complete protonation of a series of tetrabasic molecules with general formula N[(CH 2) nNH 2][(CH 2) mNH 2][(CH 2) pNH 2] (tren, pee, ppe, tpt, epb and ppb) is reported. For first time, three kinds of gas-phase proton affinities for each polybasic molecule are defined as: 'proton microaffinity (PA n, i)', 'proton macroaffinity (PA)' and 'proton overall affinity ( PA)'. The variations of calculated logPA in the series of these molecules is very similar to that of their measured log Kn. There is also a good correlation between the calculated gas-phase proton macroaffinities and proton overallaffinities with corresponding equilibrium macroconstants and overall protonation constants in solution.

Complex high affinity interactions occur between MHCI and superantigens

NASA Technical Reports Server (NTRS)

Chapes, S. K.; Herpich, A. R.; Spooner, B. S. (Principal Investigator)

1998-01-01

Staphylococcal enterotoxins A and C1 (SEA or SEC1) bound to major histocompatibility-I (MHCI) molecules with high affinity (binding constants ranging from 1.1 microM to 79 nM). SEA and SEC1 directly bound MHCI molecules that had been captured by monoclonal antibodies specific for H-2Kk, H-2Dk, or both. In addition, MHCI-specific antibodies inhibited the binding of SEC1 to LM929 cells and SEA competitively inhibited SEC1 binding; indicating that the superantigens bound to MHCI on the cell surface. The affinity and number of superantigen binding sites differed depending on whether MHCI was expressed in the membrane of LM929 cells or whether it was captured. These data support the hypothesis that MHCI molecules can serve as superantigen receptors.

[Ecological affinity and current distribution of primates (Cebidae) in Campeche, Mexico].

PubMed

Navarro Fernández, Eloísa; Pozo de la Tijera, Carmen; Escobedo Cabrera, Enrique

2003-06-01

We carried out surveys realized field work from March to September 2000 to get the current distribution of Cebids in the state of Campeche, Mexico. Based on interviews and direct observations. We defined the distribution of Ateles geoffroyi yucatanensis and Alouatta pigra and we documented the first time localities where Allouata palliata is found in the state. We made distributional maps of each species using vegetation overlays from Inventario Nacional Forestal (Inv For) and each point documented during fieldwork. We presented the distribution of species according to confiability of the verified or expected data. Using the attributes table of Inv For, we calculated the areas of distribution which were 22,735 km2 for Alouatta sp. and 18,501 km2 for A. g. yucatanensis. We also presented the area occupied by each species according to vegetation types and the relative proportion of these vegetation types in the state. We confirmed the ability of Alouatta sp. to survive in disturbed environments produced by habitat fragmentation, and the affinity of A. g. yucatanesis to well preserved habitats.

Methotrexate-loxoprofen interaction: involvement of human organic anion transporters hOAT1 and hOAT3.

PubMed

Uwai, Yuichi; Taniguchi, Risa; Motohashi, Hideyuki; Saito, Hideyuki; Okuda, Masahiro; Inui, Ken-ichi

2004-10-01

Human organic anion transporters hOAT1 (SLC22A6) and hOAT3 (SLC22A8) are responsible for renal tubular secretion of an antifolic acid methotrexate, and are considered to be involved in drug interaction of methotrexate with nonsteroidal anti-inflammatory drugs (NSAIDs). In our hospital, a delay of methotrexate elimination was experienced in a patient with Hodgkin's disease, who took loxoprofen, a commonly used NSAID in Japan, which suggested a cause. In this study, we examined the drug interaction via hOAT1 and hOAT3, using Xenopus laevis oocytes. hOAT1 and hOAT3 mediated the methotrexate transport with low affinity (K(m) of 724.0 muM) and high affinity (K(m) of 17.2 muM), respectively. Loxoprofen and its trans-OH metabolite, an active major metabolite, markedly inhibited the methotrexate transport by both transporters. Their inhibition concentrations (IC(50)) were in the range of the therapeutic levels. These findings suggest that loxoprofen retards the elimination of methotrexate, at least in part, by inhibiting hOAT1 and hOAT3.

Simultaneous addition of two ligands: a potential strategy for estimating divalent ion affinities in EF-hand proteins by isothermal titration calorimetry.

PubMed

Henzl, Michael T; Markus, Lindsey A; Davis, Meredith E; McMillan, Andrew T

2013-03-01

Capable of providing a detailed thermodynamic picture of noncovalent association reactions, isothermal titration calorimetry (ITC) has become a popular method for studying protein-ligand interactions. We routinely employ the technique to study divalent ion-binding by two-site EF-hand proteins from the parvalbumin- and polcalcin lineages. The combination of high Ca(2+) affinity and relatively low Mg(2+) affinity, and the attendant complication of parameter correlation, conspire to make the simultaneous extraction of binding constants and -enthalpies for both ions challenging. Although global analysis of multiple ITC experiments can overcome these hurdles, our current experimental protocol includes upwards of 10 titrations - requiring a substantial investment in labor, machine time, and material. This paper explores the potential for using a smaller suite of experiments that includes simultaneous titrations with Ca(2+) and Mg(2+) at different ratios of the two ions. The results obtained for four proteins, differing substantially in their divalent ion-binding properties, suggest that the approach has merit. The Ca(2+)- and Mg(2+)-binding constants afforded by the streamlined analysis are in reasonable agreement with those obtained from the standard analysis protocol. Likewise, the abbreviated analysis provides comparable values for the Ca(2+)-binding enthalpies. However, the streamlined analysis can yield divergent values for the Mg(2+)-binding enthalpies - particularly those for lower affinity sites. This shortcoming can be remedied, in large measure, by including data from a direct Ca(2+) titration in the presence of a high, fixed Mg(2+) concentration. Copyright © 2013. Published by Elsevier Inc.

Inhibition of monoamine oxidase A and B activities by imidazol(ine)/guanidine drugs, nature of the interaction and distinction from I2-imidazoline receptors in rat liver

PubMed Central

Ozaita, Andrés; Olmos, Gabriel; Assumpció Boronat, M; Miguel Lizcano, José; Unzeta, Mercedes; García-Sevilla, Jesús A

1997-01-01

I2-Imidazoline sites ([3H]-idazoxan binding) have been identified on monoamine oxidase (MAO) and proposed to modulate the activity of the enzyme through an allosteric inhibitory mechanism (Tesson et al., 1995). The main aim of this study was to assess the inhibitory effects and nature of the inhibition of imidazol(ine)/guanidine drugs on rat liver MAO-A and MAO-B isoforms and to compare their inhibitory potencies with their affinities for the sites labelled by [3H]-clonidine in the same tissue. Competition for [3H]-clonidine binding in rat liver mitochondrial fractions by imidazol(ine)/guanidine compounds revealed that the pharmacological profile of the interaction (2 - styryl - 2 - imidazoline, LSL 61112>idazoxan>2 - benzofuranyl - 2 - imidazoline, 2-BFI=cirazoline>guanabenz>oxymetazoline>>clonidine) was typical of that for I2-sites. Clonidine inhibited rat liver MAO-A and MAO-B activities with very low potency (IC50s: 700 μM and 6 mM, respectively) and displayed the typical pattern of competitive enzyme inhibition (Lineweaver-Burk plots: increased Km and unchanged Vmax values). Other imidazol(ine)/guanidine drugs also were weak MAO inhibitors with the exception of guanabenz, 2-BFI and cirazoline on MAO-A (IC50s: 4–11 μM) and 2-benzofuranyl-2-imidazol (LSL 60101) on MAO-B (IC50: 16 μM). Idazoxan was a full inhibitor, although with rather low potency, on both MAO-A and MAO-B isoenzymes (IC50s: 280 μM and 624 μM, respectively). Kinetic analyses of MAO-A inhibition by these drugs revealed that the interactions were competitive. For the same drugs acting on MAO-B the interactions were of the mixed type inhibition (increased Km and decreased Vmax values), although the greater inhibitory effects on the apparent value of Vmax/Km than on the Vmax value indicated that the competitive element of the MAO-B inhibition predominated. Competition for [3H]-Ro 41-1049 binding to MAO-A or [3H]-Ro 19-6327 binding to MAO-B in rat liver mitochondrial fractions by imidazol(ine)/guanidine compounds revealed that the drug inhibition constants (Ki values) were similar to the IC50 values displayed for the inhibition of MAO-A or MAO-B activities. In fact, very good correlations were obtained when the affinities of drugs at MAO-A or MAO-B catalytic sites were correlated with their potencies in inhibiting MAO-A (r=0.92) or MAO-B (r=0.99) activity. This further suggested a direct drug interaction with the catalytic sites of MAO-A and MAO-B isoforms. No significant correlations were found when the potencies of imidazol(ine)/guanidine drugs at the high affinity site (pKiH, nanomolar range) or the low-affinity site (pKiL, micromolar range) of I2-imidazoline receptors labelled with [3H]-clonidine were correlated with the pIC50 values of the same drugs for inhibition of MAO-A or MAO-B activity. These discrepancies indicated that I2-imidazoline receptors are not directly related to the site of action of these drugs on MAO activity in rat liver mitochondrial fractions. Although these studies cannot exclude the presence of additional binding sites on MAO that do not affect the activity of the enzyme, they would suggest that I2-imidazoline receptors represent molecular species that are distinct from MAO. PMID:9222546

Crystal structure and ligand affinity of avidin in the complex with 4‧-hydroxyazobenzene-2-carboxylic acid

NASA Astrophysics Data System (ADS)

Strzelczyk, Paweł; Bujacz, Grzegorz

2016-04-01

Avidin is a protein found in egg white that binds numerous organic compounds with high affinity, especially biotin and its derivatives. Due to its extraordinary affinity for its ligands, avidin is extensively used in biotechnology. X-ray crystallography and fluorescence-based biophysical techniques were used to show that avidin binds the dye 4‧-hydroxyazobenzene-2-carboxylic acid (HABA) with a lower affinity than biotin. The apparent dissociation constant determined for the avidin complex with HABA by microscale thermophoresis (MST) is 4.12 μM. The crystal structure of avidin-HABA complex was determined at a resolution of 2.2 Å (PDB entry 5chk). The crystals belong to a hexagonal system, in the space group P6422. In that structure, the hydrazone tautomer of HABA is bound at the bottom part of the central calyx near the polar residues. We show interactions of the dye with avidin and compare them with the previously reported avidin-biotin complex.

Interaction of phenolic acids and their derivatives with human serum albumin: Structure-affinity relationships and effects on antioxidant activity.

PubMed

Zhang, Yunyue; Wu, Simin; Qin, Yinghui; Liu, Jiaxin; Liu, Jingwen; Wang, Qingyu; Ren, Fazheng; Zhang, Hao

2018-02-01

In this study, 111 phenolic acids and their derivatives were chosen to investigate their structure-affinity relationships when binding to human serum albumin (HSA), and effects on their antioxidant activity. A comprehensive mathematical model was employed to calculate the binding constants, using a fluorescence quenching method, and this was corrected for the inner-filter effect to improve accuracy. We found that a hydroxy group at the 2-position of the benzene ring exerted a positive effect on the affinities, while a 4-hydroxy substituent had a negative influence. Both methylation of the hydroxy groups and replacing the hydroxy groups with methyl groups at the 3- and 4-positions of the benzene ring enhanced the binding affinities. Hydrophobic force and hydrogen bonding were binding forces for the phenolic acids, and their methyl esters, respectively. The antioxidant activity of the HSA-phenolic acid interaction compounds was higher than that of the phenolic acids alone. Copyright © 2017. Published by Elsevier Ltd.

PLASS: Protein-ligand affinity statistical score a knowledge-based force-field model of interaction derived from the PDB

NASA Astrophysics Data System (ADS)

Ozrin, V. D.; Subbotin, M. V.; Nikitin, S. M.

2004-04-01

We have developed PLASS (Protein-Ligand Affinity Statistical Score), a pair-wise potential of mean-force for rapid estimation of the binding affinity of a ligand molecule to a protein active site. This scoring function is derived from the frequency of occurrence of atom-type pairs in crystallographic complexes taken from the Protein Data Bank (PDB). Statistical distributions are converted into distance-dependent contributions to the Gibbs free interaction energy for 10 atomic types using the Boltzmann hypothesis, with only one adjustable parameter. For a representative set of 72 protein-ligand structures, PLASS scores correlate well with the experimentally measured dissociation constants: a correlation coefficient R of 0.82 and RMS error of 2.0 kcal/mol. Such high accuracy results from our novel treatment of the volume correction term, which takes into account the inhomogeneous properties of the protein-ligand complexes. PLASS is able to rank reliably the affinity of complexes which have as much diversity as in the PDB.

Complementary DNA display selection of high-affinity peptides binding the vacuolating toxin (VacA) of Helicobacter pylori.

PubMed

Hayakawa, Yumiko; Matsuno, Mitsuhiro; Tanaka, Makoto; Wada, Akihiro; Kitamura, Koichiro; Takei, Osamu; Sasaki, Ryuzo; Mizukami, Tamio; Hasegawa, Makoto

2015-09-01

Artificial peptides designed for molecular recognition of a bacterial toxin have been developed. Vacuolating cytotoxin A protein (VacA) is a major virulence factor of Helicobacter pylori, a gram-negative microaerophilic bacterium inhabiting the upper gastrointestinal tract, particularly the stomach. This study attempted to identify specific peptide sequences with high affinity for VacA using systematic directed evolution in vitro, a cDNA display method. A surface plasmon resonance-based biosensor and fluorescence correlation spectroscopy to examine binding of peptides with VacA identified a peptide (GRVNQRL) with high affinity. Cyclization of the peptide by attaching cysteine residues to both termini improved its binding affinity to VacA, with a dissociation constant (Kd ) of 58 nm. This study describes a new strategy for the development of artificial functional peptides, which are promising materials in biochemical analyses and medical applications. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

Na(+)-dependent transport of taurine is found only on the abluminal membrane of the blood-brain barrier.

PubMed

Rasgado-Flores, Hector; Mokashi, Ashwini; Hawkins, Richard A

2012-01-01

Luminal and abluminal plasma membranes were isolated from bovine brain microvessels and used to identify and characterize Na(+)-dependent and facilitative taurine transport. The calculated transmembrane potential was -59 mV at time 0; external Na(+) (or choline under putative zero-trans conditions) was 126 mM (T=25 °C). The apparent affinity constants of the taurine transporters were determined over a range of taurine concentrations from 0.24 μM to 11.4 μM. Abluminal membranes had both Na(+)-dependent taurine transport as well as facilitative transport while luminal membranes only had facilitative transport. The apparent K(m) for facilitative and Na(+)-dependent taurine transport were 0.06±0.02 μM and 0.7±0.1 μM, respectively. The Na(+)-dependent transport of taurine was voltage dependent over the range of voltages studied (-25 to -101 mV). The transport was over 5 times greater at -101 mV compared to when V(m) was -25 mV. The sensitivity to external osmolality of Na(+)-dependent transport was studied over a range of osmolalities (229 to 398 mOsm/kg H(2)O) using mannitol as the osmotic agent to adjust the osmolality. For these experiments the concentration of Na(+) was maintained constant at 50mM, and the calculated transmembrane potential was -59 mV. The Na(+)-dependent transport system was sensitive to osmolality with the greatest rate observed at 229 mOsm/kg H(2)O. Copyright © 2011 Elsevier Inc. All rights reserved.

On the binding affinity of macromolecular interactions: daring to ask why proteins interact

PubMed Central

Kastritis, Panagiotis L.; Bonvin, Alexandre M. J. J.

2013-01-01

Interactions between proteins are orchestrated in a precise and time-dependent manner, underlying cellular function. The binding affinity, defined as the strength of these interactions, is translated into physico-chemical terms in the dissociation constant (Kd), the latter being an experimental measure that determines whether an interaction will be formed in solution or not. Predicting binding affinity from structural models has been a matter of active research for more than 40 years because of its fundamental role in drug development. However, all available approaches are incapable of predicting the binding affinity of protein–protein complexes from coordinates alone. Here, we examine both theoretical and experimental limitations that complicate the derivation of structure–affinity relationships. Most work so far has concentrated on binary interactions. Systems of increased complexity are far from being understood. The main physico-chemical measure that relates to binding affinity is the buried surface area, but it does not hold for flexible complexes. For the latter, there must be a significant entropic contribution that will have to be approximated in the future. We foresee that any theoretical modelling of these interactions will have to follow an integrative approach considering the biology, chemistry and physics that underlie protein–protein recognition. PMID:23235262

Selection is more intelligent than design: improving the affinity of a bivalent ligand through directed evolution.

PubMed

Ahmad, Kareem M; Xiao, Yi; Soh, H Tom

2012-12-01

Multivalent molecular interactions can be exploited to dramatically enhance the performance of an affinity reagent. The enhancement in affinity and specificity achieved with a multivalent construct depends critically on the effectiveness of the scaffold that joins the ligands, as this determines their positions and orientations with respect to the target molecule. Currently, no generalizable design rules exist for construction of an optimal multivalent ligand for targets with known structures, and the design challenge remains an insurmountable obstacle for the large number of proteins whose structures are not known. As an alternative to such design-based strategies, we report here a directed evolution-based method for generating optimal bivalent aptamers. To demonstrate this approach, we fused two thrombin aptamers with a randomized DNA sequence and used a microfluidic in vitro selection strategy to isolate scaffolds with exceptionally high affinities. Within five rounds of selection, we generated a bivalent aptamer that binds thrombin with an apparent dissociation constant (K(d)) <10 pM, representing a ∼200-fold improvement in binding affinity over the monomeric aptamers and a ∼15-fold improvement over the best designed bivalent construct. The process described here can be used to produce high-affinity multivalent aptamers and could potentially be adapted to other classes of biomolecules.

Kinetics and spatial distribution of enzymes of carbon, nitrogen and phosphorus cycles in earthworm biopores

NASA Astrophysics Data System (ADS)

Hoang Thi Thu, Duyen; Razavi, Bahar S.

2016-04-01

Earthworms boost microbial activities and consequently form hotspots in soil. The distribution of enzyme activities inside the earthworm biopores is completely unknown. For the first time, we analyzed enzyme kinetics and visualized enzyme distribution inside and outside biopores by in situ soil zymography. Kinetic parameters (Vmax and Km) of 6 enzymes β-glucosidase (GLU), cellobiohydrolase (CBH), xylanase (XYL), chitinase (NAG), leucine aminopeptidase (LAP) and acid phosphatase (APT) were determined in biopores formed by Lumbricus terrestris L.. The spatial distributions of GLU, NAG and APT become visible via zymograms in comparison between earthworm-inhabited and earthworm-free soil. Zymography showed heterogeneous distribution of hotspots in the rhizosphere and biopores. The hotspot areas were 2.4 to 14 times larger in the biopores than in soil without earthworms. The significantly higher Vmax values for GLU, CBH, XYL, NAG and APT in biopores confirmed the stimulation of enzyme activities by earthworms. For CBH, XYL and NAG, the 2- to 3-fold higher Km values in biopores indicated different enzyme systems with lower substrate affinity compared to control soil. The positive effects of earthworms on Vmax were cancelled by the Km increase for CBH, XYL and NAG at a substrate concentration below 20 μmol g-1 soil. The change of enzyme systems reflected a shift in dominant microbial populations toward species with lower affinity to holo-celluloses and to N-acetylglucosamine, and with higher affinity to proteins as compared to the biopores-free soil. We conclude that earthworm biopores are microbial hotspots with much higher and dense distribution of enzyme activities compared to bulk soil. References Spohn M, Kuzyakov Y. (2014) Spatial and temporal dynamics of hotspots of enzyme activity in soil as affected by living and dead roots - a soil zymography analysis, Plant Soil 379: 67-77. Blagodatskaya, E., Kuzyakov, Y., 2013. Review paper: Active microorganisms in soil: Critical review of estimation criteria and approaches. Soil Biology & Biochemistry 67, 192-211.

Mutational scanning of the human serotonin transporter reveals fast translocating serotonin transporter mutants.

PubMed

Kristensen, Anders S; Larsen, Mads B; Johnsen, Laust B; Wiborg, Ove

2004-03-01

The serotonin transporter (SERT) belongs to a family of sodium-chloride-dependent transporters responsible for uptake of amino acids and biogenic amines from the extracellular space. SERT represents a major pharmacological target in the treatment of several clinical conditions, including depression and anxiety. In the present study we have undertaken a mutational scanning of human SERT in order to identify residues that are responsible for individual differences among related monoamine transporters. One mutant, G100A, was inactive in transport. However, ligand binding affinity was similar to wild-type, suggesting that G100A amongst different possible SERT conformations is restrained to a binding conformation. We suggest that the main role of glycine-100 is to confer structural flexibility during substrate translocation. For the two single mutants, T178A and F263C, uptake rates and K(m) values were both several-fold higher than wild-type while binding affinities and inhibitory potencies decreased considerably for several drugs. Ion dependency increased and only at hyperosmotic concentrations were K(m) values partly restored. For the double mutant, T178A/F263C, shifts in uptake kinetics and ligand affinities, as well as ion dependencies, were drastic. Effects were synergistic compared to the corresponding single mutants. In conclusion, we suggest that mutating threonine-178 to an alanine and phenylalanine-263 to a cysteine mainly alter the overall uptake kinetics of SERT by affecting the conformational equilibrium of different transporter conformations.

Inclusion of fluorophores in cyclodextrins: a closer look at the fluorometric determination of association constants by linear and nonlinear fitting procedures

NASA Astrophysics Data System (ADS)

Hutterer, Rudi

2018-01-01

The author discusses methods for the fluorometric determination of affinity constants by linear and nonlinear fitting methods. This is outlined in particular for the interaction between cyclodextrins and several anesthetic drugs including benzocaine. Special emphasis is given to the limitations of certain fits, and the impact of such studies on enzyme-substrate interactions are demonstrated. Both the experimental part and methods of analysis are well suited for students in an advanced lab.

Fluorescence resonance energy-transfer affects the determination of the affinity between ligand and proteins obtained by fluorescence quenching method

NASA Astrophysics Data System (ADS)

Xiao, Jianbo; Wei, Xinlin; Wang, Yuanfeng; Liu, Chunxi

2009-11-01

The interaction between esculin and serum albumins was investigated to illustrate that the fluorescence resonance energy-transfer (FRET) affects the determination of the binding constants obtained by fluorescence quenching method. The binding constants ( Ka) obtained by the double-logarithm curve for esculin-BSA and esculin-HSA were 1.02 × 10 7 and 2.07 × 10 4 L/mol, respectively. These results from synchronous fluorescence showed that the Tyr and Trp residues of HSA were affected more deeply than those in BSA. The excitation profile of esculin showed that in the presence of BSA and HSA, the S 0 → S 1 transition of esculin ( λexmax≈340 nm) appears, which is similar to the λemmax of BSA and HSA. The critical distance ( R0) between BSA and esculin is higher than that of HSA, which showed that the affinity of esculin and HSA should be higher than that of BSA. After centrifugation, the concentrations of esculin bound to albumins were determined by means of the fluorescence of esculin. It was found that much more esculin was bound to HSA than to BSA. However, the bound models for BSA and HSA are almost the same. The concentration of esculin bound to serum albumin at first decreased with the addition of esculin and then increased. From above results, it can be concluded that the affinity of esculin and HSA should be higher than that of esculin and BSA. This example showed that in the presence of FRET, the binding constants between ligands and proteins based on fluorescence quenching might be deviated.

Fluorescence resonance energy-transfer affects the determination of the affinity between ligand and proteins obtained by fluorescence quenching method.

PubMed

Xiao, Jianbo; Wei, Xinlin; Wang, Yuanfeng; Liu, Chunxi

2009-11-01

The interaction between esculin and serum albumins was investigated to illustrate that the fluorescence resonance energy-transfer (FRET) affects the determination of the binding constants obtained by fluorescence quenching method. The binding constants (K(a)) obtained by the double-logarithm curve for esculin-BSA and esculin-HSA were 1.02x10(7) and 2.07x10(4)L/mol, respectively. These results from synchronous fluorescence showed that the Tyr and Trp residues of HSA were affected more deeply than those in BSA. The excitation profile of esculin showed that in the presence of BSA and HSA, the S(0)-->S(1) transition of esculin (lambda(ex)(max) approximately 340nm) appears, which is similar to the lambda(em)(max) of BSA and HSA. The critical distance (R(0)) between BSA and esculin is higher than that of HSA, which showed that the affinity of esculin and HSA should be higher than that of BSA. After centrifugation, the concentrations of esculin bound to albumins were determined by means of the fluorescence of esculin. It was found that much more esculin was bound to HSA than to BSA. However, the bound models for BSA and HSA are almost the same. The concentration of esculin bound to serum albumin at first decreased with the addition of esculin and then increased. From above results, it can be concluded that the affinity of esculin and HSA should be higher than that of esculin and BSA. This example showed that in the presence of FRET, the binding constants between ligands and proteins based on fluorescence quenching might be deviated.

Low-affinity FcγR interactions can decide the fate of novel human IgG-sensitised red blood cells and platelets

PubMed Central

Armour, Kathryn L; Smith, Cheryl S; Turner, Craig P; Kirton, Christopher M; Wilkes, Anthony M; Hadley, Andrew G; Ghevaert, Cedric; Williamson, Lorna M; Clark, Michael R

2014-01-01

G1Δnab is a mutant human IgG1 constant region with a lower ability to interact with FcγR than the natural IgG constant regions. Radiolabelled RBCs and platelets sensitised with specific G1Δnab Abs were cleared more slowly from human circulation than IgG1-sensitised counterparts. However, non-destructive splenic retention of G1Δnab-coated RBCs required investigation and plasma radioactivities now suggest this also occurred for platelets sensitised with an IgG1/G1Δnab mixture. In vitro assays with human cells showed that G1Δnab-sensitised RBCs did not cause FcγRI-mediated monocyte activation, FcγRIIIa-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) or macrophage phagocytosis although they did adhere to macrophages. Thus, FcγRII was implicated in the adhesion despite the Δnab mutation reducing the already low-affinity binding to this receptor class. Additional contacts via P-selectin enhance the interaction of sensitised platelets with monocytes and this system provided evidence of FcγRII-dependent activation by G1Δnab. These results emphasise the physiological relevance of low-affinity interactions: It appears that FcγRII interactions of G1Δnab allowed splenic retention of G1Δnab-coated RBCs with inhibitory FcγRIIb binding preventing RBC destruction and that FcγRIIb engagement by G1Δnab on IgG1/G1Δnab-sensitised platelets overcame activation by IgG1. Considering therapeutic blocking Abs, G1Δnab offers lower FcγR binding and a greater bias towards inhibition than IgG2 and IgG4 constant regions. PMID:24285214

Binding Interactions of Dopamine and Apomorphine in D2High and D2Low States of Human Dopamine D2 Receptor Using Computational and Experimental Techniques.

PubMed

Durdagi, Serdar; Salmas, Ramin Ekhteiari; Stein, Matthias; Yurtsever, Mine; Seeman, Philip

2016-02-17

We have recently reported G-protein coupled receptor (GPCR) model structures for the active and inactive states of the human dopamine D2 receptor (D2R) using adrenergic crystal structures as templates. Since the therapeutic concentrations of dopamine agonists that suppress the release of prolactin are the same as those that act at the high-affinity state of the D2 receptor (D2High), D2High in the anterior pituitary gland is considered to be the functional state of the receptor. In addition, the therapeutic concentrations of anti-Parkinson drugs are also related to the dissociation constants in the D2High form of the receptor. The discrimination between the high- and low-affinity (D2Low) components of the D2R is not obvious and requires advanced computer-assisted structural biology investigations. Therefore, in this work, the derived D2High and D2Low receptor models (GPCR monomer and dimer three-dimensional structures) are used as drug-binding targets to investigate binding interactions of dopamine and apomorphine. The study reveals a match between the experimental dissociation constants of dopamine and apomorphine at their high- and low-affinity sites of the D2 receptor in monomer and dimer and their calculated dissociation constants. The allosteric receptor-receptor interaction for dopamine D2R dimer is associated with the accessibility of adjacent residues of transmembrane region 4. The measured negative cooperativity between agonist ligand at dopamine D2 receptor is also correctly predicted using the D2R homodimerization model.

Probing the electrostatics and pharmacologic modulation of sequence-specific binding by the DNA-binding domain of the ETS-family transcription factor PU.1: a binding affinity and kinetics investigation

PubMed Central

Munde, Manoj; Poon, Gregory M. K.; Wilson, W. David

2013-01-01

Members of the ETS family of transcription factors regulate a functionally diverse array of genes. All ETS proteins share a structurally-conserved but sequence-divergent DNA-binding domain, known as the ETS domain. Although the structure and thermodynamics of the ETS-DNA complexes are well known, little is known about the kinetics of sequence recognition, a facet that offers potential insight into its molecular mechanism. We have characterized DNA binding by the ETS domain of PU.1 by biosensor-surface plasmon resonance (SPR). SPR analysis revealed a striking kinetic profile for DNA binding by the PU.1 ETS domain. At low salt concentrations, it binds high-affinity cognate DNA with a very slow association rate constant (≤105 M−1 s−1), compensated by a correspondingly small dissociation rate constant. The kinetics are strongly salt-dependent but mutually balance to produce a relatively weak dependence in the equilibrium constant. This profile contrasts sharply with reported data for other ETS domains (e.g., Ets-1, TEL) for which high-affinity binding is driven by rapid association (>107 M−1 s−1). We interpret this difference in terms of the hydration properties of ETS-DNA binding and propose that at least two mechanisms of sequence recognition are employed by this family of DNA-binding domain. Additionally, we use SPR to demonstrate the potential for pharmacological inhibition of sequence-specific ETS-DNA binding, using the minor groove-binding distamycin as a model compound. Our work establishes SPR as a valuable technique for extending our understanding of the molecular mechanisms of ETS-DNA interactions as well as developing potential small-molecule agents for biotechnological and therapeutic purposes. PMID:23416556

Electrostatic repulsion, compensatory mutations, and long-range non-additive effects at the dimerization interface of the HIV capsid protein.

PubMed

del Alamo, Marta; Mateu, Mauricio G

2005-01-28

In previous studies, thermodynamic dissection of the dimerization interface in CA-C, the C-terminal domain of the capsid protein of human immunodeficiency virus type 1, revealed that individual mutation to alanine of Ser178, Glu180, Glu187 or Gln192 led to significant increases in dimerization affinity. Four related aspects derived from this observation have been now addressed, and the results can be summarized as follows: (i) thermodynamic analyses indicate the presence of an intersubunit electrostatic repulsion between both Glu180 residues. (ii) The mutation Glu180 to Ala was detected in nearly all type 2 human immunodeficiency virus variants, and in several simian immunodeficiency viruses analyzed. However, this mutation was strictly co-variant with mutations Ser178Asp in a neighboring residue, and Glu187Gln. Thermodynamic analysis of multiple mutants showed that Ser178Asp compensated, alone or together with Glu187Gln, the increase in affinity caused by the mutation Glu180Ala, and restored a lower dimerization affinity. (iii) The increase in the affinity constant caused by the multiple mutation to Ala of Ser178, Glu180, Glu187 and Gln192 was more than one order of magnitude lower than predicted if additivity were present, despite the fact that the 178/180 pair and the two other residues were located more than 10A apart. (iv) Mutations in CA-C that caused non-additive increases in dimerization affinity also caused a non-additive increase in the capacity of the isolated CA-C domain to inhibit the assembly of capsid-like HIV-1 particles in kinetic assays. In summary, the study of a protein-protein interface involved in the building of a viral capsid has revealed unusual features, including intersubunit electrostatic repulsions, co-variant, compensatory mutations that may evolutionarily preserve a low association constant, and long-range, large magnitude non-additive effects on association.

Review: correlations between oxygen affinity and sequence classifications of plant hemoglobins.

PubMed

Smagghe, Benoit J; Hoy, Julie A; Percifield, Ryan; Kundu, Suman; Hargrove, Mark S; Sarath, Gautam; Hilbert, Jean-Louis; Watts, Richard A; Dennis, Elizabeth S; Peacock, W James; Dewilde, Sylvia; Moens, Luc; Blouin, George C; Olson, John S; Appleby, Cyril A

2009-12-01

Plants express three phylogenetic classes of hemoglobins (Hb) based on sequence analyses. Class 1 and 2 Hbs are full-length globins with the classical eight helix Mb-like fold, whereas Class 3 plant Hbs resemble the truncated globins found in bacteria. With the exception of the specialized leghemoglobins, the physiological functions of these plant hemoglobins remain unknown. We have reviewed and, in some cases, measured new oxygen binding properties of a large number of Class 1 and 2 plant nonsymbiotic Hbs and leghemoglobins. We found that sequence classification correlates with distinct extents of hexacoordination with the distal histidine and markedly different overall oxygen affinities and association and dissociation rate constants. These results suggest strong selective pressure for the evolution of distinct physiological functions. The leghemoglobins evolved from the Class 2 globins and show no hexacoordination, very high rates of O(2) binding ( approximately 250 muM(-1) s(-1)), moderately high rates of O(2) dissociation ( approximately 5-15 s(-1)), and high oxygen affinity (K(d) or P(50) approximately 50 nM). These properties both facilitate O(2) diffusion to respiring N(2) fixing bacteria and reduce O(2) tension in the root nodules of legumes. The Class 1 plant Hbs show weak hexacoordination (K(HisE7) approximately 2), moderate rates of O(2) binding ( approximately 25 muM(-1) s(-1)), very small rates of O(2) dissociation ( approximately 0.16 s(-1)), and remarkably high O(2) affinities (P(50) approximately 2 nM), suggesting a function involving O(2) and nitric oxide (NO) scavenging. The Class 2 Hbs exhibit strong hexacoordination (K(HisE7) approximately 100), low rates of O(2) binding ( approximately 1 muM(-1) s(-1)), moderately low O(2) dissociation rate constants ( approximately 1 s(-1)), and moderate, Mb-like O(2) affinities (P(50) approximately 340 nM), perhaps suggesting a sensing role for sustained low, micromolar levels of oxygen.

Computational modeling approaches to quantitative structure-binding kinetics relationships in drug discovery.

PubMed

De Benedetti, Pier G; Fanelli, Francesca

2018-03-21

Simple comparative correlation analyses and quantitative structure-kinetics relationship (QSKR) models highlight the interplay of kinetic rates and binding affinity as an essential feature in drug design and discovery. The choice of the molecular series, and their structural variations, used in QSKR modeling is fundamental to understanding the mechanistic implications of ligand and/or drug-target binding and/or unbinding processes. Here, we discuss the implications of linear correlations between kinetic rates and binding affinity constants and the relevance of the computational approaches to QSKR modeling. Copyright © 2018 Elsevier Ltd. All rights reserved.

FORMAL UNCERTAINTY ANALYSIS OF A LAGRANGIAN PHOTOCHEMICAL AIR POLLUTION MODEL. (R824792)

EPA Science Inventory

This study applied Monte Carlo analysis with Latin
hypercube sampling to evaluate the effects of uncertainty
in air parcel trajectory paths, emissions, rate constants,
deposition affinities, mixing heights, and atmospheric stability
on predictions from a vertically...

Affinity interactions between natural pigments and human whole saliva.

PubMed

Yao, Jiang-Wu; Lin, Feng; Tao, Tao; Lin, Chang-Jian

2011-03-01

The aim of the present study was to assess the null hypothesis that there are no differences of affinity between pigments and human whole saliva (WS), and the affinity is not influenced by the functional groups of pigments, temperatures, pH values, and salt concentrations. The affinity constants of interactions between WS and theaflavin (TF)/curcumin (Cur)/cyanidin (Cy) were determined by surface plasmon resonance (SPR) and fluorescence quenching. Mass-uptake at various temperatures, pH values, and salt concentrations was also carried out. The order of affinity of the pigments binding to WS is TF>Cur>Cy. A large number of complexes and precipitations of pigments/proteins were formed through a quick, strong, and almost irreversible binding process. The mass-uptake of pigments was affected not only by the functional groups, but also by molecular weight of pigments, temperatures, pH values, and salt concentrations. The complex of pigments may easily and rapidly deposit onto the WS film, and are difficult to remove from the WS surface. However, the complex of pigments can be reduced by properly regulating the physicochemical conditions, such as temperatures, pH values, and salt concentrations. Copyright © 2010 Elsevier Ltd. All rights reserved.

Solitons and the energy-momentum tensor for affine Toda theory

NASA Astrophysics Data System (ADS)

Olive, D. I.; Turok, N.; Underwood, J. W. R.

1993-07-01

Following Leznov and Saveliev, we present the general solution to Toda field theories of conformal, affine or conformal affine type, associated with a simple Lie algebra g. These depend on a free massless field and on a group element. By putting the former to zero, soliton solutions to the affine Toda theories with imaginary coupling constant result with the soliton data encoded in the group element. As this requires a reformulation of the affine Kac-Moody algebra closely related to that already used to formulate the physical properties of the particle excitations, including their scattering matrices, a unified treatment of particles and solitons emerges. The physical energy—momentum tensor for a general solution is broken into a total derivative plus a part dependent only on the derivatives of the free field. Despite the non-linearity of the field equations and their complex nature the energy and momentum of the N-soliton solution is shown to be real, equalling the sum of contributions from the individual solitons. There are rank-g species of soliton, with masses given by a generalisation of a formula due to Hollowood, being proportional to the components of the left Perron-Frobenius eigenvector of the Cartan matrix of g.

Food sensing: selection and characterization of DNA aptamers to Alicyclobacillus spores for trapping and detection from orange juice.

PubMed

Hünniger, Tim; Fischer, Christin; Wessels, Hauke; Hoffmann, Antonia; Paschke-Kratzin, Angelika; Haase, Ilka; Fischer, Markus

2015-03-04

The quality of the beverage industry's products has to be constantly monitored to fulfill consumers' high expectations. The thermo-acidophilic Gram-positive Alicyclobacillus spp. are not pathogenic, but their heat-resistant endospores can survive juice-processing conditions and have become a major economic concern for the fruit juice industry. Current detection methods rely on cultivation, isolation, and organism identification, which can take up to a week, resulting in economic loss. This work presents the selection and identification of DNA aptamers targeting Alicyclobacillus spores by spore-SELEX (systematic evolution of ligands by exponential enrichment) in orange-juice-simulating buffer. The selection process was verified by various techniques, including flow cytometric binding assays, radioactive binding assays, and agarose gel electrophoresis. The subsequent aptamer characterization included the determination of dissociations constants and selectivity by different techniques, such as surface plasmon resonance spectroscopy and fluorescence microscopy. In summary, 10 different aptamers with an affinity to Alicyclobacillus spp. have been developed, analyzed, and characterized in terms of affinity and specificity.

Investigation of complexes tannic acid and myricetin with Fe(III)

NASA Astrophysics Data System (ADS)

Sungur, Şana; Uzar, Atike

2008-01-01

The pH dependence of the complexes was determined by both potentiometric and spectrophotometric studies. Stability constants and stoichiometries of the formed complexes were determined using slope ratio method. Fe(III) was formed complexes with tannic acid of various stoichiometries, which in the 1:1 molar ratio at pH < 3, in the 2:1 molar ratio at pH 3-7 and in the 4:1 molar ratio at pH > 7. Fe(III) was formed complexes with myricetin in the 1:2 molar ratio at pH 4 and 5 and in the 1:1 molar ratio at pH 6. Stability constant values were found to be 10 5 to 10 17 and 10 5 to 10 9 for Fe(III)-tannic acid complexes and Fe(III)-myricetin complexes. Both tannic acid and myricetin were possessed minimum affinities to Cu(II) and Zn(II). They had less affinity for Al(III) than for Fe(III).

Compound Selectivity and Target Residence Time of Kinase Inhibitors Studied with Surface Plasmon Resonance.

PubMed

Willemsen-Seegers, Nicole; Uitdehaag, Joost C M; Prinsen, Martine B W; de Vetter, Judith R F; de Man, Jos; Sawa, Masaaki; Kawase, Yusuke; Buijsman, Rogier C; Zaman, Guido J R

2017-02-17

Target residence time (τ) has been suggested to be a better predictor of the biological activity of kinase inhibitors than inhibitory potency (IC 50 ) in enzyme assays. Surface plasmon resonance binding assays for 46 human protein and lipid kinases were developed. The association and dissociation constants of 80 kinase inhibitor interactions were determined. τ and equilibrium affinity constants (K D ) were calculated to determine kinetic selectivity. Comparison of τ and K D or IC 50 values revealed a strikingly different view on the selectivity of several kinase inhibitors, including the multi-kinase inhibitor ponatinib, which was tested on 10 different kinases. In addition, known pan-Aurora inhibitors resided much longer on Aurora B than on Aurora A, despite having comparable affinity for Aurora A and B. Furthermore, the γ/δ-selective PI3K inhibitor duvelisib and the δ-selective drug idelalisib had similar 20-fold selectivity for δ- over γ-isoform but duvelisib resided much longer on both targets. Copyright © 2016 Elsevier Ltd. All rights reserved.

Determination of the parameters of binding between lipopolysaccharide and chitosan and its N-acetylated derivative using a gravimetric piezoquartz biosensor.

PubMed

Naberezhnykh, G A; Gorbach, V I; Kalmykova, E N; Solov'eva, T F

2015-03-01

The interaction of endotoxin (lipopolysaccharide - LPS) with low molecular weight chitosan (5.5 kDa), its N-acylated derivative and chitoliposomes was studied using a gravimetric piezoelectric quartz crystal microbalance biosensor. The optimal conditions for the formation of a biolayer based on immobilized LPS on the resonator surface and its regeneration were elaborated. The association and dissociation rate constants for LPS binding to chitosans were determined and the affinity constants (Kaf) were calculated based on the data on changes in the oscillation frequency of the quartz crystal resonator. The Kaf values correlated with the ones obtained using other methods. The affinity of N-acylated chitosan binding to LPS was higher than that of the parent chitosan binding to LPS. Based on the results obtained, we suggest that water-soluble N-acylated derivatives of chitosan with low degree of substitution of amino groups could be useful compounds for endotoxin binding and neutralization. Copyright © 2015 Elsevier B.V. All rights reserved.

DLVO Approximation Methods for Predicting the Attachment of Silver Nanoparticles to Ceramic Membranes.

PubMed

Mikelonis, Anne M; Youn, Sungmin; Lawler, Desmond F

2016-02-23

This article examines the influence of three common stabilizing agents (citrate, poly(vinylpyrrolidone) (PVP), and branched poly(ethylenimine) (BPEI)) on the attachment affinity of silver nanoparticles to ceramic water filters. Citrate-stabilized silver nanoparticles were found to have the highest attachment affinity (under conditions in which the surface potential was of opposite sign to the filter). This work demonstrates that the interaction between the electrical double layers plays a critical role in the attachment of nanoparticles to flat surfaces and, in particular, that predictions of double-layer interactions are sensitive to boundary condition assumptions (constant charge vs constant potential). The experimental deposition results can be explained when using different boundary condition assumptions for different stabilizing molecules but not when the same assumption was assumed for all three types of particles. The integration of steric interactions can also explain the experimental deposition results. Particle size was demonstrated to have an effect on the predicted deposition for BPEI-stabilized particles but not for PVP.

Real-world emission factors of fine and ultrafine aerosol particles for different traffic situations in Switzerland.

PubMed

Imhof, David; Weingartner, Ernest; Ordónez, Carlos; Gehrig, Robert; Hill, Matz; Buchmann, Brigitte; Baltensperger, Urs

2005-11-01

Extended field measurements of particle number (size distribution of particle diameters, D, in the range between 18 nm and 10 microm), surface area concentrations, and PM1 and PM10 mass concentrations were performed in Switzerland to determine traffic emissions using a comprehensive set of instruments. Measurements took place at roads with representative traffic regimes: at the kerbside of a motorway (120 km h(-1)), a highway (80-100 km h(-1)), and in an urban area with stop-and-go traffic (0-50 km h(-1)) regulated by light signals. Mean diurnal variations showed that the highest pollutant concentrations were during the morning rush hours, especially of the number density in the nanoparticle size range (D <50 nm). From the differences between up- and downwind concentrations (or differences between kerbside and background concentrations for the urban site), "real-life" emission factors were derived using NOx concentrations to calculate dilution factors. Particle number and volume emission factors of different size ranges (18-50 nm, 18-100 nm, and 18-300 nm) were derived for the total vehicle fleet and separated into a light-duty (LDV) and a heavy-duty vehicle (HDV) contribution. The total particle number emissions per vehicle were found to be about 11.7-13.5 x 10(14) particles km(-1) for constant speed (80-120 km h(-1) and 3.9 x 10(14) particles km(-1) for urban driving conditions. LDVs showed higher emission factors at constant high speed than under urban disturbed traffic flow. In contrast, HDVs emitted more air pollutants during deceleration and acceleration processes in stop-and-go traffic than with constant speed of about 80 km h(-1). On average, one HDV emits a 10-30 times higher amount of particulate air pollutants (in terms of both number and volume) than one LDV.

Utilization of satellite-satellite tracking data for determination of the geocentric gravitational constant (GM)

NASA Technical Reports Server (NTRS)

Martin, C. F.; Oh, I. H.

1979-01-01

Range rate tracking of GEOS 3 through the ATS 6 satellite was used, along with ground tracking of GEOS 3, to estimate the geocentric gravitational constant (GM). Using multiple half day arcs, a GM of 398600.52 + or - 0.12 cu km/sq sec was estimated using the GEM 10 gravity model, based on speed of light of 299792.458 km/sec. Tracking station coordinates were simultaneously adjusted, leaving geopotential model error as the dominant error source. Baselines between the adjusted NASA laser sites show better than 15 cm agreement with multiple short arc GEOS 3 solutions.

Comment on: Negative ions, molecular electron affinity and orbital structure of cata-condensed polycyclic aromatic hydrocarbons by Rustem V. Khatymov, Mars V. Muftakhov and Pavel V. Shchukin.

PubMed

Chen, Edward S; Chen, Edward C M

2018-02-15

The anion mass spectral lifetimes for several aromatic hydrocarbons reported in the subject article were related to significantly different electron affinities. The different values are rationalized using negative ion mass spectral data. Electron affinities for polycyclic aromatic hydrocarbons are reported from the temperature dependence of unpublished electron capture detector data. These are compared with published values and the largest values are assigned to the ground state. The ground state adiabatic electron affinities: (eV) pentacene, 1.41 (3); tetracene, 1.058 (5); benz(a)pyrene, 0.82 (4); benz(a) anthracene, 0.69 (2) anthracene, 0.68 (2); and pyrene, 0.59 (1) are used to assign excited state adiabatic electron affinities: (eV) tetracene: 0.88 (4); anthracene 0.53 (1); pyrene, 0.41 (1); benz(a)anthracene, 0.39 (10); chrysene, 0.32 (1); and phenanthrene, 0.12 (2) and ground state adiabatic electron affinities: (eV) dibenz(a,j)anthracene, 0.69 (3); dibenz(a,h)anthracene, 0.68 (3); benz(e)pyrene, 0.60 (3); and picene, 0.59 (3) from experimental data. The lifetime of benz(a)pyrene is predicted to be larger than 150 μs and for benzo(c)phenanthrene and picene about 40 μs, from ground state adiabatic electron affinities. The assignments of adiabatic electron affinities of aromatic hydrocarbons determined from electron capture detector and mass spectrometric data to ground and excited states are supported by constant electronegativities. A set of consistent ground state adiabatic electron affinities for 15 polycyclic aromatic hydrocarbons is related to lifetimes from the subject article. Copyright © 2017 John Wiley & Sons, Ltd.

DNA aptamers for the detection of Haemophilus influenzae type b by cell SELEX.

PubMed

Bitaraf, F S; Rasooli, I; Mousavi Gargari, S L

2016-03-01

Haemophilus influenzae type b (Hib) causes acute bacterial meningitis (ABM) in children, with a mortality rate of about 3-6 % of the affected patients. ABM can lead to death during a period of hours to several days and, hence, rapid and early detection of the infection is crucial. Aptamers, the short single-stranded DNA or RNA with high affinity to target molecules, are selected by a high-flux screening technique known as in vitro screening and systematic evolution of ligands by exponential enrichment technology (SELEX). In this study, whole-cell SELEX was applied for the selection of target-specific aptamers with high affinity to Hib. ssDNA aptamers prepared by lambda exonuclease were incubated with the target cells (Hib). The aptameric binding rate to Hib was characterized for binding affinity after seven SELEX rounds by flow cytometry. The aptamers with higher binding affinity were cloned. Four of 68 aptamer clones were selected for sequencing. The dissociation constant (Kd) of the high-affinity aptamer clones 45 and 63 were 47.10 and 28.46 pM, respectively. These aptamers did not bind to other bacterial species, including the seven meningitis-causing bacteria. They showed distinct affinity to various H. influenzae strains only. These aptamers showed the highest affinity to Hib and the lowest affinity to H. influenzae type c and to other meningitis-causing bacteria. Clone 63 could detect Hib in patients' cerebrospinal fluid (CSF) samples at 60 colony-forming units (CFU)/mL. The results indicate applicability of the aptamers for rapid and early detection of infections brought about by Hib.

H0, q0 and the local velocity field. [Hubble and deceleration constants in Big Bang expansion

NASA Technical Reports Server (NTRS)

Sandage, A.; Tammann, G. A.

1982-01-01

An attempt is made to find a systematic deviation from linearity for distances that are under the control of the Virgo cluster, and to determine the value of the mean random motion about the systematic flow, in order to improve the measurement of the Hubble and the deceleration constants. The velocity-distance relation for large and intermediate distances is studied, and type I supernovae are calibrated relatively as distance indicators and absolutely to obtain a new value for the Hubble constant. Methods of determining the deceleration constant are assessed, including determination from direct measurement, mean luminosity density, virgocentric motion, and the time scale test. The very local velocity field is investigated, and a solution is preferred with a random peculiar radial velocity of very nearby field galaxies of 90-100 km/s, and a Virgocentric motion of the local group of 220 km/s, leading to an underlying expansion rate of 55, in satisfactory agreement with the global value.

The mechanics of running in children

PubMed Central

Schepens, B; Willems, P A; Cavagna, G A

1998-01-01

The effect of age and body size on the bouncing mechanism of running was studied in children aged 2-16 years.The natural frequency of the bouncing system (fs) and the external work required to move the centre of mass of the body were measured using a force platform.At all ages, during running below ≈11 km h−1, the freely chosen step frequency (f) is about equal to fs (symmetric rebound), independent of speed, although it decreases with age from 4 Hz at 2 years to 2.5 Hz above 12 years.The decrease of step frequency with age is associated with a decrease in the mass-specific vertical stiffness of the bouncing system (k/m) due to an increase of the body mass (m) with a constant stiffness (k). Above 12 years, k/m and f remain approximately constant due to a parallel increase in both k and m with age.Above the critical speed of ≈11 km h−1, independent of age, the rebound becomes asymmetric, i.e. f < fs.The maximum running speed (V¯f,max) increases with age while the step frequency at remains constant (≈4 Hz), independent of age.At a given speed, the higher step frequency in preteens results in a mass-specific power against gravity less than that in adults. The external power required to move the centre of mass of the body is correspondingly reduced. PMID:9596810

KINETIC STUDY ON THE INHIBITION OF HEN BRAIN NEUROTOXIC ESTERASE BY MIPAFOX

EPA Science Inventory

A direct method of assaying neurotoxic esterase (NTE) activity, using 4-nitrophenyl valerate, has been described. The technique was used to determine the biomolecular rate (ki), phosphorylation (k2), and affinity (kd) constants for the reaction of hen brain microsomal NTE with mi...

Velocity dispersions in galaxies: 1: The SO galaxy NGC 7332

NASA Technical Reports Server (NTRS)

Morton, D. C.; Chevalier, R. A.

1971-01-01

A Coude spectrum of the SO galaxy NGC 7332 with 0.9 A resolution from 4186 to 4364 A was obtained with the SEC vidicon television camera and the Hale telescope. Comparisons with spectra of G and K giant stars, numerically broadened for various Maxwellian velocity distributions, give a dispersion velocity in the line of sight of 160 + or - 20 km/sec with the best fit at G8III. The dispersion appears to be constant within + or - 35 km/sec out to 1.4 kpc (H = 100 km/sec/mpc). After correction for projection, the rotation curve has a slope of 0.16 km/sec/pc at the center and a velocity of 130 km/sec at 1.4 kpc where it is still increasing. For an estimated effective radius of 3.5 kpc enclosing half the light, the virial theorem gives a mass of 1.4 x 10 to the 11th power solar masses if the mass-to-light ratio is constant throughout the galaxy. The photographic luminosity is 8.3 x 10 to the 9th power solar luminosities so that the M/L ratio is 17.

Intrinsic Thermodynamics and Structures of 2,4- and 3,4-Substituted Fluorinated Benzenesulfonamides Binding to Carbonic Anhydrases.

PubMed

Zubrienė, Asta; Smirnov, Alexey; Dudutienė, Virginija; Timm, David D; Matulienė, Jurgita; Michailovienė, Vilma; Zakšauskas, Audrius; Manakova, Elena; Gražulis, Saulius; Matulis, Daumantas

2017-01-20

The goal of rational drug design is to understand structure-thermodynamics correlations in order to predict the chemical structure of a drug that would exhibit excellent affinity and selectivity for a target protein. In this study we explored the contribution of added functionalities of benzenesulfonamide inhibitors to the intrinsic binding affinity, enthalpy, and entropy for recombinant human carbonic anhydrases (CA) CA I, CA II, CA VII, CA IX, CA XII, and CA XIII. The binding enthalpies of compounds possessing similar chemical structures and affinities were found to be very different, spanning a range from -90 to +10 kJ mol -1 , and are compensated by a similar opposing entropy contribution. The intrinsic parameters of binding were determined by subtracting the linked protonation reactions. The sulfonamide group pK a values of the compounds were measured spectrophotometrically, and the protonation enthalpies were measured by isothermal titration calorimetry (ITC). Herein we describe the development of meta- or ortho-substituted fluorinated benzenesulfonamides toward the highly potent compound 10 h, which exhibits an observed dissociation constant value of 43 pm and an intrinsic dissociation constant value of 1.1 pm toward CA IX, an anticancer target that is highly overexpressed in various tumors. Fluorescence thermal shift assays, ITC, and X-ray crystallography were all applied in this work. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effect of Cu(II), Cd(II) and Zn(II) on Pb(II) biosorption by algae Gelidium-derived materials.

PubMed

Vilar, Vítor J P; Botelho, Cidália M S; Boaventura, Rui A R

2008-06-15

Biosorption of Pb(II), Cu(II), Cd(II) and Zn(II) from binary metal solutions onto the algae Gelidium sesquipedale, an algal industrial waste and a waste-based composite material was investigated at pH 5.3, in a batch system. Binary Pb(II)/Cu(II), Pb(II)/Cd(II) and Pb(II)/Zn(II) solutions have been tested. For the same equilibrium concentrations of both metal ions (1 mmol l(-1)), approximately 66, 85 and 86% of the total uptake capacity of the biosorbents is taken by lead ions in the systems Pb(II)/Cu(II), Pb(II)/Cd(II) and Pb(II)/Zn(II), respectively. Two-metal results were fitted to a discrete and a continuous model, showing the inhibition of the primary metal biosorption by the co-cation. The model parameters suggest that Cd(II) and Zn(II) have the same decreasing effect on the Pb(II) uptake capacity. The uptake of Pb(II) was highly sensitive to the presence of Cu(II). From the discrete model it was possible to obtain the Langmuir affinity constant for Pb(II) biosorption. The presence of the co-cations decreases the apparent affinity of Pb(II). The experimental results were successfully fitted by the continuous model, at different pH values, for each biosorbent. The following sequence for the equilibrium affinity constants was found: Pb>Cu>Cd approximately Zn.

CdTe/CdSe quantum dots improve the binding affinities between α-amylase and polyphenols.

PubMed

Ni, Xiaoling

2012-03-01

People exposed to engineered nanomaterials have potential health risks associated. Human α-amylase is one of the key enzymes in the digestive system. There are few reports about the influence of quantum dots (QDs) on the digestive enzymes and their inhibition system. This work focused on the toxic effect of CdTe/CdSe QDs on the interactions between α-amylase and its natural inhibitors. Thirty-six dietary polyphenols, natural α-amylase inhibitors from food, were studied for their affinities for α-amylase in the absence and presence of CdTe/CdSe QDs by a fluorescence quenching method. The magnitudes of apparent binding constants of polyphenols for α-amylase were almost in the range of 10(5)-10(7) L mol(-1) in the presence of CdTe/CdSe QDs, which were higher than the magnitudes of apparent binding constants in the absence of CdTe/CdSe QDs (10(4)-10(6) L mol(-1)). CdTe/CdSe QDs obviously improved the affinities of dietary polyphenols for α-amylase up to 389.04 times. It is possible that the binding interaction between polyphenols and α-amylase in the presence of CdTe/CdSe QDs was mainly caused by electrostatic interactions. QDs significantly influence the digestive enzymes and their inhibition system. This journal is © The Royal Society of Chemistry 2012

Functional expression and characterization of the Trypanosoma brucei procyclic glucose transporter, THT2.

PubMed

Barrett, M P; Tetaud, E; Seyfang, A; Bringaud, F; Baltz, T

1995-12-15

The gene encoding THT2, one of two hexose-transporter isoforms present in Trypanosoma brucei, has been expressed in both Xenopus laevis oocytes and a stably transfected line of Chinese hamster ovary (CHO) cells. The heterologously expressed gene encodes a protein with pharmacological and kinetic parameters similar to those of the hexose transporter measured in procyclic-culture-form trypanosomes. The substrate recognition of the THT2 transporter differed from that of the THT1 isoform, which is expressed only in bloodstream forms, in that: (i) it has a relatively high affinity for substrate with a Km of 59 microM for 2-deoxy-D-glucose (2-DOG) and a similar high affinity for D-glucose (compared with Km of 0.5 mM for 2-DOG in bloodstream forms); (ii) the affinity for 6-deoxy-D-glucose (6-DOG) is two orders of magnitude lower than that for D-glucose, whereas the bloodstream-form transporter recognizes D-glucose and its 6-DOG analogue with similar affinity; (iii) the bloodstream-form transporter, but not THT2, recognizes 3-fluoro-3-deoxy-D-glucose. D-Fructose-transport capacity and insensitivity to D-galactose was also found in THT2-expressing CHO cells and procyclic trypanosomes. We conclude from these cumulative results that the THT2 gene encodes the transporter responsible for hexose transport in procyclic trypanosomes. The transport of 2-DOG in procyclic organisms was inhibited by both the protonophore, carbonyl cyanide 4-trifluoromethoxy phenylhydrazone (FCCP), and KCN, suggesting a requirement for a protonmotive force. However, sensitivity to these reagents depended on the external substrate concentration, with uptake being unaffected at substrate concentrations higher than 2 mM. THT2 expressed in CHO cells behaved as a facilitated transporter, and was unaffected by FCCP or KCN over the whole substrate concentration range tested.

Carrier-mediated γ-aminobutyric acid transport across the basolateral membrane of human intestinal Caco-2 cell monolayers.

PubMed

Nielsen, Carsten Uhd; Carstensen, Mette; Brodin, Birger

2012-06-01

The aim of the present study was to investigate the transport of γ-aminobutyric acid (GABA) across the basolateral membrane of intestinal cells. The proton-coupled amino acid transporter, hPAT1, mediates the influx of GABA and GABA mimetic drug substances such as vigabatrin and gaboxadol and the anticancer prodrug δ-aminolevulinic acid across the apical membrane of small intestinal enterocytes. Little is however known about the basolateral transport of these substances. We investigated basolateral transport of GABA in mature Caco-2 cell monolayers using isotope studies. Here we report that, at least two transporters seem to be involved in the basolateral transport of GABA. The basolateral uptake consisted of a high-affinity system with a K(m) of 290 μM and V(max) of 75 pmol cm(-2) min(-1) and a low affinity system with a K(m) of approximately 64 mM and V(max) of 1.6 nmol cm(-2) min(-1). The high-affinity transporter is Na(+) and Cl(-) dependent. The substrate specificity of the high-affinity transporter was further studied and Gly-Sar, Leucine, gaboxadol, sarcosine, lysine, betaine, 5-hydroxythryptophan, proline and glycine reduced the GABA uptake to approximately 44-70% of the GABA uptake in the absence of inhibitor. Other substances such as β-alanine, GABA, 5-aminovaleric acid, taurine and δ-aminolevulinic acid reduced the basolateral GABA uptake to 6-25% of the uptake in the absence of inhibitor. Our results indicate that the distance between the charged amino- and acid-groups is particular important for inhibition of basolateral GABA uptake. Thus, there seems to be a partial substrate overlap between the basolateral GABA transporter and hPAT1, which may prove important for understanding drug interactions at the level of intestinal transport. Copyright © 2012 Elsevier B.V. All rights reserved.

Role of an invariant lysine residue in folate binding on Escherichia coli thymidylate synthase: calorimetric and crystallographic analysis of the K48Q mutant

PubMed Central

Arvizu-Flores, Aldo A.; Sugich-Miranda, Rocio; Arreola, Rodrigo; Garcia-Orozco, Karina D.; Velazquez-Contreras, Enrique F.; Montfort, William R.; Maley, Frank; Sotelo-Mundo, Rogerio R.

2008-01-01

Thymidylate synthase (TS) catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) using methylene tetrahydrofolate (CH2THF) as cofactor, the glutamate tail of which forms a water-mediated hydrogen-bond with an invariant lysine residue of this enzyme. To understand the role of this interaction, we studied the K48Q mutant of Escherichia coli TS using structural and biophysical methods. The kcat of the K48Q mutant was 430 fold lower than wild-type TS in activity, while the the Km for the (R)-stereoisomer of CH2THF was 300 µM, about 30 fold larger than Km from the wild-type TS. Affinity constants were determined using isothermal titration calorimetry, which showed that binding was reduced by one order of magnitude for folate-like TS inhibitors, such as propargyl-dideaza folate (PDDF) or compounds that distort the TS active site like BW1843U89 (U89). The crystal structure of the K48Q-dUMP complex revealed that dUMP binding is not impaired in the mutamt, and that U89 in a ternary complex of K48Q-nucleotide-U89 was bound in the active site with subtle differences relative to comparable wild type complexes. PDDF failed to form ternary complexes with K48Q and dUMP. Thermodynamic data correlated with the structural determinations, since PDDF binding was dominated by enthalpic effects while U89 had an important entropic component. In conclusion, K48 is critical for catalysis since it leads to a productive CH2THF binding, while mutation at this residue does not affect much the binding of inhibitors that do not make contact with this group. PMID:18403248

Identification and Characterization of Small-Molecule Inhibitors of the R132H/R132H Mutant Isocitrate Dehydrogenase 1 Homodimer and R132H/Wild-Type Heterodimer.

PubMed

Brooks, Eric; Wu, Xiang; Hanel, Art; Nguyen, Shaun; Wang, Jing; Zhang, Jeffrey H; Harrison, Amanda; Zhang, Wentao

2014-09-01

Recurrent genetic mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) have been identified in multiple tumor types. The most frequent mutation, IDH1 R132H, is a gain-of-function mutation resulting in an enzyme-catalyzing conversion of α-ketoglutarate (α-KG) to 2-hydroxyglutarate (2-HG). A high-throughput assay quantifying consumption of NADPH by IDH1 R132H has been optimized and implemented to screen 3 million compounds in 1536-well formats. The primary high-throughput screening hits were further characterized by RapidFire-mass spectrometry measuring 2-HG directly. Multiple distinct chemotypes were identified with nanomolar potencies (6-300 nM). All inhibitors were found to be inactive against the wild-type IDH1 homodimers. An IDH1 heterodimer between wild-type and R132H mutant is capable of catalyzing conversion of α-KG to 2-HG and isocitrate to α-KG. Interestingly, one of the inhibitors, EXEL-9324, was found to inhibit both conversions by the IDH1 heterodimer. This indicates the R132H/WT heterodimer may adopt conformations distinct from that of the R132H/R132H homodimer. Further enzymatic studies support this conclusion as the heterodimer exhibited a significantly lower apparent Michaelis-Menten constant for α-KG (K(m)=110 µM) compared with the R132H homodimer (K(m)= 1200 µM). The enhanced apparent affinity for α-KG suggests R132H/WT heterodimeric IDH1 can produce 2-HG more efficiently at normal intracellular levels of α-KG (approximately 100 µM). © 2014 Society for Laboratory Automation and Screening.

Dopamine receptor contribution to the action of PCP, LSD and ketamine psychotomimetics.

PubMed

Seeman, P; Ko, F; Tallerico, T

2005-09-01

Although phencyclidine and ketamine are used to model a hypoglutamate theory of schizophrenia, their selectivity for NMDA receptors has been questioned. To determine the affinities of phencyclidine, ketamine, dizocilpine and LSD for the functional high-affinity state of the dopamine D2 receptor, D2High, their dissociation constants (Ki) were obtained on [3H]domperidone binding to human cloned dopamine D2 receptors. Phencyclidine had a high affinity for D2High with a Ki of 2.7 nM, in contrast to its low affinity for the NMDA receptor, with a Ki of 313 nM, as labeled by [3H]dizocilpine on rat striatal tissue. Ketamine also had a high affinity for D2High with a Ki of 55 nM, an affinity higher than its 3100 nM Ki for the NMDA sites. Dizocilpine had a Ki of 0.3 nM at D2High, but a Kd of 1.8 nM at the NMDA receptor. LSD had a Ki of 2 nM at D2High. Because the psychotomimetics had higher potency at D2High than at the NMDA site, the psychotomimetic action of these drugs must have a major contribution from D2 agonism. Because these drugs have a combined action on both dopamine receptors and NMDA receptors, these drugs, when given in vivo, test a combined hyperdopamine and hypoglutamate theory of psychosis.

Structural Basis of the High Affinity Interaction between the Alphavirus Nonstructural Protein-3 (nsP3) and the SH3 Domain of Amphiphysin-2.

PubMed

Tossavainen, Helena; Aitio, Olli; Hellman, Maarit; Saksela, Kalle; Permi, Perttu

2016-07-29

We show that a peptide from Chikungunya virus nsP3 protein spanning residues 1728-1744 binds the amphiphysin-2 (BIN1) Src homology-3 (SH3) domain with an unusually high affinity (Kd 24 nm). Our NMR solution complex structure together with isothermal titration calorimetry data on several related viral and cellular peptide ligands reveal that this exceptional affinity originates from interactions between multiple basic residues in the target peptide and the extensive negatively charged binding surface of amphiphysin-2 SH3. Remarkably, these arginines show no fixed conformation in the complex structure, indicating that a transient or fluctuating polyelectrostatic interaction accounts for this affinity. Thus, via optimization of such dynamic electrostatic forces, viral peptides have evolved a superior binding affinity for amphiphysin-2 SH3 compared with typical cellular ligands, such as dynamin, thereby enabling hijacking of amphiphysin-2 SH3-regulated host cell processes by these viruses. Moreover, our data show that the previously described consensus sequence PXRPXR for amphiphysin SH3 ligands is inaccurate and instead define it as an extended Class II binding motif PXXPXRpXR, where additional positive charges between the two constant arginine residues can give rise to extraordinary high SH3 binding affinity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Structure-affinity relationship of the interaction between phenolic acids and their derivatives and β-lactoglobulin and effect on antioxidant activity.

PubMed

Wu, Simin; Zhang, Yunyue; Ren, Fazheng; Qin, Yinghui; Liu, Jiaxin; Liu, Jingwen; Wang, Qingyu; Zhang, Hao

2018-04-15

In this study, 71 phenolic acids and their derivatives were used to investigate the structure-affinity relationship of β-lactoglobulin binding, and the effect of this interaction on antioxidant activity. Based on a fluorescence quenching method, an improved mathematical model was adopted to calculate the binding constants, with a correction for the inner-filter effect. Hydroxylation at the 3-position increased the affinity of the phenolic acids for β-lactoglobulin, while hydroxylation at the 2- or 4-positions had a negative effect. Complete methylation of all hydroxy groups, except at the 3-position, enhanced the binding affinity. Replacing the hydroxy groups with methyl groups at the 2-position also had a positive effect. Hydrogen bonding was one of the binding forces for the interaction. The antioxidant activity of phenolic acid-β-lactoglobulin complexes was higher than that of phenolic acids alone. These findings provide an understanding of the structure-activity relationship of the interaction between β-lactoglobulin and phenolic acids. Copyright © 2017 Elsevier Ltd. All rights reserved.

Identification of new ligands for the methionine biosynthesis transcriptional regulator (MetJ) by FAC-MS.

PubMed

Martí-Arbona, Ricardo; Teshima, Munehiro; Anderson, Penelope S; Nowak-Lovato, Kristy L; Hong-Geller, Elizabeth; Unkefer, Clifford J; Unkefer, Pat J

2012-01-01

We have developed a high-throughput approach using frontal affinity chromatography coupled to mass spectrometry (FAC-MS) for the identification and characterization of the small molecules that modulate transcriptional regulator (TR) binding to TR targets. We tested this approach using the methionine biosynthesis regulator (MetJ). We used effector mixtures containing S-adenosyl-L-methionine (SAM) and S-adenosyl derivatives as potential ligands for MetJ binding. The differences in the elution time of different compounds allowed us to rank the binding affinity of each compound. Consistent with previous results, FAC-MS showed that SAM binds to MetJ with the highest affinity. In addition, adenine and 5'-deoxy-5'-(methylthio)adenosine bind to the effector binding site on MetJ. Our experiments with MetJ demonstrate that FAC-MS is capable of screening complex mixtures of molecules and identifying high-affinity binders to TRs. In addition, FAC-MS experiments can be used to discriminate between specific and nonspecific binding of the effectors as well as to estimate the dissociation constant (K(d)) for effector-TR binding. Copyright © 2012 S. Karger AG, Basel.

Magneto-nanosensor platform for probing low-affinity protein–protein interactions and identification of a low-affinity PD-L1/PD-L2 interaction

PubMed Central

Lee, Jung-Rok; Bechstein, Daniel J. B.; Ooi, Chin Chun; Patel, Ashka; Gaster, Richard S.; Ng, Elaine; Gonzalez, Lino C.; Wang, Shan X.

2016-01-01

Substantial efforts have been made to understand the interactions between immune checkpoint receptors and their ligands targeted in immunotherapies against cancer. To carefully characterize the complete network of interactions involved and the binding affinities between their extracellular domains, an improved kinetic assay is needed to overcome limitations with surface plasmon resonance (SPR). Here, we present a magneto-nanosensor platform integrated with a microfluidic chip that allows measurement of dissociation constants in the micromolar-range. High-density conjugation of magnetic nanoparticles with prey proteins allows multivalent receptor interactions with sensor-immobilized bait proteins, more closely mimicking natural-receptor clustering on cells. The platform has advantages over traditional SPR in terms of insensitivity of signal responses to pH and salinity, less consumption of proteins and better sensitivities. Using this platform, we characterized the binding affinities of the PD-1—PD-L1/PD-L2 co-inhibitory receptor system, and discovered an unexpected interaction between the two known PD-1 ligands, PD-L1 and PD-L2. PMID:27447090

Arginine and Lysine Transporters Are Essential for Trypanosoma brucei.

PubMed

Mathieu, Christoph; Macêdo, Juan P; Hürlimann, Daniel; Wirdnam, Corina; Haindrich, Alexander C; Suter Grotemeyer, Marianne; González-Salgado, Amaia; Schmidt, Remo S; Inbar, Ehud; Mäser, Pascal; Bütikofer, Peter; Zilberstein, Dan; Rentsch, Doris

2017-01-01

For Trypanosoma brucei arginine and lysine are essential amino acids and therefore have to be imported from the host. Heterologous expression in Saccharomyces cerevisiae mutants identified cationic amino acid transporters among members of the T. brucei AAAP (amino acid/auxin permease) family. TbAAT5-3 showed high affinity arginine uptake (Km 3.6 ± 0.4 μM) and high selectivity for L-arginine. L-arginine transport was reduced by a 10-times excess of L-arginine, homo-arginine, canavanine or arginine-β-naphthylamide, while lysine was inhibitory only at 100-times excess, and histidine or ornithine did not reduce arginine uptake rates significantly. TbAAT16-1 is a high affinity (Km 4.3 ± 0.5 μM) and highly selective L-lysine transporter and of the compounds tested, only L-lysine and thialysine were competing for L-lysine uptake. TbAAT5-3 and TbAAT16-1 are expressed in both procyclic and bloodstream form T. brucei and cMyc-tagged proteins indicate localization at the plasma membrane. RNAi-mediated down-regulation of TbAAT5 and TbAAT16 in bloodstream form trypanosomes resulted in growth arrest, demonstrating that TbAAT5-mediated arginine and TbAAT16-mediated lysine transport are essential for T. brucei. Growth of induced RNAi lines could partially be rescued by supplementing a surplus of arginine or lysine, respectively, while addition of both amino acids was less efficient. Single and double RNAi lines indicate that additional low affinity uptake systems for arginine and lysine are present in T. brucei.

Arginine and Lysine Transporters Are Essential for Trypanosoma brucei

PubMed Central

Hürlimann, Daniel; Wirdnam, Corina; Haindrich, Alexander C.; Suter Grotemeyer, Marianne; González-Salgado, Amaia; Schmidt, Remo S.; Inbar, Ehud; Mäser, Pascal; Bütikofer, Peter; Zilberstein, Dan; Rentsch, Doris

2017-01-01

For Trypanosoma brucei arginine and lysine are essential amino acids and therefore have to be imported from the host. Heterologous expression in Saccharomyces cerevisiae mutants identified cationic amino acid transporters among members of the T. brucei AAAP (amino acid/auxin permease) family. TbAAT5-3 showed high affinity arginine uptake (Km 3.6 ± 0.4 μM) and high selectivity for L-arginine. L-arginine transport was reduced by a 10-times excess of L-arginine, homo-arginine, canavanine or arginine-β-naphthylamide, while lysine was inhibitory only at 100-times excess, and histidine or ornithine did not reduce arginine uptake rates significantly. TbAAT16-1 is a high affinity (Km 4.3 ± 0.5 μM) and highly selective L-lysine transporter and of the compounds tested, only L-lysine and thialysine were competing for L-lysine uptake. TbAAT5-3 and TbAAT16-1 are expressed in both procyclic and bloodstream form T. brucei and cMyc-tagged proteins indicate localization at the plasma membrane. RNAi-mediated down-regulation of TbAAT5 and TbAAT16 in bloodstream form trypanosomes resulted in growth arrest, demonstrating that TbAAT5-mediated arginine and TbAAT16-mediated lysine transport are essential for T. brucei. Growth of induced RNAi lines could partially be rescued by supplementing a surplus of arginine or lysine, respectively, while addition of both amino acids was less efficient. Single and double RNAi lines indicate that additional low affinity uptake systems for arginine and lysine are present in T. brucei. PMID:28045943

Interaction of fucoidan with proteases and inhibitors of coagulation and fibrinolysis.

PubMed

Minix, R; Doctor, V M

1997-09-01

The interactions of fucoidan with glutamic plasminogen (Glu-Plg), two-chain tissue plasminogen activator (t-PA), LMwt-urokinase, thrombin, and antithrombin III (AT-III) were investigated using fucoidan-sepharose affinity chromatography. The results showed 1) a high degree of affinity between fucoidan-sepharose and Glu-Plg; Lmwt-urokinase and thrombin while t-Pa and AT-III did not bind with fucoidan-sepharose. 2) The double reciprocal plot for the LMwt-urokinase activation of Glu-Plg showed that plasminogen activator inhibitor (PAI-1) inhibited this reaction in a noncompetitive manner and that the presence of fucoidan decreased Km for this interaction by 50% and increased Kcat by 30-fold, 3) The double reciprocal plot for the t-PA activation of Glu-Plg showed that PAI-1 inhibited this reaction in a competitive manner and that fucoidan in conjunction with 6-aminohexanoic acid (6-AH) increased Kcat for this interaction by 5-fold without affecting Km. 4) Fucoidan enhanced the interaction of thrombin with both AT-III and heparin cofactor II (HC-II) and it was more effective than unfractionated heparin of LMwt-heparin in enhancing the interaction of HC-II with thrombin.

Mineralogical, geochemical and isotopic characteristics of alkaline mafic igneous rocks from Punta delle Pietre Nere (Gargano, Southern Italy)

NASA Astrophysics Data System (ADS)

Mazzeo, F. C.; Arienzo, I.; Aulinas, M.; Casalini, M.; Di Renzo, V.; D'Antonio, M.

2018-05-01

The Punta delle Pietre Nere (Gargano, Southern Italy) igneous body is constituted by gabbroic and syenitic rocks with lamprophyric affinity of different age (58 and 62 Ma, respectively). The chemical composition of the minerals clearly indicates that there is no genetic relationship between the two lithotypes, in agreement with their significant age difference. The chemical (trace elements) and Sr-Nd-Pb-isotopic composition of these rocks highlights an "anorogenic" geochemical affinity derived from mixed DMM-HIMU-EM mantle reservoirs, similarly to other Paleogene-Oligocene magmatic provinces in the Circum-Mediterranean Area. In past literature, these features were interpreted as evidences for enriched asthenospheric mantle plume upwelling from deep regions beneath the Western Europe. Here we suggest that the HIMU-like composition of Punta delle Pietre Nere rocks is related to a lithospheric mantle source bearing amphibole-rich veins, resulting from crystallization of melts within the amphibole stability field in presence of H2O, as shown by several experimental works. Our results suggests partial melting at 70-90 km depth, which corresponds to the spinel-garnet transition (2.5-3.5 GPa) close to the amphibole stability limit ( 90-110 km and 2.5-3.5 GPa).

Population pharmacokinetics and penetration into prostatic, seminal, and vaginal fluid for ciprofloxacin, levofloxacin, and their combination.

PubMed

Bulitta, Jurgen B; Kinzig, Martina; Naber, Christoph K; Wagenlehner, Florian M E; Sauber, Christian; Landersdorfer, Cornelia B; Sörgel, Fritz; Naber, Kurt G

2011-01-01

Our objectives were to assess the pharmacokinetic interaction and body fluid penetration of ciprofloxacin and levofloxacin. This study was a single-dose open randomized three-way crossover in 15 healthy volunteers receiving 500 mg oral levofloxacin, 500 mg oral ciprofloxacin, or 250 mg levofloxacin and 250 mg ciprofloxacin co-administered. Serum, urine, and body fluid concentrations were determined by high-performance liquid chromatography and analyzed via population pharmacokinetic modeling. Modeling indicated that ciprofloxacin inhibited the renal reabsorption of levofloxacin. Ciprofloxacin increased the net renal clearance of levofloxacin by 13%, as its estimated affinity for a putative tubular reabsorption transporter was 12-fold higher (Km: 568 μM) compared to levofloxacin (Km: 6,830 μM). Levofloxacin increased the bioavailability of ciprofloxacin by 12% and achieved significantly (p < 0.05) higher concentrations at 3 h in ejaculate, prostatic, seminal, and vaginal fluid compared to ciprofloxacin. Modeling suggested that ciprofloxacin inhibited the tubular reabsorption of levofloxacin due to a 12-fold higher affinity for a putative tubular reabsorption transporter compared to levofloxacin. This pharmacokinetic interaction was not clinically relevant. Copyright © 2011 S. Karger AG, Basel.

Mechanism-based pharmacodynamic modeling of the interaction of midazolam, bretazenil, and zolpidem with ethanol.

PubMed

Tuk, Bert; van Gool, Toon; Danhof, Meindert

2002-06-01

The pharmacokinetic and pharmacodynamic interactions of ethanol with the full benzodiazepine agonist midazolam, the partial agonist bretazenil and the benzodiazepine BZ1 receptor subtype selective agonist zolpidem have been determined in the rat in vivo, using an integrated pharmacokinetic-pharmacodynamic approach. Ethanol was administered as a constant rate infusion resulting in constant plasma concentrations of 0.5 g/l. The pharmacokinetics and pharmacodynamics of midazolam, bretazenil, and zolpidem were determined following an intravenous infusion of 5.0, 2.5, and 18 mg/kg respectively. The amplitude in the 11.5-30 Hz frequency band of the EEG was used as measure of the pharmacological effect. For each of the benzodiazepines the concentration-EEG effect relationship could be described by the sigmoid Emax pharmacodynamic model. Significant differences in both EC50 and Emax were observed. The values of the EC50 were 76 +/- 11, 12 +/- 3, and 512 +/- 116 ng/ml for midazolam, bretazenil, and zolpidem respectively. The values of the Emax were 113 +/- 9, 44 +/- 3, and 175 +/- 10 microV/s. In the presence of ethanol the values of the EC50 of midazolam and zolpidem were reduced to approximately 50% of the original value. The values for Emax and Hill-factor were unchanged Due to a large interindividual variability no significant change in EC50 was observed for bretazenil. Analysis of the data on basis of a mechanism-based model showed only a decrease in the apparent affinity constant KPD for all three drugs, indicating that changes in EC50 can be explained entirely by a change in the apparent affinity constant KPD without concomitant changes in the efficacy parameter ePD and the stimulus-effect relationship. The findings of this study show that the pharmacodynamic interactions with a low dose of ethanol in vivo are qualitatively and quantitatively similar for benzodiazepine receptor full agonists, partial agonists, and benzodiazepine BZ1 receptor subtype selective agonists. This interaction can be explained entirely by a change in the affinity of the biological system for each benzodiazepine.

Straight as an arrow: humpback whales swim constant course tracks during long-distance migration

PubMed Central

Horton, Travis W.; Holdaway, Richard N.; Zerbini, Alexandre N.; Hauser, Nan; Garrigue, Claire; Andriolo, Artur; Clapham, Phillip J.

2011-01-01

Humpback whale seasonal migrations, spanning greater than 6500 km of open ocean, demonstrate remarkable navigational precision despite following spatially and temporally distinct migration routes. Satellite-monitored radio tag-derived humpback whale migration tracks in both the South Atlantic and South Pacific include constant course segments of greater than 200 km, each spanning several days of continuous movement. The whales studied here maintain these directed movements, often with better than 1° precision, despite the effects of variable sea-surface currents. Such remarkable directional precision is difficult to explain by established models of directional orientation, suggesting that alternative compass mechanisms should be explored. PMID:21508023

Straight as an arrow: humpback whales swim constant course tracks during long-distance migration.

PubMed

Horton, Travis W; Holdaway, Richard N; Zerbini, Alexandre N; Hauser, Nan; Garrigue, Claire; Andriolo, Artur; Clapham, Phillip J

2011-10-23

Humpback whale seasonal migrations, spanning greater than 6500 km of open ocean, demonstrate remarkable navigational precision despite following spatially and temporally distinct migration routes. Satellite-monitored radio tag-derived humpback whale migration tracks in both the South Atlantic and South Pacific include constant course segments of greater than 200 km, each spanning several days of continuous movement. The whales studied here maintain these directed movements, often with better than 1° precision, despite the effects of variable sea-surface currents. Such remarkable directional precision is difficult to explain by established models of directional orientation, suggesting that alternative compass mechanisms should be explored.

Relationship of nonreturn rates of dairy bulls to binding affinity of heparin to sperm

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marks, J.L.; Ax, R.L.

1985-08-01

The binding of the glycosaminoglycan (3H) heparin to bull spermatozoa was compared with nonreturn rates of dairy bulls. Semen samples from five bulls above and five below an average 71% nonreturn rate were used. Samples consisted of first and second ejaculates on a single day collected 1 d/wk for up to 5 consecutive wk. Saturation binding assays using (TH) heparin were performed to quantitate the binding characteristics of each sample. Scatchard plot analyses indicated a significant difference in the binding affinity for (TH) heparin between bulls of high and low fertility. Dissociation constants were 69.0 and 119.3 pmol for bullsmore » of high and low fertility, respectively. In contrast, the number of binding sites for (TH) heparin did not differ significantly among bulls. Differences in binding affinity of (TH) heparin to bull sperm might be used to predict relative fertility of dairy bulls.« less

Synthesis, characterization and binding affinities of rhenium(I) thiosemicarbazone complexes for the estrogen receptor (α/β).

PubMed

Núñez-Montenegro, Ara; Carballo, Rosa; Vázquez-López, Ezequiel M

2014-11-01

The binding affinities towards estrogen receptors (ERs) α and β of a set of thiosemicarbazone ligands (HL(n)) and their rhenium(I) carbonyl complexes [ReX(HL(n))(CO)3] (X=Cl, Br) were determined by a competitive standard radiometric assay with [(3)H]-estradiol. The ability of the coordinated thiosemicarbazone ligands to undergo deprotonation and the lability of the ReX bond were used as a synthetic strategy to obtain [Re(hpy)(L(n))(CO)3] (hpy=3- or 4-hydroxypyridine). The inclusion of the additional hpy ligand endows the new thiosemicarbazonate complexes with an improved affinity towards the estrogen receptors and, consequently, the values of the inhibition constant (Ki) could be determined for some of them. In general, the values of Ki for both ER subtypes suggest an appreciable selectivity towards ERα. Copyright © 2014 Elsevier Inc. All rights reserved.

Urban traffic pollution reduction for sedan cars using petrol engines by hydro-oxide gas inclusion.

PubMed

Al-Rousan, Ammar A; Alkheder, Sharaf; Musmar, Sa'ed A

2015-12-01

Petrol cars, in particular nonhybrid cars, contribute significantly to the pollution problem as compared with other types of cars. The originality of this article falls in the direction of using hydro-oxy gas to reduce pollution from petrol car engines. Experiments were performed in city areas at low real speeds, with constant engine speeds in the average of 2500 rpm and at variable velocity ratios (first speed was 10-20 km/hr, second speed was 20-35 km/hr, and third speed was 35-50 km/hr). Results indicated that through using hydro-oxy gas, a noticeable reduction in pollution was recorded. Oxygen (O2) percentage has increased by about 2.5%, and nitric oxide (NO) level has been reduced by about 500 ppm. Carbon monoxide (CO) has decreased by about 2.2%, and also CO2 has decreased by 2.1%. It's worth mentioning that for hybrid system in cars at speeds between 10 and 50 km/hr, the emission percentage change is zero. However, hybrid cars are less abundant than petrol cars. The originality of this paper falls in the direction of using hydro-oxy gas to reduce pollution from petrol car engines. Experiments were performed in city areas at low real speeds, with constant engine speeds in the average of 2500 rpm and at variable velocity ratios (first speed was 10-20 km/hr, second speed was 20-35 km/hr, and third speed was 35-50 km/h).

Catalytic properties of thermophilic lactate dehydrogenase and halophilic malate dehydrogenase at high temperature and low water activity.

PubMed

Hecht, K; Wrba, A; Jaenicke, R

1989-07-15

Thermophilic lactate dehydrogenases from Thermotoga maritima and Bacillus stearothermophilus are stable up to temperature limits close to the optimum growth temperature of their parent organisms. Their catalytic properties are anomalous in that Km shows a drastic increase with increasing temperature. At low temperatures, the effect levels off. Extreme halophilic malate dehydrogenase from Halobacterium marismortui exhibits a similar anomaly. Increasing salt concentration (NaCl) leads to an optimum curve for Km, oxaloacctate while Km, NADH remains constant. Previous claims that the activity of halophilic malate dehydrogenase shows a maximum at 1.25 M NaCl are caused by limiting substrate concentration; at substrate saturation, specific activity of halophilic malate dehydrogenase reaches a constant value at ionic strengths I greater than or equal to 1 M. Non-halophilic (mitochondrial) malate dehydrogenase shows Km characteristics similar to those observed for the halophilic enzyme. The drastic decrease in specific activity of the mitochondrial enzyme at elevated salt concentrations is caused by the salt-induced increase in rigidity of the enzyme, rather than gross structural changes.

The N-Terminal Domain of the Flo1 Flocculation Protein from Saccharomyces cerevisiae Binds Specifically to Mannose Carbohydrates ▿

PubMed Central

Goossens, Katty V. Y.; Stassen, Catherine; Stals, Ingeborg; Donohue, Dagmara S.; Devreese, Bart; De Greve, Henri; Willaert, Ronnie G.

2011-01-01

Saccharomyces cerevisiae cells possess a remarkable capacity to adhere to other yeast cells, which is called flocculation. Flocculation is defined as the phenomenon wherein yeast cells adhere in clumps and sediment rapidly from the medium in which they are suspended. These cell-cell interactions are mediated by a class of specific cell wall proteins, called flocculins, that stick out of the cell walls of flocculent cells. The N-terminal part of the three-domain protein is responsible for carbohydrate binding. We studied the N-terminal domain of the Flo1 protein (N-Flo1p), which is the most important flocculin responsible for flocculation of yeast cells. It was shown that this domain is both O and N glycosylated and is structurally composed mainly of β-sheets. The binding of N-Flo1p to d-mannose, α-methyl-d-mannoside, various dimannoses, and mannan confirmed that the N-terminal domain of Flo1p is indeed responsible for the sugar-binding activity of the protein. Moreover, fluorescence spectroscopy data suggest that N-Flo1p contains two mannose carbohydrate binding sites with different affinities. The carbohydrate dissociation constants show that the affinity of N-Flo1p for mono- and dimannoses is in the millimolar range for the binding site with low affinity and in the micromolar range for the binding site with high affinity. The high-affinity binding site has a higher affinity for low-molecular-weight (low-MW) mannose carbohydrates and no affinity for mannan. However, mannan as well as low-MW mannose carbohydrates can bind to the low-affinity binding site. These results extend the cellular flocculation model on the molecular level. PMID:21076009

The velocity structure of the lunar crust.

NASA Technical Reports Server (NTRS)

Kovach, R. L.; Watkins, J. S.

1973-01-01

Seismic refraction data, obtained at the Apollo 14 and 16 sites, when combined with other lunar seismic data, allow a compressional wave velocity profile of the lunar near-surface and crust to be derived. The regolith, although variable in thickness over the lunar surface, possesses surprisingly similar seismic properties. Underlying the regolith at both the Apollo 14 Fra Mauro site and the Apollo 16 Descartes site is low-velocity brecciated material or impact derived debris. Key features of the lunar seismic velocity profile are: (1) velocity increases from 100 to 300 m/sec in the upper 100 m to about 4 km/sec at 5 km depth, (2) a more gradual increase from about 4 km/sec to about 6 km/sec at 25 km depth,(3) a discontinuity at a depth of 25 km, and (4) a constant value of about 7 km/sec at depths from 25 km to about 60 km.

Synthetic cannabinoids: In silico prediction of the cannabinoid receptor 1 affinity by a quantitative structure-activity relationship model.

PubMed

Paulke, Alexander; Proschak, Ewgenij; Sommer, Kai; Achenbach, Janosch; Wunder, Cora; Toennes, Stefan W

2016-03-14

The number of new synthetic psychoactive compounds increase steadily. Among the group of these psychoactive compounds, the synthetic cannabinoids (SCBs) are most popular and serve as a substitute of herbal cannabis. More than 600 of these substances already exist. For some SCBs the in vitro cannabinoid receptor 1 (CB1) affinity is known, but for the majority it is unknown. A quantitative structure-activity relationship (QSAR) model was developed, which allows the determination of the SCBs affinity to CB1 (expressed as binding constant (Ki)) without reference substances. The chemically advance template search descriptor was used for vector representation of the compound structures. The similarity between two molecules was calculated using the Feature-Pair Distribution Similarity. The Ki values were calculated using the Inverse Distance Weighting method. The prediction model was validated using a cross validation procedure. The predicted Ki values of some new SCBs were in a range between 20 (considerably higher affinity to CB1 than THC) to 468 (considerably lower affinity to CB1 than THC). The present QSAR model can serve as a simple, fast and cheap tool to get a first hint of the biological activity of new synthetic cannabinoids or of other new psychoactive compounds. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Characterization and regulation of glycine transport in Fusarium oxysporum var. lini.

PubMed

Castro, I M; Lima, A A; Nascimento, A F; Ruas, M M; Nicoli, J R; Brandão, R L

1996-08-01

Glycine was transported in Fusarium oxysporum cells, grown on glycine as the sole source of carbon and nitrogen, by a facilitated diffusion transport system with a half-saturation constant (Ks) of 11 mM and a maximum velocity (Vmax) of 1.2 mM (g dry weight)-1 h-1 at pH 5.0 and 26 degrees C. Under conditions of nitrogen starvation, the same system was present together with a high-affinity one (Ks) of about 47 microM and Vmax of about 60 microM (g dry weight)-1 h-1). The low-affinity system was more specific than the high-affinity system. Cells grown on gelatine showed the same behavior. In cells grown on glucose-gelatine medium, the low-affinity system was poorly expressed even after carbon and nitrogen starvation. Moreover, addition of glucose to cells grown on glycine and resuspended in mineral medium caused an increase of the glycine transport probably due to a boost in protein synthesis. This stimulation did not affect the Ks of the low-affinity system. These results demonstrate that, as is the case for other eukaryotic systems, F. oxysporum glycine transport is under control of nitrogen sources but its regulation by carbon sources appears to be more complex.

Allosteric Models for Cooperative Polymerization of Linear Polymers

PubMed Central

Miraldi, Emily R.; Thomas, Peter J.; Romberg, Laura

2008-01-01

In the cytoskeleton, unfavorable nucleation steps allow cells to regulate where, when, and how many polymers assemble. Nucleated polymerization is traditionally explained by a model in which multistranded polymers assemble cooperatively, whereas linear, single-stranded polymers do not. Recent data on the assembly of FtsZ, the bacterial homolog of tubulin, do not fit either category. FtsZ can polymerize into single-stranded protofilaments that are stable in the absence of lateral interactions, but that assemble cooperatively. We developed a model for cooperative polymerization that does not require polymers to be multistranded. Instead, a conformational change allows subunits in oligomers to associate with high affinity, whereas a lower-affinity conformation is favored in monomers. We derive equations for calculating polymer concentrations, subunit conformations, and the apparent affinity of subunits for polymer ends. Certain combinations of equilibrium constants produce the sharp critical concentrations characteristic of cooperative polymerization. In these cases, the low-affinity conformation predominates in monomers, whereas virtually all polymers are composed of high-affinity subunits. Our model predicts that the three routes to forming HH dimers all involve unstable intermediates, limiting nucleation. The mathematical framework developed here can represent allosteric assembly systems with a variety of biochemical interpretations, some of which can show cooperativity, and others of which cannot. PMID:18502809

Inhibitory effects of nitrite on the reactions of bovine carbonic anhydrase II with CO2 and bicarbonate consistent with zinc-bound nitrite.

PubMed

Nielsen, Per M; Fago, Angela

2015-08-01

Carbonic anhydrase (CA) is a zinc enzyme that catalyzes hydration of carbon dioxide (CO2) and dehydration of bicarbonate in red blood cells, thus facilitating CO2 transport and excretion. Bovine CA II may also react with nitrite to generate nitric oxide, although nitrite is a known inhibitor of the CO2 hydration reaction. To address the potential in vivo interference of these reactions and the nature of nitrite binding to the enzyme, we here investigate the inhibitory effect of 10-30 mM nitrite on Michaelis-Menten kinetics of CO2 hydration and bicarbonate dehydration by stopped-flow spectroscopy. Our data show that nitrite significantly affects the apparent dissociation constant KM for CO2 (11 mM) and bicarbonate (221 mM), and the turnover number kcat for the CO2 hydration (1.467 × 10(6) s(-1)) but not for the bicarbonate dehydration (7.927 × 10(5) s(-1)). These effects demonstrate mixed and competitive inhibition for the reaction with CO2 and bicarbonate, respectively, and are consistent with nitrite binding to the active site zinc. The high apparent dissociation constant found here for CO2, bicarbonate and nitrite (16-120 mM) are all overall consistent with published data and reveal a large capacity of free enzyme available for binding each of the three substrates at their in vivo levels, with little or no significant interference among reactions. The low affinity of the enzyme for nitrite suggests that the in vivo interaction between red blood cell CA II and nitrite requires compartmentalization at the anion exchanger protein of the red cell membrane to be physiologically relevant. Copyright © 2015 Elsevier Inc. All rights reserved.

Arginine kinase from the Tardigrade, Macrobiotus occidentalis: molecular cloning, phylogenetic analysis and enzymatic properties.

PubMed

Uda, Kouji; Ishida, Mikako; Matsui, Tohru; Suzuki, Tomohiko

2010-10-01

Arginine kinase (AK), which catalyzes the reversible transfer of phosphate from ATP to arginine to yield phosphoarginine and ADP, is widely distributed throughout the invertebrates. We determined the cDNA sequence of AK from the tardigrade (water bear) Macrobiotus occidentalis, cloned the sequence into pET30b plasmid, and expressed it in Escherichia coli as a 6x His-tag—fused protein. The cDNA is 1377 bp, has an open reading frame of 1080 bp, and has 5′- and 3′-untranslated regions of 116 and 297 bp, respectively. The open reading frame encodes a 359-amino acid protein containing the 12 residues considered necessary for substrate binding in Limulus AK. This is the first AK sequence from a tardigrade. From fragmented and non-annotated sequences available from DNA databases, we assembled 46 complete AK sequences: 26 from arthropods (including 19 from Insecta), 11 from nematodes, 4 from mollusks, 2 from cnidarians and 2 from onychophorans. No onychophoran sequences have been reported previously. The phylogenetic trees of 104 AKs indicated clearly that Macrobiotus AK (from the phylum Tardigrada) shows close affinity with Epiperipatus and Euperipatoides AKs (from the phylum Onychophora), and therefore forms a sister group with the arthropod AKs. Recombinant 6x His-tagged Macrobiotus AK was successfully expressed as a soluble protein, and the kinetic constants (K(m), K(d), V(ma) and k(cat)) were determined for the forward reaction. Comparison of these kinetic constants with those of AKs from other sources (arthropods, mollusks and nematodes) indicated that Macrobiotus AK is unique in that it has the highest values for k(cat) and K(d)K(m) (indicative of synergistic substrate binding) of all characterized AKs.

Static Stretching Alters Neuromuscular Function and Pacing Strategy, but Not Performance during a 3-Km Running Time-Trial

PubMed Central

Damasceno, Mayara V.; Duarte, Marcos; Pasqua, Leonardo A.; Lima-Silva, Adriano E.; MacIntosh, Brian R.; Bertuzzi, Rômulo

2014-01-01

Purpose Previous studies report that static stretching (SS) impairs running economy. Assuming that pacing strategy relies on rate of energy use, this study aimed to determine whether SS would modify pacing strategy and performance in a 3-km running time-trial. Methods Eleven recreational distance runners performed a) a constant-speed running test without previous SS and a maximal incremental treadmill test; b) an anthropometric assessment and a constant-speed running test with previous SS; c) a 3-km time-trial familiarization on an outdoor 400-m track; d and e) two 3-km time-trials, one with SS (experimental situation) and another without (control situation) previous static stretching. The order of the sessions d and e were randomized in a counterbalanced fashion. Sit-and-reach and drop jump tests were performed before the 3-km running time-trial in the control situation and before and after stretching exercises in the SS. Running economy, stride parameters, and electromyographic activity (EMG) of vastus medialis (VM), biceps femoris (BF) and gastrocnemius medialis (GA) were measured during the constant-speed tests. Results The overall running time did not change with condition (SS 11:35±00:31 s; control 11:28±00:41 s, p = 0.304), but the first 100 m was completed at a significantly lower velocity after SS. Surprisingly, SS did not modify the running economy, but the iEMG for the BF (+22.6%, p = 0.031), stride duration (+2.1%, p = 0.053) and range of motion (+11.1%, p = 0.0001) were significantly modified. Drop jump height decreased following SS (−9.2%, p = 0.001). Conclusion Static stretch impaired neuromuscular function, resulting in a slow start during a 3-km running time-trial, thus demonstrating the fundamental role of the neuromuscular system in the self-selected speed during the initial phase of the race. PMID:24905918

A sensitive glucose biosensor based on Ag@C core-shell matrix.

PubMed

Zhou, Xuan; Dai, Xingxin; Li, Jianguo; Long, Yumei; Li, Weifeng; Tu, Yifeng

2015-04-01

Nano-Ag particles were coated with colloidal carbon (Ag@C) to improve its biocompatibility and chemical stability for the preparation of biosensor. The core-shell structure was evidenced by transmission electron microscope (TEM) and the Fourier transfer infrared (FTIR) spectra revealed that the carbon shell is rich of function groups such as -OH and -COOH. The as-prepared Ag@C core-shell structure can offer favorable microenvironment for immobilizing glucose oxidase and the direct electrochemistry process of glucose oxidase (GOD) at Ag@C modified glassy carbon electrode (GCE) was realized. The modified electrode exhibited good response to glucose. Under optimum experimental conditions the biosensor linearly responded to glucose concentration in the range of 0.05-2.5mM, with a detection limit of 0.02mM (S/N=3). The apparent Michaelis-Menten constant (KM(app)) of the biosensor is calculated to be 1.7mM, suggesting high enzymatic activity and affinity toward glucose. In addition, the GOD-Ag@C/Nafion/GCE shows good reproducibility and long-term stability. These results suggested that core-shell structured Ag@C is an ideal matrix for the immobilization of the redox enzymes and further the construction of the sensitive enzyme biosensor. Copyright © 2015 Elsevier B.V. All rights reserved.

LeCPK1, a Calcium-Dependent Protein Kinase from Tomato. Plasma Membrane Targeting and Biochemical Characterization1

PubMed Central

Rutschmann, Frank; Stalder, Urs; Piotrowski, Markus; Oecking, Claudia; Schaller, Andreas

2002-01-01

The cDNA of LeCPK1, a calcium-dependent protein kinase, was cloned from tomato (Lycopersicon esculentum Mill.). LeCPK1 was expressed in Escherichia coli and purified from bacterial extracts. The recombinant protein was shown to be a functional protein kinase using a synthetic peptide as the substrate (syntide-2, Km = 85 μm). Autophosphorylation of LeCPK1 was observed on threonine and serine residues, one of which was identified as serine-439. Kinase activity was shown to be Ca2+ dependent and required the C-terminal, calmodulin-like domain of LeCPK1. Two classes of high- and low-affinity Ca2+-binding sites were observed, exhibiting dissociation constants of 0.6 and 55 μm, respectively. LeCPK1 was found to phosphorylate the regulatory C-terminal domain of the plasma membrane H+-ATPase in vitro. A potential role in the regulation of proton pump activity is corroborated by the apparent colocalization of the plasma membrane H+-ATPase and LeCPK1 in vivo. Upon transient expression in suspension-cultured cells, a C-terminal fusion of LeCPK1 with the green fluorescent protein was targeted to the plasma membrane. Myristoylation of the LeCPK1 N terminus was found to be required for plasma membrane targeting. PMID:12011347

Cloning of a Novel Aldo-Keto Reductase Gene from Klebsiella sp. Strain F51-1-2 and Its Functional Expression in Escherichia coli▿

PubMed Central

Jiang, Hong; Yang, Chao; Qu, Hong; Liu, Zheng; Fu, Q. S.; Qiao, Chuanling

2007-01-01

A soil bacterium capable of metabolizing organophosphorus compounds by reducing the P=S group in the molecules was taxonomically identified as Klebsiella sp. strain F51-1-2. The gene involved in the reduction of organophosphorus compounds was cloned from this strain by the shotgun technique, and the deduced protein (named AKR5F1) showed homology to members of the aldo-keto reductase (AKR) superfamily. The intact coding region for AKR5F1 was subcloned into vector pET28a and overexpressed in Escherichia coli BL21(DE3). Recombinant His6-tagged AKR5F1 was purified in one step using Ni-nitrilotriacetic acid affinity chromatography. Assays for cofactor specificity indicated that reductive transformation of organophosphorus compounds by the recombinant AKR5F1 specifically required NADH. The kinetic constants of the purified recombinant AKR5F1 toward six thion organophosphorus compounds were determined. For example, the Km and kcat values of reductive transformation of malathion by the purified recombinant AKR5F1 are 269.5 ± 47.0 μΜ and 25.7 ± 1.7 min−1, respectively. Furthermore, the reductive transformation of organophosphorus compounds can be largely explained by structural modeling. PMID:17575004

Environmental Fate Studies on Certain Munition Wastewater Constituents - Literature Review

DTIC Science & Technology

1980-03-01

gram-negative bacteria , actinaycetes, yeasts, and fungi. They found that TNT at 50 Mgtter severely inhibited the growth of these organisms in wost...i , conditions . I Biodegradation ,. Chambers et al. (1963) reported evidence of degradation of 2,4-DNT with phenol-adapted bacteria and 100 mg/liter...coefficient based on organic carbon contentoc H - Henry’s Law constant kA - Hydrolysis rate constant under acidic conditions k.M - Hydrolysis rate constant

The significance of the source of zinc and its anti-VSC effect.

PubMed

Rölla, G; Jonski, G; Young, A

2002-06-01

The anti-VSC (volatile sulphur compounds) effect of zinc is known to be associated with free zinc ions. To examine whether zinc salts with low stability constants were more suitable as sources of zinc in zinc lozenges than zinc salts with high stability constants. The former provide free zinc ions upon dissolution in water, whereas the latter provide few such ions. Identical lozenges were produced which contained either zinc acetate, zinc gluconate (low stability constants), zinc citrate or amino-acid chelated zinc (extremely high stability constants). All the lozenges contained 0.1 per cent of zinc. A test panel of 10 volunteers used the different lozenges randomly. VSC were measured by GC. The lozenge with the highest stability constant was as effective as those with very low stability constants. The anti-VSC effect was thus not related to this constant. These findings may be explained by the possibility that alternative ligands with stronger affinity for zinc than the original ligands in the lozenges may be present in the oral cavity. An in vitro experiment indicated that the sulphide ion (S2-) may be such a ligand.

The cost of transport of human running is not affected, as in walking, by wide acceleration/deceleration cycles.

PubMed

Minetti, Alberto E; Gaudino, Paolo; Seminati, Elena; Cazzola, Dario

2013-02-15

Although most of the literature on locomotion energetics and biomechanics is about constant-speed experiments, humans and animals tend to move at variable speeds in their daily life. This study addresses the following questions: 1) how much extra metabolic energy is associated with traveling a unit distance by adopting acceleration/deceleration cycles in walking and running, with respect to constant speed, and 2) how can biomechanics explain those metabolic findings. Ten males and ten females walked and ran at fluctuating speeds (5 ± 0, ± 1, ± 1.5, ± 2, ± 2.5 km/h for treadmill walking, 11 ± 0, ± 1, ± 2, ± 3, ± 4 km/h for treadmill and field running) in cycles lasting 6 s. Field experiments, consisting of subjects following a laser spot projected from a computer-controlled astronomic telescope, were necessary to check the noninertial bias of the oscillating-speed treadmill. Metabolic cost of transport was found to be almost constant at all speed oscillations for running and up to ±2 km/h for walking, with no remarkable differences between laboratory and field results. The substantial constancy of the metabolic cost is not explained by the predicted cost of pure acceleration/deceleration. As for walking, results from speed-oscillation running suggest that the inherent within-stride, elastic energy-free accelerations/decelerations when moving at constant speed work as a mechanical buffer for among-stride speed fluctuations, with no extra metabolic cost. Also, a recent theory about the analogy between sprint (level) running and constant-speed running on gradients, together with the mechanical determinants of gradient locomotion, helps to interpret the present findings.

MODELING OF METAL BINDING ON HUMIC SUBSTANCES USING THE NIST DATABASE: AN A PRIORI FUNCTIONAL GROUP APPROACH

EPA Science Inventory

Various modeling approaches have been developed for metal binding on humic substances. However, most of these models are still curve-fitting exercises-- the resulting set of parameters such as affinity constants (or the distribution of them) is found to depend on pH, ionic stren...

Identification of human antibody fragment clones specific for tetanus toxoid in a bacteriophage. lambda. immunoexpression library

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mullinax, R.L.; Gross, E.A.; Amberg, J.R.

1990-10-01

The authors have applied a molecular biology approach to the identification of human monoclonal antibodies. Human peripheral blood lymphocyte mRNA was converted to cDNA and a select subset was amplified by the polymerase chain reaction. These products, containing coding sequences for numerous immunoglobulin heavy- and {kappa} light-chain variable and constant region domains, were inserted into modified bacteriophase {lambda} expression vectors and introduced into Escherichia coli by infection to yield a combinatorial immunoexpression library. Clones with binding activity to tetanus toxoid were identified by filter hybridization with radiolabeled antigen and appeared at a frequency of 0.2{percent} in the library. These humanmore » antigen binding fragments, consisting of a heavy-chain fragment covalently linked to a light chain, displayed high affinity of binding to tetanus toxoid with equilibrium constants in the nanomolar range but did not cross-react with other proteins tested. They estimate that this human immunoexpression library contains 20,000 clones with high affinity and specificity to our chosen antigen.« less

Using the Concept of Transient Complex for Affinity Predictions in CAPRI Rounds 20–27 and Beyond

PubMed Central

Qin, Sanbo; Zhou, Huan-Xiang

2013-01-01

Predictions of protein-protein binders and binding affinities have traditionally focused on features pertaining to the native complexes. In developing a computational method for predicting protein-protein association rate constants, we introduced the concept of transient complex after mapping the interaction energy surface. The transient complex is located at the outer boundary of the bound-state energy well, having near-native separation and relative orientation between the subunits but not yet formed most of the short-range native interactions. We found that the width of the binding funnel and the electrostatic interaction energy of the transient complex are among the features predictive of binders and binding affinities. These ideas were very promising for the five affinity-related targets (T43–45, 55, and 56) of CAPRI rounds 20–27. For T43, we ranked the single crystallographic complex as number 1 and were one of only two groups that clearly identified that complex as a true binder; for T44, we ranked the only design with measurable binding affinity as number 4. For the nine docking targets, continuing on our success in previous CAPRI rounds, we produced 10 medium-quality models for T47 and acceptable models for T48 and T49. We conclude that the interaction energy landscape and the transient complex in particular will complement existing features in leading to better prediction of binding affinities. PMID:23873496

Determination of dissociation constant of the NFκB p50/p65 heterodimer using fluorescence cross-correlation spectroscopy in the living cell

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tiwari, Manisha; Mikuni, Shintaro; Muto, Hideki

Highlights: •We used two-laser-beam FCCS to determine the dissociation constant (K{sub d}) of IPT domain of p50/p65 heterodimer in living cell. •Interaction of p50 and p65 was analyzed in the cytoplasm and nucleus of single living cell. •Binding affinity of p50/p65 heterodimer is higher in cytoplasm than that of nucleus. -- Abstract: Two-laser-beam fluorescence cross-correlation spectroscopy (FCCS) is promising technique that provides quantitative information about the interactions of biomolecules. The p50/p65 heterodimer is the most abundant and well understood of the NFκB dimers in most cells. However, the quantitative value of affinity, namely the K{sub d}, for the heterodimer inmore » living cells is not known yet. To quantify the heterodimerization of the IPT domain of p50/p65 in the living cell, we used two-laser-beam FCCS. The K{sub d} values of mCherry{sub 2}- and EGFP-fused p50 and p65 were determined to be 0.46 μM in the cytoplasm and 1.06 μM in the nucleus of the living cell. These results suggest the different binding affinities of the p50/p65 heterodimer in the cytoplasm and nucleus of the living cell and different complex formation in each region.« less

Highly affine and selective aptamers against cholera toxin as capture elements in magnetic bead-based sandwich ELAA.

PubMed

Frohnmeyer, Esther; Frisch, Farina; Falke, Sven; Betzel, Christian; Fischer, Markus

2018-03-10

Aptamers are single-stranded DNA or RNA oligonucleotides, which have been emerging as recognition elements in disease diagnostics and food control, including the detection of bacterial toxins. In this study, we employed the semi-automated just in time-selection to identify aptamers that bind to cholera toxin (CT) with high affinity and specificity. CT is the main virulence factor of Vibrio cholerae and the causative agent of the eponymous disease. For the selected aptamers, dissociation constants in the low nanomolar range (23-56 nM) were determined by fluorescence-based affinity chromatography and cross-reactivity against related proteins was evaluated by direct enzyme-linked aptamer assay (ELAA). Aptamer CT916 has a dissociation constant of 48.5 ± 0.5 nM and shows negligible binding to Shiga-like toxin 1B, protein A and BSA. This aptamer was chosen to develop a sandwich ELAA for the detection of CT from binding buffer and local tap water. Amine-C6- or biotin-modified CT916 was coupled to magnetic beads to serve as the capture element. Using an anti-CT polyclonal antibody as the reporter, detection limits of 2.1 ng/ml in buffer and 2.4 ng/ml in tap water, with a wide log-linear dynamic range from 1 ng/ml to 1000 ng/ml and 500 ng/ml, respectively, were achieved. Copyright © 2018 Elsevier B.V. All rights reserved.

Transport mechanism for lovastatin acid in bovine kidney NBL-1 cells: kinetic evidences imply involvement of monocarboxylate transporter 4.

PubMed

Nagasawa, Kazuki; Nagai, Katsuhito; Ishimoto, Atsushi; Fujimoto, Sadaki

2003-08-27

We previously indicated that lovastatin acid, a 3-hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, was transported by a monocarboxylate transporter (MCT) in cultured rat mesangial cells. In this study, to identify the MCT isoform(s) responsible for the lovastatin acid uptake, the transport mechanism was investigated using bovine kidney NBL-1 cells, which have been reported to express only MCT4 at the protein level. On RT-PCR analysis, the message of mRNAs for MCT1 and MCT4 was detected in the NBL-1 cells used in this study, which was confirmed by kinetic analysis of [14C]L-lactic acid uptake, consisting of high- and low-affinity components corresponding to MCT1 and MCT4, respectively. The lovastatin acid uptake depended on an inwardly directed H+-gradient, and was inhibited by representative monocarboxylates, but not by inhibitors/substrates for organic anion transporting polypeptides and organic anion transporters. In addition, L-lactic acid competitively inhibited the uptake of lovastatin acid and lovastatin acid inhibited the low affinity component of [14C]L-lactic acid uptake dose dependently. The inhibition constant of L-lactic acid for lovastatin acid uptake was almost the same as the Michaelis constant for [14C]L-lactic acid uptake by the low-affinity component. These kinetic evidences imply that lovastatin acid was taken up into NBL-1 cells via MCT4.

Selecting for Fast Protein-Protein Association As Demonstrated on a Random TEM1 Yeast Library Binding BLIP.

PubMed

Cohen-Khait, Ruth; Schreiber, Gideon

2018-04-27

Protein-protein interactions mediate the vast majority of cellular processes. Though protein interactions obey basic chemical principles also within the cell, the in vivo physiological environment may not allow for equilibrium to be reached. Thus, in vitro measured thermodynamic affinity may not provide a complete picture of protein interactions in the biological context. Binding kinetics composed of the association and dissociation rate constants are relevant and important in the cell. Therefore, changes in protein-protein interaction kinetics have a significant impact on the in vivo activity of the proteins. The common protocol for the selection of tighter binders from a mutant library selects for protein complexes with slower dissociation rate constants. Here we describe a method to specifically select for variants with faster association rate constants by using pre-equilibrium selection, starting from a large random library. Toward this end, we refine the selection conditions of a TEM1-β-lactamase library against its natural nanomolar affinity binder β-lactamase inhibitor protein (BLIP). The optimal selection conditions depend on the ligand concentration and on the incubation time. In addition, we show that a second sort of the library helps to separate signal from noise, resulting in a higher percent of faster binders in the selected library. Fast associating protein variants are of particular interest for drug development and other biotechnological applications.

The Hubble Constant.

PubMed

Jackson, Neal

2007-01-01

I review the current state of determinations of the Hubble constant, which gives the length scale of the Universe by relating the expansion velocity of objects to their distance. In the last 20 years, much progress has been made and estimates now range between 60 and 75 km s -1 Mpc -1 , with most now between 70 and 75 km s -1 Mpc -1 , a huge improvement over the factor-of-2 uncertainty which used to prevail. Further improvements which gave a generally agreed margin of error of a few percent rather than the current 10% would be vital input to much other interesting cosmology. There are several programmes which are likely to lead us to this point in the next 10 years.

Impaired binding affinity of electronegative low-density lipoprotein (LDL) to the LDL receptor is related to nonesterified fatty acids and lysophosphatidylcholine content.

PubMed

Benítez, Sonia; Villegas, Virtudes; Bancells, Cristina; Jorba, Oscar; González-Sastre, Francesc; Ordóñez-Llanos, Jordi; Sánchez-Quesada, José Luis

2004-12-21

The binding characteristics of electropositive [LDL(+)] and electronegative LDL [LDL(-)] subfractions to the LDL receptor (LDLr) were studied. Saturation kinetic studies in cultured human fibroblasts demonstrated that LDL(-) from normolipemic (NL) and familial hypercholesterolemic (FH) subjects had lower binding affinity than their respective LDL(+) fractions (P < 0.05), as indicated by higher dissociation constant (K(D)) values. FH-LDL(+) also showed lower binding affinity (P < 0.05) than NL-LDL(+) (K(D), sorted from lower to higher affinity: NL-LDL(-), 33.0 +/- 24.4 nM; FH-LDL(-), 24.4 +/- 7.1 nM; FH-LDL(+), 16.6 +/- 7.0 nM; NL-LDL(+), 10.9 +/- 5.7 nM). These results were confirmed by binding displacement studies. The impaired affinity binding of LDL(-) could be attributed to altered secondary and tertiary structure of apolipoprotein B, but circular dichroism (CD) and tryptophan fluorescence (TrpF) studies revealed no structural differences between LDL(+) and LDL(-). To ascertain the role of increased nonesterified fatty acids (NEFA) and lysophosphatidylcholine (LPC) content in LDL(-), LDL(+) was enriched in NEFA or hydrolyzed with secretory phospholipase A(2). Modification of LDL gradually decreased the affinity to LDLr in parallel to the increasing content of NEFA and/or LPC. Modified LDLs with a NEFA content similar to that of LDL(-) displayed similar affinity. ApoB structure studies of modified LDLs by CD and TrpF showed no difference compared to LDL(+) or LDL(-). Our results indicate that NEFA loading or phospholipase A(2) lipolysis of LDL leads to changes that affect the affinity of LDL to LDLr with no major effect on apoB structure. Impaired affinity to the LDLr shown by LDL(-) is related to NEFA and/or LPC content rather than to structural differences in apolipoprotein B.

Synthesis of Mono- and Di-Glucosides of Zearalenone and α-/β-Zearalenol by Recombinant Barley Glucosyltransferase HvUGT14077

PubMed Central

Michlmayr, Herbert; Varga, Elisabeth; Lupi, Francesca; Malachová, Alexandra; Hametner, Christian; Berthiller, Franz; Adam, Gerhard

2017-01-01

Zearalenone (ZEN) is an estrogenic mycotoxin occurring in Fusarium-infected cereals. Glucosylation is an important plant defense mechanism and generally reduces the acute toxicity of mycotoxins to humans and animals. Toxicological information about ZEN-glucosides is limited due to the unavailability of larger amounts required for animal studies. HvUGT14077, a recently-validated ZEN-conjugating barley UDP-glucosyltransferase was expressed in Escherichia coli, affinity purified, and characterized. HvUGT14077 possesses high affinity (Km = 3 µM) and catalytic efficiency (kcat/Km = 190 s−1·mM−1) with ZEN. It also efficiently glucosylates the phase-I ZEN-metabolites α-zearalenol and β-zearalenol, with kcat/Km of 40 and 74 s−1·mM−1, respectively. HvUGT14077 catalyzes O-glucosylation at C-14 and C-16 with preference of 14-glucoside synthesis. Furthermore, relatively slow consecutive formation of 14,16-di-glucosides was observed; their structures were tentatively identified by mass spectrometry and for ZEN-14,16-di-glucoside confirmed by nuclear magnetic resonance spectroscopy. Recombinant HvUGT14077 allowed efficient preparative synthesis of ZEN-glucosides, yielding about 90% ZEN-14-glucoside and 10% ZEN-16-glucoside. The yield of ZEN-16-glucoside could be increased to 85% by co-incubation with a β-glucosidase highly selective for ZEN-14-glucoside. Depletion of the co-substrate UDP-glucose was counteracted by a sucrose synthase based regeneration system. This strategy could also be of interest to increase the yield of minor glucosides synthesized by other glucosyltransferases. PMID:28208765

Effects of asparagine mutagenesis of conserved aspartic acids in helix two (D2.50) and three (D3.32) of M1 – M4 muscarinic receptors on the irreversible binding of nitrogen mustard analogs of acetylcholine and McN-A-343

PubMed Central

Suga, Hinako; Ehlert, Frederick J.

2013-01-01

We investigated how asparagine mutagenesis of conserved aspartic acids in helix two (D2.50) and three (D3.32) of M1 – M4 muscarinic receptors alters the irreversible binding of acetylcholine mustard and BR384 (4-[(2-bromoethyl)methyl-amino]-2-butynyl N-(3-chlorophenyl)carbamate), a nitrogen mustard derivative of McN-A-343 ([4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyl] trimethylammonium chloride). The D2.50N mutation moderately increased the affinity of the aziridinium ions of acetylcholine mustard and BR384 for M2 – M4 receptors and had little effect on the rate constant for receptor alkylation. The D3.32N mutation greatly reduced the rate constant for receptor alkylation by acetylcholine mustard, but not by BR384, although the affinity of BR384 was reduced. The combination of both mutations (D2.50N/D3.32N) substantially reduced the rate constant for receptor alkylation by BR384 relative to wild type and mutant D2.50N and D3.32N receptors. The change in binding affinity caused by the mutations suggests that the D2.50N mutation alters the interaction of acetylcholine mustard with D3.32 of M1 and M3 receptors, but not that of the M4 receptor. BR384 exhibited the converse relationship. The simplest explanation is that acetylcholine mustard and BR384 alkylate at least two residues on M1 – M4 receptors and that the D2.50N mutation alters the rate of alkylation of D3.32 relative to another residue, perhaps D2.50 itself. PMID:23826889

Low-affinity FcγR interactions can decide the fate of novel human IgG-sensitised red blood cells and platelets.

PubMed

Armour, Kathryn L; Smith, Cheryl S; Turner, Craig P; Kirton, Christopher M; Wilkes, Anthony M; Hadley, Andrew G; Ghevaert, Cedric; Williamson, Lorna M; Clark, Michael R

2014-03-01

G1Δnab is a mutant human IgG1 constant region with a lower ability to interact with FcγR than the natural IgG constant regions. Radiolabelled RBCs and platelets sensitised with specific G1Δnab Abs were cleared more slowly from human circulation than IgG1-sensitised counterparts. However, non-destructive splenic retention of G1Δnab-coated RBCs required investigation and plasma radioactivities now suggest this also occurred for platelets sensitised with an IgG1/G1Δnab mixture. In vitro assays with human cells showed that G1Δnab-sensitised RBCs did not cause FcγRI-mediated monocyte activation, FcγRIIIa-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) or macrophage phagocytosis although they did adhere to macrophages. Thus, FcγRII was implicated in the adhesion despite the Δnab mutation reducing the already low-affinity binding to this receptor class. Additional contacts via P-selectin enhance the interaction of sensitised platelets with monocytes and this system provided evidence of FcγRII-dependent activation by G1Δnab. These results emphasise the physiological relevance of low-affinity interactions: It appears that FcγRII interactions of G1Δnab allowed splenic retention of G1Δnab-coated RBCs with inhibitory FcγRIIb binding preventing RBC destruction and that FcγRIIb engagement by G1Δnab on IgG1/G1Δnab-sensitised platelets overcame activation by IgG1. Considering therapeutic blocking Abs, G1Δnab offers lower FcγR binding and a greater bias towards inhibition than IgG2 and IgG4 constant regions. © 2013 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Modified high-affinity binding of Ni2+, Ca2+ and Zn2+ to natural mutants of human serum albumin and proalbumin.

PubMed

Kragh-Hansen, U; Brennan, S O; Minchiotti, L; Galliano, M

1994-07-01

High-affinity binding of radioactive Ni2+, Ca2+ and Zn2+ to six genetic albumin variants and to normal albumin isolated from the same heterozygote carriers was studied by equilibrium dialysis at pH 7.4. The three cations bind differently to albumin. Ni2+ binds to a site in the N-terminal region of the protein which is partially blocked by the presence of a propeptide as in proalbumin (proAlb) Varese (Arg-2-->His), proAlb Christchurch (Arg-1-->Gln) and proAlb Blenheim (Asp1-->Val) and by the presence of only an extra Arg residue (Arg-1) as in Arg-Alb and albumin (Alb) Redhill. The association constants are decreased by more than one order of magnitude in these cases, suggesting biological consequences for the ligand. The additional structural changes in Alb Redhill have no effect on Ni2+ binding. Finally, the modification of Alb Blenheim (Asp1-->Val) reduces the binding constant to 50%. Ca2+ binding is decreased to about 60-80% by the presence of a propeptide and the mutation Asp1-->Val. Arg-1 alone does not affect binding, whereas Alb Redhill binds Ca2+ more strongly than the normal protein (125%). In contrast with binding of Ni2+ and Ca2+, albumin shows heterogeneity with regard to binding of Zn2+, i.e. the number of high-affinity sites was calculated to be, on average, 0.43. The binding constant for Zn2+ is increased to 125% in the case of proAlb Varese, decreased to 50-60% for proAlb Christchurch and Alb Redhill but is normal for proAlb Blenheim, Alb Blenheim and Arg-Alb. The effects of the mutations on binding of Ca2+ and Zn2+ indicate that primary binding, when operative, is to as yet unidentified sites in domain I of the albumin molecule.

Rhodamine Inhibitors of P-glycoprotein: An Amide/Thioamide “Switch” for ATPase Activity

PubMed Central

Gannon, Michael K.; Holt, Jason J.; Bennett, Stephanie M.; Wetzel, Bryan R.; Loo, Tip W.; Bartlett, M. Claire; Clarke, David M.; Sawada, Geri A.; Higgins, J. William; Tombline, Gregory; Raub, Thomas J.; Detty, Michael R.

2012-01-01

We have examined 46 tetramethylrosamine/rhodamine derivatives with structural diversity in the heteroatom of the xanthylium core, the amino substituents of the 3- and 6-positions, and the alkyl, aryl, or heteroaryl group at the 9-substituent. These compounds were examined for affinity and ATPase stimulation in isolated MDR3 CL P-gp and human P-gp-His10, for their ability to promote uptake of calcein AM and vinblastine in multidrug-resistant MDCKII-MDR1 cells, and for transport in monolayers of MDCKII-MDR1 cells. Thioamide 31-S gave KM of 0.087 μM in human P-gp. Small changes in structure among this set of compounds affected affinity as well as transport rate (or flux) even though all derivatives examined were substrates for P-gp. With isolated protein, tertiary amide groups dictate high affinity and high stimulation while tertiary thioamide groups give high affinity and inhibition of ATPase activity. In MDCKII-MDR1 cells, the tertiary thioamide-containing derivatives promote uptake of calcein AM and have very slow passive, absorptive, and secretory rates of transport relative to transport rates for tertiary amide-containing derivatives. Thioamide 31-S promoted uptake of calcein AM and inhibited efflux of vinblastine with IC50’s of ~2 μM in MDCKII-MDR1 cells. PMID:19402665

Recovery of Uranium from Seawater: Preparation and Development of Polymer-Supported Extractants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spiro, Alexandratos

2013-12-01

A new series of polymer-supported extractants is proposed for the removal and recovery of uranium from seawater. The objective is to produce polymers with improved stability, loading capacity, and sorption kinetics compared to what is found with amidoximes. The target ligands are diphosphonates and aminomethyldiphosphonates. Small molecule analogues, especially of aminomethyldiphos-phonates, have exceptionally high stability constants for the uranyl ion. The polymeric diphosphonates will have high affinities due to their ability to form six-membered rings with the uranyl ion while the aminomethyldiphosphonates may have yet higher affinities due to possible tridentate coordination and their greater acidity. A representative set ofmore » the polymers to be prepared are indicated.« less

On the structure of arithmetic sums of Cantor sets with constant ratios of dissection

NASA Astrophysics Data System (ADS)

Anisca, Razvan; Chlebovec, Christopher

2009-09-01

We investigate conditions which imply that the topological structure of the arithmetic sum of two Cantor sets with constant ratios of dissection at each step is either: a Cantor set, a finite union of closed intervals, or three mixed models (L, R and M-Cantorval). We obtain general results that apply in particular for the case of homogeneous Cantor sets, thus generalizing the results of Mendes and Oliveira. The method used here is new in this context. We also produce results regarding the arithmetic sum of two affine Cantor sets of a special kind.

Perturbative Aspects of Low-Dimensional Quantum Field Theory

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wardaya, Asep Y.; Theoretical Physics Laboratory, Theoretical High Energy Physics and Instrumentation Research Group, FMIPA, Institut Teknologi Bandung, Jl. Ganesha 10 Bandung 40132; Zen, Freddy P.

We investigate the low-dimensional applications of Quantum Field Theory (QFT), namely Chern-Simons-Witten Theory (CSWT) and Affine Toda Field Theory (ATFT) in 3- and 2- dimensions. We discuss the perturbative aspects of both theories and compare the results to the exact solutions obtained nonperturbatively. For the three dimensions CSWT case, the perturbative term agree with the nonperturbative polynomial invariants up to third order of the coupling constant 1/k. In the two dimensions ATFT, we investigate the perturbative aspect of S-matrices for A{sub 1}{sup (1)} case in eighth order of the coupling constant {beta}.

Functional and Biochemical Characterization of Cucumber Genes Encoding Two Copper ATPases CsHMA5.1 and CsHMA5.2*

PubMed Central

Migocka, Magdalena; Posyniak, Ewelina; Maciaszczyk-Dziubinska, Ewa; Papierniak, Anna; Kosieradzaka, Anna

2015-01-01

Plant copper P1B-type ATPases appear to be crucial for maintaining copper homeostasis within plant cells, but until now they have been studied mostly in model plant systems. Here, we present the molecular and biochemical characterization of two cucumber copper ATPases, CsHMA5.1 and CsHMA5.2, indicating a different function for HMA5-like proteins in different plants. When expressed in yeast, CsHMA5.1 and CsHMA5.2 localize to the vacuolar membrane and are activated by monovalent copper or silver ions and cysteine, showing different affinities to Cu+ (Km ∼1 or 0.5 μm, respectively) and similar affinity to Ag+ (Km ∼2.5 μm). Both proteins restore the growth of yeast mutants sensitive to copper excess and silver through intracellular copper sequestration, indicating that they contribute to copper and silver detoxification. Immunoblotting with specific antibodies revealed the presence of CsHMA5.1 and CsHMA5.2 in the tonoplast of cucumber cells. Interestingly, the root-specific CsHMA5.1 was not affected by copper stress, whereas the widely expressed CsHMA5.2 was up-regulated or down-regulated in roots upon copper excess or deficiency, respectively. The copper-induced increase in tonoplast CsHMA5.2 is consistent with the increased activity of ATP-dependent copper transport into tonoplast vesicles isolated from roots of plants grown under copper excess. These data identify CsHMA5.1 and CsHMA5.2 as high affinity Cu+ transporters and suggest that CsHMA5.2 is responsible for the increased sequestration of copper in vacuoles of cucumber root cells under copper excess. PMID:25963145

The pH dependence of the allosteric response of human liver pyruvate kinase to fructose-1,6-bisphosphate, ATP, and alanine

PubMed Central

Fenton, Aron W.; Hutchinson, Myra

2009-01-01

The allosteric regulation of human liver pyruvate kinase (hL-PYK) by fructose-1,6-bisphosphate (Fru-1,6-BP; activator), ATP (inhibitor) and alanine (Ala; inhibitor) was monitored over a pH range from 6.5 to 8.0 at 37°C. As a function of increasing pH, hL-PYK's affinity for the substrate phosphoenolpyruvate (PEP), and for Fru-1,6-BP decreases, while affinities for ATP and Ala slightly increases. At pH 6.5, Fru-1,6-BP and ATP elicit only small allosteric impacts on PEP affinity. As pH increases, Fru-1,6-BP and ATP elicit greater allosteric responses, but the response to Ala is relatively constant. Since the magnitudes of the allosteric coupling for ATP and for Ala inhibition are different and the pH dependences of these magnitudes are not similar, these inhibitors likely elicit their responses using different molecular mechanisms. In addition, our results fail to support a general correlation between pH dependent changes in effector affinity and pH dependent changes in the corresponding allosteric response. PMID:19467627

Molecularly Imprinted Polymers with DNA Aptamer Fragments as Macromonomers.

PubMed

Zhang, Zijie; Liu, Juewen

2016-03-01

Molecularly imprinted polymers (MIPs) are produced in the presence of a template molecule. After removing the template, the cavity can selectively rebind the template. MIPs are attractive functional materials with a low cost and high stability, but traditional MIPs often suffer from low binding affinity. This study employs DNA aptamer fragments as macromonomers to improve MIPs. The DNA aptamer for adenosine was first split into two halves, fluorescently labeled, and copolymerized into MIPs. With a fluorescence quenching assay, the importance of imprinting was confirmed. Further studies were carried out using isothermal titration calorimetry (ITC). Compared to the mixture of the free aptamer fragments, their MIPs doubled the binding affinity. Each free aptamer fragment alone cannot bind adenosine, whereas MIPs containing each fragment are effective binders. We further shortened one of the aptamer fragments, and the DNA length was pushed to as short as six nucleotides, yielding MIPs with a dissociation constant of 27 μM adenosine. This study provides a new method for preparing functional MIP materials by combining high-affinity biopolymer fragments with low-cost synthetic monomers, allowing higher binding affinity and providing a method for signaling binding based on DNA chemistry.

Kv11.1 (hERG)-induced cardiotoxicity: a molecular insight from a binding kinetics study of prototypical Kv11.1 (hERG) inhibitors

PubMed Central

Yu, Z; IJzerman, A P; Heitman, L H

2015-01-01

Background and Purpose Drug-induced arrhythmia due to blockade of the Kv11.1 channel (also known as the hERG K+ channel) is a frequent side effect. Previous studies have primarily focused on equilibrium parameters, i.e. affinity or potency, of drug candidates at the channel. The aim of this study was to determine the kinetics of the interaction with the channel for a number of known Kv11.1 blockers and to explore a possible correlation with the affinity or physicochemical properties of these compounds. Experimental Approach The affinity and kinetic parameters of 15 prototypical Kv11.1 inhibitors were evaluated in a number of [3H]-dofetilide binding assays. The lipophilicity (logKW-C8) and membrane partitioning (logKW-IAM) of these compounds were determined by means of HPLC analysis. Key Results A novel [3H]-dofetilide competition association assay was set up and validated, which allowed us to determine the binding kinetics of the Kv11.1 blockers used in this study. Interestingly, the compounds' affinities (Ki values) were correlated to their association rates rather than dissociation rates. Overall lipophilicity or membrane partitioning of the compounds were not correlated to their affinity or rate constants for the channel. Conclusions and Implications A compound's affinity for the Kv11.1 channel is determined by its rate of association with the channel, while overall lipophilicity and membrane affinity are not. In more general terms, our findings provide novel insights into the mechanism of action for a compound's activity at the Kv11.1 channel. This may help to elucidate how Kv11.1-induced cardiotoxicity is governed and how it can be circumvented in the future. PMID:25296617

Exploring Thermoresponsive Affinity Agents to Enhance Microdialysis Sampling Efficiency of Proteins

NASA Astrophysics Data System (ADS)

Vasicek, Thaddeus

Affinity agents increase microdialysis protein relative recovery, yet they have not seen widespread use within the microdialysis community due to their additional instrumentation requirements and prohibitive cost. This dissertation describes new affinity agents for microdialysis that require no additional instrumentation to use, have nearly 100% particle recovery, are 7 times more cost efficient than alternatives, and have low specificity enabling their use for a wide variety of proteins. Initially gold nanoparticles were chosen as an affinity ligand support due to their high surface area/volume ratio and colloidal stability. Poly (N-isopropylacrylamide) was immobilized to the gold nanoparticles, which served to sterically stabilize the particles and to act as a generic, reversible protein capture agent. A method was developed to reproducibly vary and quantify poly (N-isopropylacrylamide) graft density from 0.09 to 0.40 ligands/nm2 on gold nanoparticles. During characterization of the polymer coated gold nanoparticles, irreversible particle agglomeration was observed at low polymer graft density in ionic solutions, which prevented further development as a protein capture agent. Poly (N-isopropylacrylamide) nanogels, which have low nonspecific adsorption, low interparticle attractive forces owing to the low curvature of the particle, and a low Hamaker constant, were synthesized to overcome the agglomeration problem. A generic protein affinity ligand cibacron blue, was immobilized to the nanogels, which enabled rapid determination of particle recovery. The perfusion of the nanogels through a microdialysis probe was optimized yielding 100% particle recovery using a combination of a syringe and peristaltic pump. The microdialysis collection efficiency of CCL2, a physiologically relevant cytokine, was increased 3-fold with addition of the nanogel to the microdialysis perfusion fluid. The reduction in instrumentation requirements, low cost, and low specificity obtained with the new affinity agents will lead to increased affinity agent use for microdiaylsis protein sampling.

Measuring Norfloxacin Binding to Trypsin Using a Fluorescence Quenching Assay in an Upper-Division, Integrated Laboratory Course

ERIC Educational Resources Information Center

Hicks, Katherine A.

2016-01-01

Fluorescence quenching assays are often used to measure dissociation constants that quantify the binding affinity between small molecules and proteins. In an upper-division undergraduate laboratory course, where students work on projects using a guided inquiry-based approach, a binding titration experiment at physiological pH is performed to…

The growth of continents and some consequences since 1.5 Ga

NASA Technical Reports Server (NTRS)

Howell, David G.

1988-01-01

The budget of Earth's oceanic sediment masses was discussed in terms of crustal growth and recycling. Based on estimates of the volume of oceanic sediments and the average age of oceanic crust, a continental denudation rate of 1.65 cu km/yr was computed. This crudely balances estimated crustal production rates of about 1 cu km/yr, but the efficiency of sediment loss via subduction, for example, must be considered. It was argued, on the basis of earthquake focal solutions, imagery of subduction zones, and plate kinematic reconstructions that little, if any, sediment was lost in this way. This yields a present day crustal growth rate of about 1 cu km/yr. The volume of continents to 1.5 Ga ago was discussed, assuming constant continental thickness and freeboard, and a constant hydrosphere volume. It was concluded that ocean ridge length was a factor of about 1.75 greater 1.5 Ga ago, but a major uncertainty is the average spreading rate in the past.

Thermodynamic stability of carbonic anhydrase: measurements of binding affinity and stoichiometry using ThermoFluor.

PubMed

Matulis, Daumantas; Kranz, James K; Salemme, F Raymond; Todd, Matthew J

2005-04-05

ThermoFluor (a miniaturized high-throughput protein stability assay) was used to analyze the linkage between protein thermal stability and ligand binding. Equilibrium binding ligands increase protein thermal stability by an amount proportional to the concentration and affinity of the ligand. Binding constants (K(b)) were measured by examining the systematic effect of ligand concentration on protein stability. The precise ligand effects depend on the thermodynamics of protein stability: in particular, the unfolding enthalpy. An extension of current theoretical treatments was developed for tight binding inhibitors, where ligand effect on T(m) can also reveal binding stoichiometry. A thermodynamic analysis of carbonic anhydrase by differential scanning calorimetry (DSC) enabled a dissection of the Gibbs free energy of stability into enthalpic and entropic components. Under certain conditions, thermal stability increased by over 30 degrees C; the heat capacity of protein unfolding was estimated from the dependence of calorimetric enthalpy on T(m). The binding affinity of six sulfonamide inhibitors to two isozymes (human type 1 and bovine type 2) was analyzed by both ThermoFluor and isothermal titration calorimetry (ITC), resulting in a good correlation in the rank ordering of ligand affinity. This combined investigation by ThermoFluor, ITC, and DSC provides a detailed picture of the linkage between ligand binding and protein stability. The systematic effect of ligands on stability is shown to be a general tool to measure affinity.

Manipulation of a DNA aptamer-protein binding site through arylation of internal guanine residues.

PubMed

Van Riesen, Abigail J; Fadock, Kaila L; Deore, Prashant S; Desoky, Ahmed; Manderville, Richard A; Sowlati-Hashjin, Shahin; Wetmore, Stacey D

2018-05-23

Chemically modified aptamers have the opportunity to increase aptamer target binding affinity and provide structure-activity relationships to enhance our understanding of molecular target recognition by the aptamer fold. In the current study, 8-aryl-2'-deoxyguanosine nucleobases have been inserted into the G-tetrad and central TGT loop of the thrombin binding aptamer (TBA) to determine their impact on antiparallel G-quadruplex (GQ) folding and thrombin binding affinity. The aryl groups attached to the dG nucleobase vary greatly in aryl ring size and impact on GQ stability (∼20 °C change in GQ thermal melting (Tm) values) and thrombin binding affinity (17-fold variation in dissociation constant (Kd)). At G8 of the central TGT loop that is distal from the aptamer recognition site, the probes producing the most stable GQ structure exhibited the strongest thrombin binding affinity. However, within the G-tetrad, changes to the electron density of the dG component within the modified nucleobase can diminish thrombin binding affinity. Detailed molecular dynamics (MD) simulations on the modified TBA (mTBA) and mTBA-protein complexes demonstrate how the internal 8-aryl-dG modification can manipulate the interactions between the DNA nucleobases and the amino acid residues of thrombin. These results highlight the potential of internal fluorescent nuclobase analogs (FBAs) to broaden design options for aptasensor development.

Characterization of Gly-D-Phe, Gly-L-Leu, and D-Phe as affinity ligands to thermolysin.

PubMed

Yasukawa, Kiyoshi; Kusano, Masayuki; Nakamura, Koji; Inouye, Kuniyo

2006-04-01

In this study, glycyl-D-phenylalanine (Gly-D-Phe), glycyl-L-leucine (Gly-L-Leu), and D-phenylalanine (D-Phe) were characterized for their abilities as affinity ligands to thermolysin. Each of the ligands was immobilized to the resin. The optimum pH for adsorption of thermolysin is 5.0-6.0 for each of the ligands. By the affinity column chromatography in which 2mg thermolysin was applied onto 4 ml volume of the resins at pH 5.5, the adsorption ratios based on casein hydrolysis activity were 100% for each of the ligands. However, the adsorption ratios of the resins containing Gly-L-Leu and D-Phe, unlike that of Gly-D-Phe, were progressively decreased with increasing the amounts of thermolysin applied to the column. Measurement of adsorption isotherms showed that the association constant to thermolysin at pH 5.5 of the resins containing Gly-D-Phe was (3.3+/-0.8)x10(5)M(-1), while those of Gly-L-Leu and D-Phe were approximately ten times less. This result is coincident with the observations of performances in affinity column chromatography. On the other hand, maximum thermolysin binding capacities were almost the same among the resins examined. These results indicate that Gly-D-Phe is more suitable than Gly-L-Leu and D-Phe as an affinity ligand for purification of thermolysin.

A cryptic Gondwana-forming orogen located in Antarctica.

PubMed

Daczko, Nathan R; Halpin, Jacqueline A; Fitzsimons, Ian C W; Whittaker, Joanne M

2018-05-30

The most poorly exposed and least understood Gondwana-forming orogen lies largely hidden beneath ice in East Antarctica. Called the Kuunga orogen, its interpolation between scattered outcrops is speculative with differing and often contradictory trends proposed, and no consensus on the location of any sutures. While some discount a suture altogether, paleomagnetic data from Indo-Antarctica and Australo-Antarctica do require 3000-5000 km relative displacement during Ediacaran-Cambrian Gondwana amalgamation, suggesting that the Kuunga orogen sutured provinces of broadly Indian versus Australian affinity. Here we use compiled data from detrital zircons offshore of East Antarctica that fingerprint two coastal subglacial basement provinces between 60 and 130°E, one of Indian affinity with dominant ca. 980-900 Ma ages (Indo-Antarctica) and one of Australian affinity with dominant ca. 1190-1140 and ca. 1560 Ma ages (Australo-Antarctica). We combine this offshore compilation with existing and new onshore U-Pb geochronology and previous geophysical interpretations to delimit the Indo-Australo-Antarctic boundary at a prominent geophysical lineament which intersects the coast east of Mirny at ~94°E.

Isolation and characterization of a dual function protein from Allium sativum bulbs which exhibits proteolytic and hemagglutinating activities.

PubMed

Parisi, Mónica G; Moreno, Silvia; Fernández, Graciela

2008-04-01

A dual function protein was isolated from Allium sativum bulbs and was characterized. The protein had a molecular mass of 25-26 kDa under non-reducing conditions, whereas two polypeptide chains of 12.5+/-0.5 kDa were observed under reducing conditions. E-64 and leupeptin inhibited the proteolytic activity of the protein, which exhibited characteristics similar to cysteine peptidase. The enzyme exhibited substrate specificity and hydrolyzed natural substrates such as alpha-casein (K(m): 23.0 microM), azocasein, haemoglobin and gelatin. It also showed a high affinity for synthetic peptides such as Cbz-Ala-Arg-Arg-OMe-beta-Nam (K(m): 55.24 microM, k(cat): 0.92 s(-1)). The cysteine peptidase activity showed a remarkable stability after incubation at moderate temperatures (40-50 degrees C) over a pH range of 5.5-6.5. The N-terminus of the protein displayed a 100% sequence similarity to the sequences of a mannose-binding lectin isolated from garlic bulbs. Moreover, the purified protein was retained in the chromatographic column when Con-A Sepharose affinity chromatography was performed and the protein was able to agglutinate trypsin-treated rabbit red cells. Therefore, our results indicate the presence of an additional cysteine peptidase activity on a lectin previously described.

Residues in the H+ Translocation Site Define the pKa for Sugar Binding to LacY†

PubMed Central

Smirnova, Irina; Kasho, Vladimir; Sugihara, Junichi; Choe, Jun-Yong; Kaback, H. Ronald

2009-01-01

A remarkably high pKa of approximately 10.5 has been determined for sugar-binding affinity to the lactose permease of Escherichia coli (LacY), indicating that, under physiological conditions, substrate binds to fully protonated LacY. We have now systematically tested site-directed replacements for the residues involved in sugar binding, as well as H+ translocation and coupling, in order to determine which residues may be responsible for this alkaline pKa. Mutations in the sugar-binding site (Glu126, Trp151, Glu269) markedly decrease affinity for sugar but do not alter the pKa for binding. In contrast, replacements for residues involved in H+ translocation (Arg302, Tyr236, His322, Asp240, Glu325, Lys319) exhibit pKa values for sugar binding that are either shifted toward neutral pH or independent of pH. Values for the apparent dissociation constant for sugar binding (Kdapp) increase greatly for all mutants except neutral replacements for Glu325 or Lys319, which are characterized by remarkably high affinity sugar binding (i.e., low Kdapp) from pH 5.5 to pH 11. The pH dependence of the on- and off-rate constants for sugar binding measured directly by stopped-flow fluorometry implicates koff as a major factor for the affinity change at alkaline pH and confirms the effects of pH on Kdapp inferred from steady-state fluorometry. These results indicate that the high pKa for sugar binding by wild-type LacY cannot be ascribed to any single amino acid residue but appears to reside within a complex of residues involved in H+ translocation. There is structural evidence for water bound in this complex, and the water could be the site of protonation responsible for the pH dependence of sugar binding. PMID:19689129

Functional characterization of the dimerization domain of the ferric uptake regulator (Fur) of Pseudomonas aeruginosa

PubMed Central

Bai, Erdeni; Rosell, Federico I.; Lige, Bao; Mauk, Marcia R.; Lelj-Garolla, Barbara; Moore, Geoffrey R.; Mauk, A. Grant

2006-01-01

The functional properties of the recombinant C-terminal dimerization domain of the Pseudomonas aeruginosa Fur (ferric uptake regulator) protein expressed in and purified from Escherichia coli have been evaluated. Sedimentation velocity measurements demonstrate that this domain is dimeric, and the UV CD spectrum is consistent with a secondary structure similar to that observed for the corresponding region of the crystallographically characterized wild-type protein. The thermal stability of the domain as determined by CD spectroscopy decreases significantly as pH is increased and increases significantly as metal ions are added. Potentiometric titrations (pH 6.5) establish that the domain possesses a high-affinity and a low-affinity binding site for metal ions. The high-affinity (sensory) binding site demonstrates association constants (KA) of 10(±7)×106, 5.7(±3)×106, 2.0(±2)×106 and 2.0(±3)×104 M−1 for Ni2+, Zn2+, Co2+ and Mn2+ respectively, while the low-affinity (structural) site exhibits association constants of 1.3(±2)×106, 3.2(±2)×104, 1.76(±1)×105 and 1.5(±2)×103 M−1 respectively for the same metal ions (pH 6.5, 300 mM NaCl, 25 °C). The stability of metal ion binding to the sensory site follows the Irving–Williams order, while metal ion binding to the partial sensory site present in the domain does not. Fluorescence experiments indicate that the quenching resulting from binding of Co2+ is reversed by subsequent titration with Zn2+. We conclude that the domain is a reasonable model for many properties of the full-length protein and is amenable to some analyses that the limited solubility of the full-length protein prevents. PMID:16928194

A bambusuril macrocycle that binds anions in water with high affinity and selectivity.

PubMed

Yawer, Mirza Arfan; Havel, Vaclav; Sindelar, Vladimir

2015-01-02

Synthetic receptors that function in water are important for the qualitative and quantitative detection of anions, which may act as pollutants in the environment or play important roles in biological processes. Neutral receptors are particularly appealing because they are often more selective than positively charged receptors; however, their affinity towards anions in pure water is only in range of 1-10(3)  L mol(-1) . The anion-templated synthesis of a water-soluble bambusuril derivative is shown to be an outstanding receptor for various inorganic anions in pure water, with association constants of up to 10(7)  L mol(-1) . Furthermore, the macrocycle discriminates between anions with unprecedented selectivity (up to 500 000-fold). We anticipate that the combination of remarkable affinity and selectivity of this macrocycle will enable the efficient detection and isolation of diverse anions in aqueous solutions, which is not possible with current supramolecular systems. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Insensitivity of cerebral oxygen transport to oxygen affinity of hemoglobin-based oxygen carriers.

PubMed

Koehler, Raymond C; Fronticelli, Clara; Bucci, Enrico

2008-10-01

The cerebrovascular effects of exchange transfusion of various cell-free hemoglobins that possess different oxygen affinities are reviewed. Reducing hematocrit by transfusion of a non-oxygen-carrying solution dilates pial arterioles on the brain surface and increases cerebral blood flow to maintain a constant bulk oxygen transport to the brain. In contrast, transfusion of hemoglobins with P50 of 4-34 Torr causes constriction of pial arterioles that offsets the decrease in blood viscosity to maintain cerebral blood flow and oxygen transport. The autoregulatory constriction is dependent on synthesis of 20-HETE from arachidonic acid. This oxygen-dependent reaction is apparently enhanced by facilitated oxygen diffusion from the red cell to the endothelium arising from increased plasma oxygen solubility in the presence of low or high-affinity hemoglobin. Exchange transfusion of recombinant hemoglobin polymers with P50 of 3 and 18 Torr reduces infarct volume from experimental stroke. Cell-free hemoglobins do not require a P50 as high as red blood cell hemoglobin to facilitate oxygen delivery.

A mechanism for the activation of the Na/H exchanger NHE-1 by cytoplasmic acidification and mitogens

PubMed Central

Lacroix, Jérôme; Poët, Mallorie; Maehrel, Céline; Counillon, Laurent

2004-01-01

Eukaryotic cells constantly have to fight against internal acidification. In mammals, this task is mainly performed by the ubiquitously expressed electroneutral Na+/H+ exchanger NHE-1, which activates in a cooperative manner when cells become acidic. Despite its biological importance, the mechanism of this activation is still poorly understood, the most commonly accepted hypothesis being the existence of a proton-sensor site on the internal face of the transporter. This work uncovers mutations that lead to a nonallosteric form of the exchanger and demonstrates that NHE-1 activation is best described by a Monod–Wyman–Changeux concerted mechanism for a dimeric transporter. During intracellular acidification, a low-affinity form of NHE-1 is converted into a form possessing a higher affinity for intracellular protons, with no requirement for an additional proton-sensor site on the protein. This new mechanism also explains the activation of the exchanger by growth signals, which shift the equilibrium towards the high-affinity form. PMID:14710192

Application of molecularly imprinted polymers to selective removal of clofibric acid from water.

PubMed

Dai, Chaomeng; Zhang, Juan; Zhang, Yalei; Zhou, Xuefei; Liu, Shuguang

2013-01-01

A new molecularly imprinted polymer (MIP) adsorbent for clofibric acid (CA) was prepared by a non-covalent protocol. Characterization of the obtained MIP was achieved by scanning electron microscopy (SEM) and nitrogen sorption. Sorption experimental results showed that the MIP had excellent binding affinity for CA and the adsorption of CA by MIP was well described by pseudo-second-order model. Scatchard plot analysis revealed that two classes of binding sites were formed in the MIP with dissociation constants of 7.52 ± 0.46 mg L(-1) and 114 ± 4.2 mg L(-1), respectively. The selectivity of MIP demonstrated higher affinity for CA over competitive compound than that of non-imprinted polymers (NIP). The MIP synthesized was used to remove CA from spiked surface water and exhibited significant binding affinity towards CA in the presence of total dissolved solids (TDS). In addition, MIP reusability was demonstrated for at least 12 repeated cycles without significant loss in performance.

Application of Molecularly Imprinted Polymers to Selective Removal of Clofibric Acid from Water

PubMed Central

Dai, Chaomeng; Zhang, Juan; Zhang, Yalei; Zhou, Xuefei; Liu, Shuguang

2013-01-01

A new molecularly imprinted polymer (MIP) adsorbent for clofibric acid (CA) was prepared by a non-covalent protocol. Characterization of the obtained MIP was achieved by scanning electron microscopy (SEM) and nitrogen sorption. Sorption experimental results showed that the MIP had excellent binding affinity for CA and the adsorption of CA by MIP was well described by pseudo-second-order model. Scatchard plot analysis revealed that two classes of binding sites were formed in the MIP with dissociation constants of 7.52±0.46 mg L−1 and 114±4.2 mg L−1, respectively. The selectivity of MIP demonstrated higher affinity for CA over competitive compound than that of non-imprinted polymers (NIP). The MIP synthesized was used to remove CA from spiked surface water and exhibited significant binding affinity towards CA in the presence of total dissolved solids (TDS). In addition, MIP reusability was demonstrated for at least 12 repeated cycles without significant loss in performance. PMID:24205143

New potentiometric sensor based on molecularly imprinted nanoparticles for cocaine detection.

PubMed

Smolinska-Kempisty, K; Ahmad, O Sheej; Guerreiro, A; Karim, K; Piletska, E; Piletsky, S

2017-10-15

Here we present a potentiometric sensor for cocaine detection based on molecularly imprinted polymer nanoparticles (nanoMIPs) produced by the solid-phase imprinting method. The composition of polymers with high affinity for cocaine was optimised using molecular modelling. Four compositions were selected and polymers prepared using two protocols: chemical polymerisation in water and UV-initiated polymerisation in organic solvent. All synthesised nanoparticles had very good affinity to cocaine with dissociation constants between 0.6nM and 5.3nM. Imprinted polymers produced in organic solvent using acrylamide as a functional monomer demonstrated the highest yield and affinity, and so were selected for further sensor development. For this, nanoparticles were incorporated within a PVC matrix which was then used to prepare an ion-selective membrane integrated with a potentiometric transducer. It was demonstrated that the sensor was able to quantify cocaine in blood serum samples in the range of concentrations between 1nM and 1mM. Copyright © 2017 Elsevier B.V. All rights reserved.

An integrated catch-and-hold mechanism activates nicotinic acetylcholine receptors.

PubMed

Jadey, Snehal; Auerbach, Anthony

2012-07-01

In neuromuscular acetylcholine (ACh) receptor channels (AChRs), agonist molecules bind with a low affinity (LA) to two sites that can switch to high affinity (HA) and increase the probability of channel opening. We measured (by using single-channel kinetic analysis) the rate and equilibrium constants for LA binding and channel gating for several different agonists of adult-type mouse AChRs. Almost all of the variation in the equilibrium constants for LA binding was from differences in the association rate constants. These were consistently below the limit set by diffusion and were substantially different even though the agonists had similar sizes and the same charge. This suggests that binding to resting receptors is not by diffusion alone and, hence, that each binding site can undergo two conformational changes ("catch" and "hold") that connect three different structures (apo-, LA-bound, and HA-bound). Analyses of ACh-binding protein structures suggest that this binding site, too, may adopt three discrete structures having different degrees of loop C displacement ("capping"). For the agonists we tested, the logarithms of the equilibrium constants for LA binding and LA↔HA gating were correlated. Although agonist binding and channel gating have long been considered to be separate processes in the activation of ligand-gated ion channels, this correlation implies that the catch-and-hold conformational changes are energetically linked and together comprise an integrated process having a common structural basis. We propose that loop C capping mainly reflects agonist binding, with its two stages corresponding to the formation of the LA and HA complexes. The catch-and-hold reaction coordinate is discussed in terms of preopening states and thermodynamic cycles of activation.

An integrated catch-and-hold mechanism activates nicotinic acetylcholine receptors

PubMed Central

Jadey, Snehal

2012-01-01

In neuromuscular acetylcholine (ACh) receptor channels (AChRs), agonist molecules bind with a low affinity (LA) to two sites that can switch to high affinity (HA) and increase the probability of channel opening. We measured (by using single-channel kinetic analysis) the rate and equilibrium constants for LA binding and channel gating for several different agonists of adult-type mouse AChRs. Almost all of the variation in the equilibrium constants for LA binding was from differences in the association rate constants. These were consistently below the limit set by diffusion and were substantially different even though the agonists had similar sizes and the same charge. This suggests that binding to resting receptors is not by diffusion alone and, hence, that each binding site can undergo two conformational changes (“catch” and “hold”) that connect three different structures (apo-, LA-bound, and HA-bound). Analyses of ACh-binding protein structures suggest that this binding site, too, may adopt three discrete structures having different degrees of loop C displacement (“capping”). For the agonists we tested, the logarithms of the equilibrium constants for LA binding and LA↔HA gating were correlated. Although agonist binding and channel gating have long been considered to be separate processes in the activation of ligand-gated ion channels, this correlation implies that the catch-and-hold conformational changes are energetically linked and together comprise an integrated process having a common structural basis. We propose that loop C capping mainly reflects agonist binding, with its two stages corresponding to the formation of the LA and HA complexes. The catch-and-hold reaction coordinate is discussed in terms of preopening states and thermodynamic cycles of activation. PMID:22732309

Frequency-constant Q, unity and disorder

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hargreaves, N.D.

1995-12-31

In exploration geophysics we obtain information about the earth by observing its response to different types of applied force. The response can cover the full range of possible Q values (where Q, the quality factor, is a measure of energy dissipation), from close to infinity in the case of deep crustal seismic to close to 0 in the case of many electromagnetic methods. When Q is frequency-constant, however, the various types of response have a common scaling behavior and can be described as being self-affine. The wave-equation then takes on a generalised form, changing from the standard wave-equation at Qmore » = {infinity} to the diffusion equation at Q = 0, via lossy, diffusive, propagation at intermediate Q values. Solutions of this wave-diffusion equation at any particular Q value can be converted to an equivalent set of results for any other Q value. In particular it is possible to convert from diffusive to wave propagation by a mapping from Q < {infinity} to Q = {infinity}. In the context of seismic sounding this is equivalent to applying inverse Q-filtering; in a more general context the mapping integrates different geophysical observations by referencing them to the common result at Q = {infinity}. The self-affinity of the observations for frequency-constant Q is an expression of scale invariance in the fundamental physical properties of the medium of propagation, this being the case whether the mechanism of diffusive propagation is scattering of intrinsic attenuation. Scale invariance, or fractal scaling, is a general property of disordered systems; the assumption of frequency-constant Q not only implies a unity between different geophysical observations, but also suggests that it is the disordered nature of the earth`s sub-surface that is the unifying factor.« less

‘And then there were three’: highly efficient uptake of potassium by foliar trichomes of epiphytic bromeliads

PubMed Central

Winkler, Uwe; Zotz, Gerhard

2010-01-01

Background and Aims Vascular epiphytes have to acquire nutrients from atmospheric wash out, stem-flow, canopy soils and trapped litter. Physiological studies on the adaptations to nutrient acquisition and plant utilization of nutrients have focused on phosphorus and nitrogen; potassium, as a third highly abundant nutrient element, has received minor attention. In the present study, potassium uptake kinetics by leaves, within-plant distribution and nutrient accumulation were analysed to gain an improved understanding of physiological adaptations to non-terrestrial nutrient supply of plants. Methods Radioactively labelled 86RbCl was used as an analogue to study uptake kinetics of potassium absorbed from tanks of epiphytes, its plant distribution and the correlation between uptake efficiency and abundance of trichomes, functioning as uptake organs of leaves. Potassium in leaves was additionally analysed by atomic absorption spectroscopy to assess plant responses to potassium deficiency. Key Results Labelled rubidium was taken up from tanks over a wide range of concentrations, 0·01–90 mm, which was achieved by two uptake systems. In four tank epiphytes, the high-affinity transporters had average Km values of 41·2 µm, and the low-affinity transporters average Km values of 44·8 mm. Further analysis in Vriesea splenriet showed that high-affinity uptake of rubidium was an ATP-dependent process, while low-affinity uptake was mediated by a K+-channel. The kinetic properties of both types of transporters are comparable with those of potassium transporters in roots of terrestrial plants. Specific differences in uptake velocities of epiphytes are correlated with the abundance of trichomes on their leaf surfaces. The main sinks for potassium were fully grown leaves. These leaves thus function as internal potassium sources, which allow growth to be maintained during periods of low external potassium availability. Conclusions Vascular epiphytes possess effective mechanisms to take up potassium from both highly diluted and highly concentrated solutions, enabling the plant to incorporate this nutrient element quickly and almost quantitatively from tank solutions. A surplus not needed for current metabolism is stored, i.e. plants show luxury consumption. PMID:20542886

Troposphere-stratosphere (surface-55 km) monthly winter general circulation statistics for the Northern Hemisphere Interannual variations

NASA Technical Reports Server (NTRS)

Geller, M. A.; Wu, M.-F.; Gelman, M. E.

1984-01-01

Individual monthly mean general circulation statistics for the Northern Hemisphere winters of 1978-79, 1979-80, 1980-81, and 1981-82 are examined for the altitude region from the earth's surface to 55 km. Substantial interannual variability is found in the mean zonal geostrophic wind; planetary waves with zonal wavenumber one and two; the heat and momentum fluxes; and the divergence of the Eliassen-Palm flux. These results are compared with previous studies by other workers. This variability in the monthly means is examined further by looking at both time-latitude sections at constant pressure levels and time-height sections at constant latitudes. The implications of this interannual variability for verifying models and interpreting observations are discussed.

Invertase immobilization onto radiation-induced graft copolymerized polyethylene pellets

NASA Astrophysics Data System (ADS)

de Queiroz, Alvaro Antonio Alencar; Vitolo, Michele; de Oliveira, Rômulo Cesar; Higa, Olga Zazuco

1996-06-01

The graft copolymer poly(ethylene-g-acrylic acid) (LDPE-g-AA) was prepared by radiation-induced graft copolymerization of acrylic acid onto low density polyethylene (LDPE) pellets, and characterized by infrared photoacoustic spectroscopy and scanning electron microscopy (SEM). The presence of the grafted poly(acrylic acid) (PAA) was established. Invertase was immobilized onto the graft polymer and the thermodynamic parameters of the soluble and immobilized enzyme were determined. The Michaelis constant, Km, and the maximum reaction velocity, Vmax, were determined for the free and the immobilized invertase. The Michaelis constant, Km was larger for the immobilized invertase than for the free enzyme, whereas Vmax was smaller for the immobilized invertase. The thermal stability of the immobilized invertase was higher than that of the free enzyme.

Anti-epidermal growth factor receptor conjugated mesoporous zinc oxide nanofibers for breast cancer diagnostics

NASA Astrophysics Data System (ADS)

Ali, Md. Azahar; Mondal, Kunal; Singh, Chandan; Dhar Malhotra, Bansi; Sharma, Ashutosh

2015-04-01

We report the fabrication of an efficient, label-free, selective and highly reproducible immunosensor with unprecedented sensitivity (femto-molar) to detect a breast cancer biomarker for early diagnostics. Mesoporous zinc oxide nanofibers (ZnOnFs) are synthesized by electrospinning technique with a fiber diameter in the range of 50-150 nm. Fragments of ZnOnFs are electrophoretically deposited on an indium tin oxide glass substrate and conjugated via covalent or electrostatic interactions with a biomarker (anti-ErbB2; epidermal growth factor receptor 2). Oxygen plasma treatment of the carbon doped ZnOnFs generates functional groups (-COOH, -OH, etc.) that are effective for the conjugation of anti-ErbB2. ZnOnFs without plasma treatment that conjugate via electrostatic interactions were also tested for comparison. Label-free detection of the breast cancer biomarker by this point-of-care device is achieved by an electrochemical impedance technique that has high sensitivity (7.76 kΩ μM-1) and can detect 1 fM (4.34 × 10-5 ng mL-1) concentration. The excellent impedimetric response of this immunosensor provides a fast detection (128 s) in a wide detection test range (1.0 fM-0.5 μM). The oxy-plasma treated ZnOnF immunoelectrode shows a higher association constant (404.8 kM-1 s-1) indicating a higher affinity towards the ErbB2 antigen compared to the untreated ZnOnF immunoelectrode (165.6 kM-1 s-1). This sensor is about an order of magnitude more sensitive than the best demonstrated in the literature based on different nanomaterials and about three orders of magnitude better than the ELISA standard for breast cancer biomarker detection. This proposed point-of-care cancer diagnostic offers several advantages, such as higher stability, rapid monitoring, simplicity, cost-effectiveness, etc., and should prove to be useful for the detection of other bio- and cancer markers.We report the fabrication of an efficient, label-free, selective and highly reproducible immunosensor with unprecedented sensitivity (femto-molar) to detect a breast cancer biomarker for early diagnostics. Mesoporous zinc oxide nanofibers (ZnOnFs) are synthesized by electrospinning technique with a fiber diameter in the range of 50-150 nm. Fragments of ZnOnFs are electrophoretically deposited on an indium tin oxide glass substrate and conjugated via covalent or electrostatic interactions with a biomarker (anti-ErbB2; epidermal growth factor receptor 2). Oxygen plasma treatment of the carbon doped ZnOnFs generates functional groups (-COOH, -OH, etc.) that are effective for the conjugation of anti-ErbB2. ZnOnFs without plasma treatment that conjugate via electrostatic interactions were also tested for comparison. Label-free detection of the breast cancer biomarker by this point-of-care device is achieved by an electrochemical impedance technique that has high sensitivity (7.76 kΩ μM-1) and can detect 1 fM (4.34 × 10-5 ng mL-1) concentration. The excellent impedimetric response of this immunosensor provides a fast detection (128 s) in a wide detection test range (1.0 fM-0.5 μM). The oxy-plasma treated ZnOnF immunoelectrode shows a higher association constant (404.8 kM-1 s-1) indicating a higher affinity towards the ErbB2 antigen compared to the untreated ZnOnF immunoelectrode (165.6 kM-1 s-1). This sensor is about an order of magnitude more sensitive than the best demonstrated in the literature based on different nanomaterials and about three orders of magnitude better than the ELISA standard for breast cancer biomarker detection. This proposed point-of-care cancer diagnostic offers several advantages, such as higher stability, rapid monitoring, simplicity, cost-effectiveness, etc., and should prove to be useful for the detection of other bio- and cancer markers. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr00194c

Acoustic monitoring of the tide height and slope-water intrusion at the New Jersey Shelf in winter conditions.

PubMed

Turgut, Altan; Orr, Marshall; Pasewark, Bruce

2007-05-01

Waveguide invariant theory is used to describe the frequency shifts of constant acoustic intensity level curves in broadband signal spectrograms measured at the New Jersey Shelf during the winter of 2003. The broadband signals (270-330 Hz) were transmitted from a fixed source and received at three fixed receivers, located at 10, 20, and 30 km range along a cross-shelf propagation track. The constant acoustic intensity level curves of the received signals indicate regular frequency shifts that can be well predicted by the change in water depth observed through tens of tidal cycles. A second pattern of frequency shifts is observed at only 30 km range where significant variability of slope-water intrusion was measured. An excellent agreement between observed frequency shifts of the constant acoustic intensity levels and those predicted by the change in tide height and slope water elevations suggests the capability of long-term acoustic monitoring of tide and slope water intrusions in winter conditions.

The alpha3(betaMet222Ser/Tyr345Trp)3gamma subcomplex of the TF1-ATPase does not hydolyze ATP at a significant rate until the substrate binds to the catalytic site of the lowest affinity.

PubMed

Ren, Huimiao; Bandyopadhyay, Sanjay; Allison, William S

2006-05-16

The alpha(3)(betaM(222)S/Y(345)W)(3)gamma double-mutant subcomplex of the F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)), free of endogenous nucleotides, does not entrap inhibitory MgADP in a catalytic site during turnover. It hydrolyzes 100 nM-2 mM ATP with a K(m) of 31 microM and a k(cat) of 220 s(-)(1). Fluorescence titrations of the introduced tryptophans with MgADP or MgATP revealed that both Mg-nucleotide complexes bind to the catalytic site of the highest affinity with K(d)()1 values of less than 1 nM and bind to the site of intermediate affinity with a common K(d)2 value of about 12 nM. The K(d)3 values obtained for the catalytic site of the lowest affinity from titrations with MgADP and MgATP are 25 and 37 microM, respectively. The double mutant hydrolyzes 200 nM ATP with a first-order rate of 1.5 s(-)(1), which is 0.7% of k(cat). Hence, it does not hydrolyze ATP at a significant rate when the catalytic site of intermediate affinity is saturated and the catalytic site of the lowest affinity is minimally occupied. After the addition of stoichiometric MgATP to the alpha(3)(betaM(222)S/Y(345)W)(3)gamma subcomplex, one-third of the tryptophan fluorescence remains quenched after 10 min. The product [(3)H]ADP remains bound when the wild-type and double-mutant subcomplexes hydrolyze substoichiometric [(3)H]ATP. In contrast, (32)P(i) is not retained when the wild-type subcomplex hydrolyzes substoichiometric [gamma-(32)P]ATP. This precludes assessment of the equilibrium at the high-affinity catalytic site when the wild-type TF(1) subcomplex hydrolyzes substoichiometric ATP.

An HLA-B27 Homodimer Specific Antibody Recognizes a Discontinuous Mixed-Disulfide Epitope as Identified by Affinity-Mass Spectrometry

NASA Astrophysics Data System (ADS)

Iuraşcu, Marius-Ionuţ; Marroquin Belaunzanar, Osiris; Cozma, Claudia; Petrausch, Ulf; Renner, Christoph; Przybylski, Michael

2016-06-01

HLA-B27 homodimer formation is believed to be a hallmark of HLA-B27 associated spondyloarthritides. Recently, we have generated a homodimer-specific monoclonal antibody (HD6) and have demonstrated that HLA-B27 homodimer complexes are present on monocytes of healthy HLA-B27 gene carriers at low levels, with significantly increased levels at active disease. The capability of the HD6 antibody to discriminate between correctly formed HLA-B27 heterotrimers and pathology-associated homodimers is striking and cannot be explained by the primary structure of HLA-B27. We hypothesized that HD6 accesses a unique epitope and used affinity-mass spectrometry for its identification. The HD6 antibody was immobilized on an activated sepharose affinity column, and HLA-B27 homodimer characterized for affinity. The epitope was identified by proteolytic epitope excision and MALDI mass spectrometry, and shown to comprise a discontinuous Cys-203- 257-Cys mixed-disulfide peptide structure that is not accessible in HLA-B27 heterotrimers due to protection by noncovalently linked β2-microglobulin. The epitope peptides were synthesized by solid phase peptide synthesis, and the two monomeric peptide components, HLA-B27(203-219) and HLA-B27(257-273), as well as the homo- and hetero-dimeric disulfide linked combinations prepared. The affinity binding constants KD towards the antibodies were determined using a surface acoustic wave (SAW) biosensor, and showed the highest affinity with a KD of approximately 40 nM to the HD6 antibody for the (203-219)-SS-(257-273) mixed disulfide epitope.

Two classes of binding sites for [3H]substance P in rat cerebral cortex.

PubMed

Geraghty, D P; Burcher, E

1993-01-22

The binding characteristics of [3H]substance P ([3H]SP) were investigated in membranes prepared from rat cerebral cortex. Binding of [3H]SP reached equilibrium after 50 min at 25 degrees C and was saturable at 8 nM. Saturation data could be resolved into high affinity (equilibrium dissociation constant, Kd, 0.22 nM) and low affinity sites (Kd, 2.65 nM). The low affinity sites were more numerous than the high affinity sites, with a ratio of 4:1. The non-hydrolyzable GTP analogue GppNHp had no effect on binding, indicating that the high and low affinity sites are not guanine nucleotide-regulated states of the same (NK-1) receptor. The low affinity sites are unlikely to represent NK-3 receptors since coincubation with the selective NK-3 receptor agonist senktide did not alter the biphasic nature of [3H]SP binding. The rank order of potency for inhibition of [3H]SP (2 nM) binding was SP > or = [Sar9, Met(O2)11]-SP > or = physalaemin > SP(3-11) > NP gamma = [Ala3]-SP > or = SP(4-11) > or = NPK > or = SP(5-11) > or = NKB approximately NKA > SP(1-9), compatible with binding to an NK-1 site. N-terminal fragments and non-amidated analogues were ineffective competitors for [3H]SP binding. However, competition data for several peptides including substance P (SP) and the NK-1 selective agonist [Sar9, Met(O2)11]-SP could be resolved into two components.(ABSTRACT TRUNCATED AT 250 WORDS)

Na and K Dependence of the Na/K Pump in Cystic Fibrosis Fibroblasts

NASA Astrophysics Data System (ADS)

Reznik, Vivian M.; Schneider, Jerry A.; Mendoza, Stanley A.

1981-11-01

The Na and K dependence of the Na/K pump was measured in skin fibroblasts from patients with cystic fibrosis and age/sex-matched controls. Under basal conditions, there was no difference between control and cystic fibrosis cells in protein per cell, intracellular Na and K content, or Na/K pump activity (measured as ouabain-sensitive 86Rb uptake). There was no difference in the Na dependence of the Na/K pump between cystic fibrosis cells and control cells. In cells from patients with cystic fibrosis, the Na/K pump had a significantly lower affinity for K (Km = 1.6 mM) when compared to normals (Km = 0.9 mM). This difference was demonstrated by using two independent experimental designs.

[Planar molecular arrangements aid the design of MHC class II binding peptides].

PubMed

Cortés, A; Coral, J; McLachlan, C; Benítez, R; Pinilla, L

2017-01-01

The coupling between peptides and MHC-II proteins in the human immune system is not well understood. This work presents an evidence-based hypothesis of a guiding intermolecular force present in every human MHC-II protein (HLA-II). Previously, we examined the spatial positions of the fully conserved residues in all HLA-II protein types. In each one, constant planar patterns were revealed. These molecular planes comprise of amino acid groups of the same chemical species (for example, Gly) distributed across the protein structure. Each amino acid plane has a unique direction and this directional element offers spatial selectivity. Constant within all planes, too, is the presence of an aromatic residue possessing electrons in movement, leading the authors to consider that the planes generate electromagnetic fields that could serve as an attractive force in a single direction. Selection and attraction between HLA-II molecules and antigen peptides would, therefore, be non-random, resulting in a coupling mechanism as effective and rapid as is clearly required in the immune response. On the basis of planar projections onto the HLA-II groove, modifications were made by substituting the key residues in the class II-associated invariant chain peptide-a peptide with a universal binding affinity-resulting in eight different modified peptides with affinities greater than that of the unmodified peptide. Accurate and reliable prediction of MHC class II-binding peptides may facilitate the design of universal vaccine-peptides with greatly enhanced binding affinities. The proposed mechanisms of selection, attraction and coupling between HLA-II and antigen peptides are explained further in the paper.

Interaction of two-dimensional magnetoexcitons

NASA Astrophysics Data System (ADS)

Dumanov, E. V.; Podlesny, I. V.; Moskalenko, S. A.; Liberman, M. A.

2017-04-01

We study interaction of the two-dimensional magnetoexcitons with in-plane wave vector k→∥ = 0 , taking into account the influence of the excited Landau levels (ELLs) and of the external electric field perpendicular to the surface of the quantum well and parallel to the external magnetic field. It is shown that the account of the ELLs gives rise to the repulsion between the spinless magnetoexcitons with k→∥ = 0 in the Fock approximation, with the interaction constant g decreasing inverse proportional to the magnetic field strength B (g (0) ∼ 1 / B) . In the presence of the perpendicular electric field the Rashba spin-orbit coupling (RSOC), Zeeman splitting (ZS) and nonparabolicity of the heavy-hole dispersion law affect the Landau quantization of the electrons and holes. They move along the new cyclotron orbits, change their Coulomb interactions and cause the interaction between 2D magnetoexcitons with k→∥ = 0 . The changes of the Coulomb interactions caused by the electrons and by the holes moving with new cyclotron orbits are characterized by some coefficients, which in the absence of the electric field turn to be unity. The differences between these coefficients of the electron-hole pairs forming the magnetoexcitons determine their affinities to the interactions. The interactions between the homogeneous, semihomogeneous and heterogeneous magnetoexcitons forming the symmetric states with the same signs of their affinities are attractive whereas in the case of different sign affinities are repulsive. In the heterogeneous asymmetric states the interactions have opposite signs in comparison with the symmetric states. In all these cases the interaction constant g have the dependence g (0) 1 /√{ B} .

Recombinant glycoprotein G analog for determination of specific immunoglobulins to herpes simplex virus type 2 by ELISA.

PubMed

Korshun, Ludmila; Vudmaska, Mariya; Moysa, Larissa; Kovtonjuk, Galina; Mikhalap, Svetlana; Ganova, Larissa; Spivak, Nikolay

2013-12-01

In order to the detection of type-specific IgG to herpes simplex virus type 2 (HSV-2) in human serum or plasma the recombinant analog of HSV-2 glycoprotein G (gG2) was created. To construct an expression vector the DNA fragment with a sequence identical to immunodominant regions of HSV-2 gG2 was cloned into modified vector pET28a containing of the glutation-S-transferase sequence (pET28-GST). Escherichia coli BL21 (DE3) were transformed with the recombinant plasmid. The target protein was expressed mainly in soluble form. Chromatographic purification of soluble GST-gG2 protein was performed taking into account the features of its primary structure that are 6His-tag and GST-tag. To determine the affinity constant of the specific IgG to GST-gG2 we used the method proposed by Friguet et al. (1985). The affinity constants were within the range of 10(7)-10(8)M(-1) proving their high-affinity. The purified recombinant HSV-2 antigen was used to design a diagnostic ELISA kit, which was evaluated with referent controls and standard panels of sera containing and/or not containing anti-HSV-2 IgG. Comparative evaluation of this kit and the commercially available "HSV-Type 2 IgG-ELISA" (NovaTec, Dietzenbach, Germany) kit was performed. There was no significant difference (P>0.05). It allows to use developed ELISA kit for clinical diagnosis of HSV-2 infection. Copyright © 2013 Elsevier B.V. All rights reserved.

Monoclonal antibodies against human angiotensinogen, their characterization and use in an angiotensinogen enzyme linked immunosorbent assay.

PubMed

Rubin, I; Lykkegaard, S; Olsen, A A; Selmer, J; Ballegaard, M

1988-01-01

Monoclonal antibodies were produced against human angiotensinogen. An enzyme linked immunosorbent assay (ELISA) was developed using a high affinity monoclonal antibody as catching antibody and a polyclonal rabbit anti human angiotensinogen antibody as detecting antibody in a "sandwich" ELISA. Linear range of the ELISA was 15-450 pmol/l of human angiotensinogen. Intra- and inter- assay variation coefficients were in the range of 2% to 8%. A correlation coefficient, r = 0.97, (n = 20), with values obtained by radioimmunoassay. This correlation coefficient, obtained by using both normal and pregnant sera, confirmed that the ELISA fulfill the requirements for clinical useful assay. Characterization of the antibodies were performed with respect to affinity constant and epitopes.

Evaluation of galectin binding by frontal affinity chromatography (FAC).

PubMed

Iwaki, Jun; Hirabayashi, Jun

2015-01-01

Frontal affinity chromatography (FAC) is a simple and versatile procedure enabling quantitative determination of diverse biological interactions in terms of dissociation constants (K d), even though these interactions are relatively weak. The method is best applied to glycans and their binding proteins, with the analytical system operating on the basis of highly reproducible isocratic elution by liquid chromatography. Its application to galectins has been successfully developed to characterize their binding specificities in detail. As a result, their minimal requirements for recognition of disaccharides, i.e., β-galactosides, as well as characteristic features of individual galectins, have been elucidated. In this chapter, we describe standard procedures to determine the K d's for interactions between a series of standard glycans and various galectins.

When the Hegemony Studies the Minority--An Israeli Jewish Researcher Studies Druze Women: Transformations of Power, Alienation, and Affinity in the Field

ERIC Educational Resources Information Center

Weiner-Levy, Naomi

2009-01-01

This study describes the positionality and power relations revealed during research on women in Israel's Druze minority, conducted by a Jewish woman in the country's hegemonic society. Although the researcher's position and power appear obvious, changes took place constantly, reflecting her unstable position as a stranger, "outsider" or…

A three-compartment model for micropollutants sorption in sludge: methodological approach and insights.

PubMed

Barret, Maialen; Patureau, Dominique; Latrille, Eric; Carrère, Hélène

2010-01-01

In sludge resulting from wastewater treatment, organic micropollutants sorb to particles and to dissolved/colloidal matter (DCM). Both interactions may influence their physical and biological fate throughout the wastewater treatment processes. To our knowledge, sludge has never been considered as a three-compartment matrix, in which micropollutants coexist in three states: freely dissolved, sorbed-to-particles and sorbed-to-DCM. A methodology is proposed to concomitantly determine equilibrium constants of sorption to particles (K(part)) and to DCM (K(DCM)). Polycyclic Aromatic Hydrocarbons (PAHs) were chosen as model compounds for the experiments. The logarithm of estimated equilibrium constants ranged from 3.1 to 4.3 and their usual correlation to PAH hydrophobicity was verified. Moreover, PAH affinities for particles and for DCM could be compared. Affinity for particles was found to be stronger, probably due to their physical and chemical characteristics. This work provided a useful tool to assess the freely dissolved, sorbed-to-particles and sorbed-to-DCM concentrations of contaminants, which are necessary to accurately predict their fate. Besides, guidelines to investigate the link between sorption and the fundamental concept of bioavailability were proposed. (c) 2009 Elsevier Ltd. All rights reserved.

Mechanism of Metal Ion Activation of the Diphtheria Toxin Repressor DtxR

NASA Astrophysics Data System (ADS)

D'Aquino, J. Alejandro; Ringe, Dagmar

2006-08-01

The diphtheria toxin repressor, DtxR, is a metal ion-activated transcriptional regulator that has been linked to the virulence of Corynebacterium diphtheriae. Structure determination has shown that there are two metal ion binding sites per repressor monomer, and site-directed mutagenesis has demonstrated that binding site 2 (primary) is essential for recognition of the target DNA repressor, leaving the role of binding site 1 (ancillary) unclear (1 - 3). Calorimetric techniques have demonstrated that while binding site 1 (ancillary) has high affinity for metal ion with a binding constant of 2 × 10-7, binding site 2 (primary) is a low affinity binding site with a binding constant of 6.3 × 10-4. These two binding sites act independently and their contribution can be easily dissected by traditional mutational analysis. Our results clearly demonstrate that binding site 1 (ancillary) is the first one to be occupied during metal ion activation, playing a critical role in stabilization of the repressor. In addition, structural data obtained for the mutants Ni-DtxR(H79A,C102D), reported here and the previously reported DtxR(H79A) (4) has allowed us to propose a mechanism of metal ion activation for DtxR.

Prediction of selectivity for enantiomeric separations of uncharged compounds by capillary electrophoresis involving dual cyclodextrin systems.

PubMed

Abushoffa, Adel M; Fillet, Marianne; Hubert, Phillipe; Crommen, Jacques

2002-03-01

The single-isomer polyanionic cyclodextrin (CD) derivative heptakis-6-sulfato-beta-cyclodextrin (HSbetaCD) has been tested as chiral additive for the enantioseparation of non-steroidal anti-inflammatory drugs, such as fenoprofen, flurbiprofen, ibuprofen and ketoprofen, in capillary electrophoresis, using a pH 2.5 phosphoric acid-triethanolamine buffer in the reversed polarity mode. In most cases, the enantiomers of these acidic compounds, present in uncharged form at that pH, were only poorly resolved with HSbetaCD alone. However, the use of HSbetaCD in combination with the neutral CD derivative, heptakis-(2,3,6-tri-O-methyl)-beta-cyclodextrin (TMbetaCD), which has a particularly high enantioselectivity towards these compounds, has led to complete enantioresolution in reasonably low migration times in most cases. Affinity constants for the enantiomers with the two cyclodextrins were determined, using linear regression in a two-step approach. Affinity constants with the charged HSbetaCD were first calculated in single systems while those with the neutral TMbetaCD were determined in dual systems. Selectivity for the enantiomeric separation of these compounds in dual CD systems could be predicted using recently developed mathematical models.

Preliminary selection and evaluation of the binding of aptamers against a Hantavirus antigen using fluorescence spectroscopy and modeling

NASA Astrophysics Data System (ADS)

Missailidis, Sotiris; de Oliveira, Renata Carvalho; Silva, Dilson; Cortez, Célia Martins; Guterres, Alexandro; Vicente, Luciana Helena Bassan; de Godoy, Daniela Tupy; Lemos, Elba

2015-12-01

In this study we have aimed to develop novel aptamers against the Hantavirus nucleoprotein N, a valid antigen already used in the Hantavirus reference laboratory of the Institute Oswaldo Cruz in Rio de Janeiro, Brazil. Such aptamers, if they are found to bind with high affinity and specificity for the selected hantavirus antigen, they could be translated into novel diagnostic assays with the ability to provide early detection for hantaviroses and their related disease syndromes. In a preliminary screening, we have managed to identify three aptamer species. We have analyzed a short and a long version of these aptamer using fluorescence spectroscopy and modelled their binding. We have identified Stern-Volmer constants for the selected aptamers, which have shown affinity for their target, with a different binding between the short and the long versions of them. Short aptamers have shown to have a higher Stern-Volmer constant and the ability to potentially bind to more than one binding site on the antigen. The information provided by the spectroscopic screening has been invaluable in allowing us to define candidates for further development into diagnostic assays.

Affinity capillary electrophoresis and fluorescence spectroscopy for studying enantioselective interactions between omeprazole enantiomer and human serum albumin.

PubMed

Xu, Yujing; Hong, Tingting; Chen, Xueping; Ji, Yibing

2017-05-01

Baseline separation of omeprazole (OME) enantiomers was achieved by affinity capillary electrophoresis (ACE), using human serum albumin (HSA) as the chiral selector. The influence of several experimental variables such as HSA concentration, the type and content of organic modifiers, applied voltage and running buffer concentration on the separation was evaluated. The binding of esomeprazole (S-omeprazole, S-OME) and its R-enantiomer (R-omeprazole, R-OME) to HSA under simulated physiological conditions was studied by ACE and fluorescence spectroscopy which was considered as a reference method. ACE studies demonstrated that the binding constants of the two enantiomers and HSA were 3.18 × 10 3 M -1 and 5.36 × 10 3 M -1 , respectively. The binding properties including the fluorescence quenching mechanisms, binding constants, binding sites and the number of binding sites were obtained by fluorescence spectroscopy. Though the ACE method could not get enough data when compared with the fluorescence spectrum method, the separation and binding studies of chiral drugs could be achieved simultaneously via this method. This study is of great significance for the investigation and clinical application of chiral drugs. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Tautomeric transformation of temozolomide, their proton affinities and chemical reactivities: A theoretical approach.

PubMed

Sang-Aroon, Wichien; Ruangpornvisuti, Vithaya; Amornkitbamrung, Vittaya

2016-05-01

The gas-phase geometry optimizations of bare, mono- and dihydrated complexes of temozolomide isomers were carried out using density functional calculation at the M06-2X/6-31+G(d,p) level of the theory. The structures and protonation energies of protonated species of temozolomide are reported. Chemical indices of all isomers and protonated species are also reported. Energies, thermodynamic quantities, rate constants and equilibrium constants of tautomeric and rotameric transformations of all isomers I1↔TZM↔HIa↔HIb↔I2↔I3 in bare and hydrated systems were obtained. Copyright © 2016 Elsevier Inc. All rights reserved.

Quartz crystal microbalance for the cardiac markers/antibodies binding kinetic measurements in the plasma samples

NASA Astrophysics Data System (ADS)

Agafonova, L. E.; Shumyantseva, V. V.; Archakov, A. I.

2014-06-01

The quartz crystal microbalance (QCM) was exploited for cardiac markers detection and kinetic studies of immunochemical reaction of cardiac troponin I (cTnI) and human heart fatty acid binding protein (H-FABP) with the corresponding monoclonal antibodies in undiluted plasma (serum) and standard solutions. The QCM technique allowed to dynamically monitor the kinetic differences in specific interactions and nonspecific sorption, without multiple labeling procedures and separation steps. The affinity binding process was characterized by the association (ka) and the dissociation (kd) kinetic constants and the equilibrium association (K) constant, all of which were obtained from experimental data.

Water miscible mono alcohols' effect on the proteolytic performance of Bacillus clausii serine alkaline protease.

PubMed

Duman, Yonca Avci; Kazan, Dilek; Denizci, Aziz Akin; Erarslan, Altan

2014-01-01

In this study, our investigations showed that the increasing concentrations of all examined mono alcohols caused a decrease in the Vm, kcat and kcat/Km values of Bacillus clausii GMBE 42 serine alkaline protease for casein hydrolysis. However, the Km value of the enzyme remained almost the same, which was an indicator of non-competitive inhibition. Whereas inhibition by methanol was partial non-competitive, inhibition by the rest of the alcohols tested was simple non-competitive. The inhibition constants (KI) were in the range of 1.32-3.10 M, and the order of the inhibitory effect was 1-propanol>2-propanol>methanol>ethanol. The ΔG(≠) and ΔG(≠)E-T values of the enzyme increased at increasing concentrations of all alcohols examined, but the ΔG(≠)ES value of the enzyme remained almost the same. The constant Km and ΔG(≠)ES values in the presence and absence of mono alcohols indicated the existence of different binding sites for mono alcohols and casein on enzyme the molecule. The kcat of the enzyme decreased linearly by increasing log P and decreasing dielectric constant (D) values, but the ΔG(≠) and ΔG(≠)E-T values of the enzyme increased by increasing log P and decreasing D values of the reaction medium containing mono alcohols.

Co-solvent effects on reaction rate and reaction equilibrium of an enzymatic peptide hydrolysis.

PubMed

Wangler, A; Canales, R; Held, C; Luong, T Q; Winter, R; Zaitsau, D H; Verevkin, S P; Sadowski, G

2018-04-25

This work presents an approach that expresses the Michaelis constant KaM and the equilibrium constant Kth of an enzymatic peptide hydrolysis based on thermodynamic activities instead of concentrations. This provides KaM and Kth values that are independent of any co-solvent. To this end, the hydrolysis reaction of N-succinyl-l-phenylalanine-p-nitroanilide catalysed by the enzyme α-chymotrypsin was studied in pure buffer and in the presence of the co-solvents dimethyl sulfoxide, trimethylamine-N-oxide, urea, and two salts. A strong influence of the co-solvents on the measured Michaelis constant (KM) and equilibrium constant (Kx) was observed, which was found to be caused by molecular interactions expressed as activity coefficients. Substrate and product activity coefficients were used to calculate the activity-based values KaM and Kth for the co-solvent free reaction. Based on these constants, the co-solvent effect on KM and Kx was predicted in almost quantitative agreement with the experimental data. The approach presented here does not only reveal the importance of understanding the thermodynamic non-ideality of reactions taking place in biological solutions and in many technological applications, it also provides a framework for interpreting and quantifying the multifaceted co-solvent effects on enzyme-catalysed reactions that are known and have been observed experimentally for a long time.

Geology of the saucer-shaped sill near Mahad, western Deccan Traps, India, and its significance to the Flood Basalt Model

NASA Astrophysics Data System (ADS)

Duraiswami, Raymond A.; Shaikh, Tahira N.

2013-07-01

An ˜22-m-thick saucer-shaped sill occurs near Mahad and is exposed as a curvilinear, miniature ridge within the Deccan Traps. The sill has variable dips (42-55°). It has a 7.1-km long axis and 5.3 km short axis (aspect ratio of 1.4) and is larger than the MV sill of the Golden Valley sill complex, South Africa and the Panton sill, Australia. The sill has distinct glassy upper and lower chilled margins with a coarse-grained highly jointed core. The samples from the margin are invariably fractured and iron stained because of deuteric alteration. The rock from the sill is plagioclase-phyric basalt. At least three thick sill-like apophyses emanate from the base of the main sill. The apophyses change direction because of bending and thinning from a horizontal concordant sheet at the top to a discordant inclined form that bends again to pass into a lower horizontal concordant sheet. We interpret such features as `nascent saucer-shaped sills' that did not inflate to form nested sills. Geochemically, the sill consists of poorly differentiated tholeiitic basalt that has a restricted geochemical range. Critical trace element ratios and primitive mantle normalised trace and REE patterns indicate that the sills have geochemical affinities to the Poladpur chemical type and that the pahoehoe flow they intrude belongs to the Bushe Formation. Calculated magmatic overpressures during sill emplacement range from 8.4 to 11.3 MPa (for Young's modulus E = 5 GPa) and 16.7 to 22.5 MPa (for E=10 GPa) and depth to magma chamber ranges from 8.5 to 11.5 km ( E = 5 GPa) and 17.1 to 22.9 km ( E = 10 GPa), consistent with petrological and gravity modelling. The volume of the Mahad sill is approximately 276 km3 and is constant irrespective of the variations in the values of host-rock Young's modulus. In 1980, Cox (J Petrol 21:629-650, 1980) proposed a conceptual model of the crust-mantle section beneath the Karoo CFB which is considered as the fundamental model for flood basalt volcanism. Our paper confirms the presence of a sill plus the inferred substructure beneath Mahad that are compatible with predictions of that model. In LIPS, saucer-shaped sills are formed in areas experiencing extensional tectonics where processes such as the Cook-Gordon delamination and Dundurs elastic extensional mismatch between layered sedimentary rocks or lava flows are responsible for the deflection of dykes into sills. A similar process is envisaged for the formation of the Mahad sill.

Purification and partial biochemical characterization of polyphenol oxidase from mango (Mangifera indica cv. Manila).

PubMed

Palma-Orozco, Gisela; Marrufo-Hernández, Norma A; Sampedro, José G; Nájera, Hugo

2014-10-08

Polyphenol oxidase (PPO) is an enzyme widely distributed in the plant kingdom that has been detected in most fruits and vegetables. PPO was extracted and purified from Manila mango (Mangifera indica), and its biochemical properties were studied. PPO was purified 216-fold by hydrophobic interaction and ion exchange chromatography. PPO was purified to homogeneity, and the estimated PPO molecular weight (MW) by SDS-PAGE was ≈31.5 kDa. However, a MW of 65 kDa was determined by gel filtration, indicating a dimeric structure for the native PPO. The isolated PPO showed the highest affinity to pyrogallol (Km = 2.77 mM) followed by 4-methylcatechol (Km = 3.14 mM) and catechol (Km = 15.14 mM). The optimum pH for activity was 6.0. PPO was stable in the temperature range of 20-70 °C. PPO activity was completely inhibited by tropolone, ascorbic acid, sodium metabisulfite, and kojic acid at 0.1 mM.

Carbon Source Preference in Chemosynthetic Hot Spring Communities

PubMed Central

Urschel, Matthew R.; Kubo, Michael D.; Hoehler, Tori M.; Peters, John W.

2015-01-01

Rates of dissolved inorganic carbon (DIC), formate, and acetate mineralization and/or assimilation were determined in 13 high-temperature (>73°C) hot springs in Yellowstone National Park (YNP), Wyoming, in order to evaluate the relative importance of these substrates in supporting microbial metabolism. While 9 of the hot spring communities exhibited rates of DIC assimilation that were greater than those of formate and acetate assimilation, 2 exhibited rates of formate and/or acetate assimilation that exceeded those of DIC assimilation. Overall rates of DIC, formate, and acetate mineralization and assimilation were positively correlated with spring pH but showed little correlation with temperature. Communities sampled from hot springs with similar geochemistries generally exhibited similar rates of substrate transformation, as well as similar community compositions, as revealed by 16S rRNA gene-tagged sequencing. Amendment of microcosms with small (micromolar) amounts of formate suppressed DIC assimilation in short-term (<45-min) incubations, despite the presence of native DIC concentrations that exceeded those of added formate by 2 to 3 orders of magnitude. The concentration of added formate required to suppress DIC assimilation was similar to the affinity constant (Km) for formate transformation, as determined by community kinetic assays. These results suggest that dominant chemoautotrophs in high-temperature communities are facultatively autotrophic or mixotrophic, are adapted to fluctuating nutrient availabilities, and are capable of taking advantage of energy-rich organic substrates when they become available. PMID:25819970

Partially reduced graphene oxide-gold nanorods composite based bioelectrode of improved sensing performance.

PubMed

Nirala, Narsingh R; Abraham, Shiju; Kumar, Vinod; Pandey, Shobhit A; Yadav, Umakant; Srivastava, Monika; Srivastava, S K; Singh, Vidya Nand; Kayastha, Arvind M; Srivastava, Anchal; Saxena, Preeti S

2015-11-01

The present work proposes partially reduced graphene oxide-gold nanorods supported by chitosan (CH-prGO-AuNRs) as a potential bioelectrode material for enhanced glucose sensing. Developed on ITO substrate by immobilizing glucose oxidase on CH-prGO-AuNRs composite, these CH-prGO-AuNRs/ITO bioelectrodes demonstrate high sensitivity of 3.2 µA/(mg/dL)/cm(2) and linear range of 25-200 mg/dL with an ability to detect as low as 14.5 mg/dL. Further, these CH-prGO-AuNRs/ITO based electrodes attest synergistiacally enhanced sensing properties when compared to simple graphene oxide based CH-GO/ITO electrode. This is evident from one order higher electron transfer rate constant (Ks) value in case of CH-prGO-AuNRs modified electrode (12.4×10(-2) cm/s), in contrast to CH-GO/ITO electrode (6×10(-3) cm/s). Additionally, very low Km value [15.4 mg/dL(0.85 mM)] ensures better binding affinity of enzyme to substrate which is desirable for good biosensor stability and resistance to environmental interferences. Hence, with better loading capacity, kinetics and stability, the proposed CH-prGO-AuNRs composite shows tremendous potential to detect several bio-analytes in the coming future. Copyright © 2015 Elsevier B.V. All rights reserved.

Lectins as endocytic ligands: an assessment of lectin binding and uptake to rabbit conjunctival epithelial cells.

PubMed

Qaddoumi, Mohamed; Lee, Vincent H L

2004-07-01

To investigate the binding and uptake pattern of three plant lectins in rabbit conjunctival epithelial cells (RCECs) with respect to their potential for enhancing cellular macromolecular uptake. Three fluorescein-labeled plant lectins (Lycoperison esculentum, TL; Solanum tuberosum, STL; and Ulex europaeus 1, UEA-1) were screened with respect to time-, concentration-, and temperature-dependent binding and uptake. Chitin (30 mg/ml) and L-alpha-fucose (10 mM) were used as inhibitory sugars to correct for nonspecific binding of TL or STL and UEA-1, respectively. Confocal microscopy was used to confirm internalization of STL. The binding and uptake of all three lectins in RCECs was time-dependent (reaching a plateau at 1-2 h period) and saturable at 1-h period. The rank order of affinity constants (km) was STL>TL>UEA-1 with values of 0.39>0.48>4.81 microM, respectively. However, maximal, specific binding/uptake potential was in the order UEA-1>STL>TL with values of 53.7, 52.3, and 15.0 nM/mg of cell protein, respectively. Lectins showed temperature dependence in their uptake, with STL exhibiting the highest endocytic capacity. Internalized STL was visualized by confocal microscopy to be localized to the cell membrane and cytoplasm. Based on favorable binding and uptake characteristics, potato lectin appears to be a useful candidate for further investigation as an ocular drug delivery system.

Cloning, expression and mutation of a triazophos hydrolase gene from Burkholderia sp. SZL-1.

PubMed

Zhang, Hao; Li, Qiang; Guo, Su-Hui; Cheng, Ming-Gen; Zhao, Meng-Jun; Hong, Qing; Huang, Xing

2016-06-01

Triazophos is a broad-spectrum and highly effective insecticide, and the residues of triazophos have been frequently detected in the environment. A triazophos-degrading bacterium, Burkholderia sp. SZL-1, was isolated from a long-term triazophos-polluted soil. Strain SZL-1 could hydrolyze triazophos to 1-phenyl-3-hydroxy-1,2,4-triazole, which was further utilized as the carbon sources for growth. The triazophos hydrolase gene trhA, cloned from strain SZL-1, was expressed and homogenously purified using Ni-nitrilotriacetic acid affinity chromatography. TrhA is 55 kDa and displays maximum activity at 25°C, pH 8.0. This enzyme still has nearly 60% activity at the range of 15°C-50°C for 30 min. TrhA was mutated by sequential error prone PCR and screened for improved activity for triazophos degradation. One purified variant protein (Val89-Gly89) named TrhA-M1 showed up to 3-fold improvement in specific activity against triazophos, and the specificity constants of Kcat and Kcat/Km for TrhA-M1 were improved up to 2.3- and 8.28-fold, respectively, compared to the wild-type enzyme. The results in this paper provided potential material for the contaminated soil remediation and hydrolase genetic structure research. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Identification of a novel phosphatase with high affinity for nucleotides monophosphate from common bean (Phaseolus vulgaris).

PubMed

Cabello-Díaz, Juan Miguel; Quiles, Francisco Antonio; Lambert, Rocío; Pineda, Manuel; Piedras, Pedro

2012-04-01

Common bean (Phaseolus vulgaris) seedlings accumulate ureides derived from purines after germination. The first step in the conversion of purines to ureides is the removal of the 5'-phosphate group by a phosphatase that has not been established yet. Two main phosphatase activities were detected in the embryonic axes of common bean using inosine monophosphate as substrate in an in-gel assay. Both activities differed in their sensitive to the common phosphatase inhibitor molybdate, with the molybdate-resistant as the first enzyme induced after radicle protrusion. The molybdate-resistant phosphatase has been purified to electrophoretic homogeneity and this is the first enzyme which shows this resistance purified and characterized from plant tissues. The native enzyme was a monomer of 55 kDa and it showed highest activity with nucleotides as substrates, with the K(m) values in the micromolar range. Among nucleotides, the highest specific constant (V(max)/K(m)) was observed for adenosine monophosphate. Furthermore, the enzyme was inhibited by nucleosides, the products of the enzymatic reaction, with maximum effect for adenosine. Common bean seedlings imbibed in the presence of adenosine monophosphate in vivo showed the highest molybdate-resistant phosphatase activity in the axes in addition to increased ureide content. The data presented suggests that purified phosphatase is involved in nucleotide metabolism in embryonic axes from common bean. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Constraints on universe models with cosmological constant from cosmic microwave background anisotropy

NASA Astrophysics Data System (ADS)

Sugiyama, Naoshi; Gouda, Naoteru; Sasaki, Misao

1990-12-01

Thorough numerical calculations of the fluctuations in the cosmic microwave background radiation using the gage-invariant formalism are carried out for various cosmological models with the cosmological constant. It is shown that a spatially flat cold dark matter-dominated universe of Omega(0) = 0.1 to about 0.4 and H(0) = 50 to about 100 km/s per Mpc with adiabatic perturbations has the possibility of giving the final answer to cosmological puzzles. It is also found that the introduction of the cosmological constant may revive pure baryonic universe models.

A high affinity monoclonal antibody recognizing the light chain of human coagulating factor VII.

PubMed

Sarial, Sheila; Asadi, Farzad; Jeddi-Tehrani, Mahmood; Hadavi, Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Taghizadeh-Jahed, Masoud; Shokri, Fazel; Rabbani, Hodjattallah

2012-12-01

Factor VII (FVII) is a serine protease-coagulating element responsible for the initiation of an extrinsic pathway of clot formation. Here we generated and characterized a high affinity monoclonal antibody that specifically recognizes human FVII. Recombinant human FVII (rh-FVII) was used for the production of a monoclonal antibody using BALB/c mice. The specificity of the antibody was determined by Western blot using plasma samples from human, mouse, sheep, goat, bovine, rabbit, and rat. Furthermore, the antibody was used to detect transiently expressed rh-FVII in BHK21 cell line using Western blot and sandwich ELISA. A mouse IgG1 (kappa chain) monoclonal antibody clone 1F1-B11 was produced against rh-FVII. The affinity constant (K(aff)) of the antibody was calculated to be 6.4×10(10) M(-1). The antibody could specifically recognize an epitope on the light chain of hFVII, with no reactivity with factor VII from several other animals. In addition, transiently expressed rh-FVII in BHK21 cells was recognized by 1F1-B11. The high affinity as well as the specificity of 1F1-B11 for hFVII will facilitate the affinity purification of hFVII and also production of FVII deficient plasma and minimizes the risk of bovine FVII contamination when fetal bovine serum-supplemented media are used for production and subsequent purification of rh-FVII.

Acid-base properties of humic substances from composted and thermally-dried sewage sludges and amended soils as determined by potentiometric titration and the NICA-Donnan model.

PubMed

Fernández, José M; Plaza, César; Senesi, Nicola; Polo, Alfredo

2007-09-01

The acid-base properties of humic acids (HAs) and fulvic acids (FAs) isolated from composted sewage sludge (CS), thermally-dried sewage sludge (TS), soils amended with either CS or TS at a rate of 80 t ha(-1)y(-1) for 3y and the corresponding unamended soil were investigated by use of potentiometric titrations. The non-ideal competitive adsorption (NICA)-Donnan model for a bimodal distribution of proton binding sites was fitted to titration data by use of a least-squares minimization method. The main fitting parameters of the NICA-Donnan model obtained for each HA and FA sample included site densities, median affinity constants and widths of affinity distributions for proton binding to low and high affinity sites, which were assumed to be, respectively, carboxylic- and phenolic-type groups. With respect to unamended soil HA and FA, the HAs and FAs from CS, and especially TS, were characterized by smaller acidic functional group contents, larger proton binding affinities of both carboxylic- and phenolic-type groups, and smaller heterogeneity of carboxylic and phenolic-type groups. Amendment with CS or TS led to a decrease of acidic functional group contents and a slight increase of proton binding affinities of carboxylic- and phenolic-type groups of soil HAs and FAs. These effects were more evident in the HA and FA fractions from CS-amended soil than in those from TS-amended soil.

A rhodium(III) complex for high-affinity DNA base-pair mismatch recognition

PubMed Central

Junicke, Henrik; Hart, Jonathan R.; Kisko, Jennifer; Glebov, Oleg; Kirsch, Ilan R.; Barton, Jacqueline K.

2003-01-01

A rhodium(III) complex, rac-[Rh(bpy)2phzi]3+ (bpy, 2,2′-bipyridine; phzi, benzo[a]phenazine-5,6-quinone diimine) has been designed as a sterically demanding intercalator targeted to destabilized mismatched sites in double-helical DNA. The complex is readily synthesized by condensation of the phenazine quinone with the corresponding diammine complex. Upon photoactivation, the complex promotes direct strand scission at single-base mismatch sites within the DNA duplex. As with the parent mismatch-specific reagent, [Rh(bpy)2(chrysi)]3+ [chrysene-5,6-quinone diimine (chrysi)], mismatch selectivity depends on the helix destabilization associated with mispairing. Unlike the parent chrysi complex, the phzi analogue binds and cleaves with high affinity and efficiency. The specific binding constants for CA, CC, and CT mismatches within a 31-mer oligonucleotide duplex are 0.3, 1, and 6 × 107 M−1, respectively; site-specific photocleavage is evident at nanomolar concentrations. Moreover, the specificity, defined as the ratio in binding affinities for mispaired vs. well paired sites, is maintained. The increase in affinity is attributed to greater stability in the mismatched site associated with stacking by the heterocyclic aromatic ligand. The high-affinity complex is also applied in the differential cleavage of DNA obtained from cell lines deficient in mismatch repair vs. those proficient in mismatch repair. Agreement is found between photocleavage by the mismatch-specific probes and deficiency in mismatch repair. This mismatch-specific targeting, therefore, offers a potential strategy for new chemotherapeutic design. PMID:12610209

Runout and fine-sediment deposits of axisymmetric turbidity currents

NASA Astrophysics Data System (ADS)

Dade, W. Brian; Huppert, Herbert E.

1995-09-01

We develop a model that describes the runout behavior and resulting deposit of a radially spreading, suspension-driven gravity current on a surface of negligible slope. Our analysis considers the separate cases of constant-volume and constant-flux sources. It incorporates expressions for the conservation of volume, a Froude number condition at the current front, and the evolution of the driving suspension due to settling of particles to the underlying bed. The model captures the key features of a range of experimental observations. The analysis also provides important scaling relationships between the geometry of a deposit and the source conditions for the deposit-forming flow, as well as explicit expressions for flow speed and deposit thickness as functions of radial distance from the source. Among the results of our study we find that, in the absence of information regarding flow history, the geometries of relatively well-sorted deposits generated by flows with source conditions of constant volume or constant flux are virtually indistinguishable. The results of our analysis can be used by geologists in the interpretation of some geologically important gravity-surge deposits. Using our analytical results, we consider three previously studied, radially symmetric turbidites of the Hispaniola-Caicos basin in the western Atlantic Ocean. From gross geometry and grain size of the turbidites alone we estimate for the respective deposit-forming events that upon entry into the basin the initial sediment concentrations were approximately 3% by volume and the total volumes were roughly between 30 km3 and 100 km3. Each of the suspension-driven flows is inferred to have spread into the basin with a characteristic speed of 3-5 m s-1, and reached its ultimate runout length of about 60-75 km while laying down a deposit over a period of about 10-12 hours.

Urodilatin: binding properties and stimulation of cGMP generation in rat kidney cells.

PubMed

Saxenhofer, H; Fitzgibbon, W R; Paul, R V

1993-02-01

Urodilatin (URO) [ANP-(95-126)] is an analogue of atrial natriuretic peptide (alpha-ANP) [ANP-(99-126)] that was first isolated from human urine. In rat mesangial cells, URO competed with high affinity for non-guanylate cyclase-coupled ANPR-C receptors [concentration at which 50% labeled ligand is displaced (IC50) approximately 70 pM], but with lesser affinity to the guanylate cyclase-linked ANPR-A receptors (IC50 approximately 800 pM). alpha-ANP bound to both receptors with similar affinity [dissociation constant (Kd) approximately 150 pM]. In papillary collecting duct homogenates, which possess only ANPR-A receptors, the apparent Kd value averaged 229 pM for alpha-ANP and 2.7 nM for URO. Intravenous URO was at least as potent and effective as alpha-ANP in inducing diuresis and natriuresis in anesthetized rats, but URO was approximately 10-fold less potent in stimulating guanosine 3',5'-cyclic monophosphate generation in mesangial and inner medullary collecting duct cells. We conclude that URO has a lesser affinity than alpha-ANP for guanylate cyclase-coupled ANP receptors in the kidney and that the relative natriuretic potency of URO in vivo cannot be directly attributed to its binding characteristics with ANPR-A receptors.

Low density and high affinity of platelet [3H]paroxetine binding in women with bulimia nervosa.

PubMed

Ekman, Agneta; Sundblad-Elverfors, Charlotta; Landén, Mikael; Eriksson, Tomas; Eriksson, Elias

2006-06-15

Impaired serotonin transmission has been suggested to be implicated in the pathophysiology of bulimia nervosa. As an indirect measure of brain serotonergic activity, the binding of tritiated ligands to platelet serotonin transporters has been studied in bulimia nervosa as well as in other putatively serotonin-related psychiatric disorders. In this study, the density and affinity of platelet serotonin transporters were assessed in 20 women meeting the DSM-IV criteria for bulimia nervosa and in 14 controls without previous or ongoing eating disorder using [(3)H]paroxetine as a ligand. In comparison to controls, women with bulimia nervosa had a significantly reduced number of platelet binding sites (B(max) = 721 +/- 313 vs. 1145 +/- 293 fmol/mg protein) and an increase in the affinity for the ligand demonstrated by a lower dissociaton constant (K(d) = 33 +/- 10 vs. 44 +/- 10 pM). A significant correlation between B(max) and K(d) values was found in patients but not in controls. Our results support the notion that bulimia nervosa is associated with a reduction in platelet serotonin transporter density. In addition, our study is the first to report that this reduced transporter density in women with bulimia nervosa is accompanied by an increase in the affinity of the transporter for the ligand.

Localization in human interleukin 2 of the binding site to the alpha chain (p55) of the interleukin 2 receptor.

PubMed Central

Sauvé, K; Nachman, M; Spence, C; Bailon, P; Campbell, E; Tsien, W H; Kondas, J A; Hakimi, J; Ju, G

1991-01-01

Human interleukin 2 (IL-2) analogs with defined amino acid substitutions were used to identify specific residues that interact with the 55-kDa subunit (p55) or alpha chain of the human IL-2 receptor. Analog proteins containing specific substitutions for Lys-35, Arg-38, Phe-42, or Lys-43 were inactive in competitive binding assays for p55. All of these analogs retained substantial competitive binding to the intermediate-affinity p70 subunit (beta chain) of the receptor complex. The analogs varied in ability to interact with the high-affinity p55/p70 receptor. Despite the lack of binding to p55, all analogs exhibited significant biological activity, as assayed on the murine CTLL cell line. The dissociation constants of Arg-38 and Phe-42 analogs for p70 were consistent with intermediate-affinity binding; the Kd values were not significantly affected by the presence of p55 in binding to the high-affinity IL-2 receptor complex. These results confirm the importance of the B alpha-helix in IL-2 as the locus for p55-receptor binding and support a revised model of IL-2-IL-2 receptor interaction. PMID:2052547

The Src SH2 domain interacts dynamically with the focal adhesion kinase binding site as demonstrated by paramagnetic NMR spectroscopy.

PubMed

Lindfors, Hanna E; Drijfhout, Jan Wouter; Ubbink, Marcellus

2012-06-01

The interaction between the tyrosine kinases Src and focal adhesion kinase (FAK) is a key step in signaling processes from focal adhesions. The phosphorylated tyrosine residue 397 in FAK is able to bind the Src SH2 domain. To establish the extent of the FAK binding motif, the binding affinity of the SH2 domain for phosphorylated and unphosphorylated FAK-derived peptides of increasing length was determined and compared with that of the internal Src SH2 binding site. It is shown that the FAK peptides have higher affinity than the internal binding site and that seven negative residues adjacent to the core SH2 binding motif increase the binding constant 30-fold. A rigid spin-label incorporated in the FAK peptides was used to establish on the basis of paramagnetic relaxation enhancement whether the peptide-protein complex is well defined. A large spread of the paramagnetic effects on the surface of the SH2 domain suggests that the peptide-protein complex exhibits dynamics, despite the high affinity of the peptide. The strong electrostatic interaction between the positive side of the SH2 domain and the negative peptide results in a high affinity but may also favor a dynamic interaction. Copyright © 2012 Wiley Periodicals, Inc.

Decrease in the red cell cofactor 2,3-diphosphoglycerate increases hemoglobin oxygen affinity in the hibernating brown bear Ursus arctos.

PubMed

Revsbech, Inge G; Malte, Hans; Fröbert, Ole; Evans, Alina; Blanc, Stéphane; Josefsson, Johan; Fago, Angela

2013-01-01

During winter hibernation, brown bears (Ursus arctos) reduce basal O(2) consumption rate to ∼25% compared with the active state, while body temperature decreases moderately (to ∼30°C), suggesting a temperature-independent component in their metabolic depression. To establish whether changes in O(2) consumption during hibernation correlate with changes in blood O(2) affinity, we took blood samples from the same six individuals of hibernating and nonhibernating free-ranging brown bears during winter and summer, respectively. A single hemoglobin (Hb) component was detected in all samples, indicating no switch in Hb synthesis. O(2) binding curves measured on red blood cell lysates at 30°C and 37°C showed a less temperature-sensitive O(2) affinity than in other vertebrates. Furthermore, hemolysates from hibernating bears consistently showed lower cooperativity and higher O(2) affinity than their summer counterparts, regardless of the temperature. We found that this increase in O(2) affinity was associated with a significant decrease in the red cell Hb-cofactor 2,3-diphosphoglycerate (DPG) during hibernation to approximately half of the summer value. Experiments performed on purified Hb, to which DPG had been added to match summer and winter levels, confirmed that the low DPG content was the cause of the left shift in the Hb-O(2) equilibrium curve during hibernation. Levels of plasma lactate indicated that glycolysis is not upregulated during hibernation and that metabolism is essentially aerobic. Calculations show that the increase in Hb-O(2) affinity and decrease in cooperativity resulting from decreased red cell DPG may be crucial in maintaining a fairly constant tissue oxygen tension during hibernation in vivo.

Characterization of Receptor Binding Profiles of Influenza A Viruses Using An Ellipsometry-Based Label-Free Glycan Microarray Assay Platform

PubMed Central

Fei, Yiyan; Sun, Yung-Shin; Li, Yanhong; Yu, Hai; Lau, Kam; Landry, James P.; Luo, Zeng; Baumgarth, Nicole; Chen, Xi; Zhu, Xiangdong

2015-01-01

A key step leading to influenza viral infection is the highly specific binding of a viral spike protein, hemagglutinin (HA), with an extracellular glycan receptor of a host cell. Detailed and timely characterization of virus-receptor binding profiles may be used to evaluate and track the pandemic potential of an influenza virus strain. We demonstrate a label-free glycan microarray assay platform for acquiring influenza virus binding profiles against a wide variety of glycan receptors. By immobilizing biotinylated receptors on a streptavidin-functionalized solid surface, we measured binding curves of five influenza A virus strains with 24 glycans of diverse structures and used the apparent equilibrium dissociation constants (avidity constants, 10–100 pM) as characterizing parameters of viral receptor profiles. Furthermore by measuring binding kinetic constants of solution-phase glycans to immobilized viruses, we confirmed that the glycan-HA affinity constant is in the range of 10 mM and the reaction is enthalpy-driven. PMID:26193329

Characterization of Receptor Binding Profiles of Influenza A Viruses Using An Ellipsometry-Based Label-Free Glycan Microarray Assay Platform.

PubMed

Fei, Yiyan; Sun, Yung-Shin; Li, Yanhong; Yu, Hai; Lau, Kam; Landry, James P; Luo, Zeng; Baumgarth, Nicole; Chen, Xi; Zhu, Xiangdong

2015-07-16

A key step leading to influenza viral infection is the highly specific binding of a viral spike protein, hemagglutinin (HA), with an extracellular glycan receptor of a host cell. Detailed and timely characterization of virus-receptor binding profiles may be used to evaluate and track the pandemic potential of an influenza virus strain. We demonstrate a label-free glycan microarray assay platform for acquiring influenza virus binding profiles against a wide variety of glycan receptors. By immobilizing biotinylated receptors on a streptavidin-functionalized solid surface, we measured binding curves of five influenza A virus strains with 24 glycans of diverse structures and used the apparent equilibrium dissociation constants (avidity constants, 10-100 pM) as characterizing parameters of viral receptor profiles. Furthermore by measuring binding kinetic constants of solution-phase glycans to immobilized viruses, we confirmed that the glycan-HA affinity constant is in the range of 10 mM and the reaction is enthalpy-driven.

Investigation of flavonoids bearing different substituents on ring C and their cu2+ complex binding with bovine serum albumin: structure-affinity relationship aspects.

PubMed

Shi, Shuyun; Zhang, Yuping; Chen, Xiaoqin; Peng, Mijun

2011-10-12

The effects of 1:1 flavonoid-Cu(2+) complexes of four flavonoids with different C-ring substituents, quercetin (QU), luteolin (LU), taxifolin (TA), and (+)-catechin (CA), on bovine serum albumin (BSA) were investigated and compared with corresponding free flavonoids by spectroscopic analysis in an attempt to characterize the chemical association taking place. The results indicated that all of the quenching mechanisms were based on static quenching combined with nonradiative energy transfer. Cu(2+) chelation changed the binding constants for BSA depending on the structures of flavonoids and the detected concentrations. The reduced hydroxyl groups, increased steric hindrance, and hydrophilicity of Cu(2+) chelation may be the main reasons for the reduced binding constants, whereas the formation of stable flavonoid-Cu(2+) complexes and synergistic action could increase the binding constants. The changed trends of critical energy transfer distance (R(0)) for Cu(2+) chelation were contrary to those of binding constants.

Cloning and expression of Saccharomyces cerevisiae SUC2 gene in yeast platform and characterization of recombinant enzyme biochemical properties.

PubMed

Mohandesi, Nooshin; Siadat, Seyed Omid Ranaei; Haghbeen, Kamahldin; Hesampour, Ardeshir

2016-12-01

Invertase (EC.3.2.1.26) catalyzes the hydrolysis of sucrose to an equimolar mixture of D-glucose and D-fructose which is of interest for various industrial applications. In this research, Saccharomyces cerevisiae invertase gene (SUC2) was optimized based on Pichia pastoris codon preference. The synthetic gene was introduced into the methylotrophic yeast Pichia pastoris under the control of the inducible AOX1 promoter. High level of the extracellular recombinant invertase (R-inv) production was achieved via methanol induction for 4 days and purified by His-Tag affinity chromatography which appeared to be a mixture of glycosylated proteins with various sizes of 85-95 kDa on SDS-PAGE. Deglycosylation of the proteins by Endo-H resulted in the proteins with average molecular weight of 60 kDa. The purified recombinant invertase biochemical properties and kinetic parameters determined a pH and temperature optimum at 4.8 and 60 °C, respectively, which in comparison with native S. cerevisiae invertase, thermal stability of recombinant invertase is highly increased in different heating treatment experiments. The purification of recombinant invertase resulted in an enzyme with specific activity of 178.56 U/mg with 3.83-fold of purification and the kinetic constants for enzyme were Km value of 19 mM and Vmax value of 300 μmol min -1  mg -1 With kinetic efficiency (Kcat/Km) of 13.15 s -1  mmol -1 it can be concluded that recombinant P. pastoris invertase can be more effective for industrial quality criteria. We conclude that recombinant P. pastoris enzyme with broad pH stability, substrate specificity and proper thermal stability can fulfil a series of predefined industrial quality criteria to be used in food, pharmaceutical and bio ethanol production industries.

Characterizing Isozymes of Chlorite Dismutase for Water Treatment

PubMed Central

Mobilia, Kellen C.; Hutchison, Justin M.; Zilles, Julie L.

2017-01-01

This work investigated the potential for biocatalytic degradation of micropollutants, focusing on chlorine oxyanions as model contaminants, by mining biology to identify promising biocatalysts. Existing isozymes of chlorite dismutase (Cld) were characterized with respect to parameters relevant to this high volume, low-value product application: kinetic parameters, resistance to catalytic inactivation, and stability. Maximum reaction velocities (Vmax) were typically on the order of 104 μmol min-1 (μmol heme)-1. Substrate affinity (Km) values were on the order of 100 μM, except for the Cld from Candidatus Nitrospira defluvii (NdCld), which showed a significantly lower affinity for chlorite. NdCld also had the highest susceptibility to catalytic inactivation. In contrast, the Cld from Ideonella dechloratans was least susceptible to catalytic inactivation, with a maximum turnover number of approximately 150,000, more than sevenfold higher than other tested isozymes. Under non-reactive conditions, Cld was quite stable, retaining over 50% of activity after 30 days, and most samples retained activity even after 90–100 days. Overall, Cld from I. dechloratans was the most promising candidate for environmental applications, having high affinity and activity, a relatively low propensity for catalytic inactivation, and excellent stability. PMID:29312158

The thermal structure and dynamics of the atmosphere of Venus between 70 and 90 km from the Galileo-NIMS spectra

NASA Technical Reports Server (NTRS)

Roos-Serote, M.; Drossart, P.; Encrenaz, TH.; Lellouch, E.; Carlson, R. W.; Baines, K. H.; Taylor, F. W.; Calcutt, S. B.

1995-01-01

An analysis of thermal profiles and dynamics over a wide range of latitudes for the venusian atmosphere between 70 and 90 km is presented based on high spatial resolution infrared spectra of the night side obtained by the near infrared mapping spectrometer (NIMS) experiment during the Galileo-Venus encounter in February 1990. Using the 4.3-micrometer CO2 absorption band, the temperature profile is retrieved in the 75- to 91-km altitude region over a latitudinal range of -59 deg to +64 deg. Compared to earlier observations from the Pioneer Venus mission, the temperature at 91 km is about 10 K higher and between 74 and 83 km about 3.6 K colder. An equator to pole warming at constant pressure levels is found and implications for the zonal wind profiles are drawn under the assumption that the atmosphere is in cyclostrophic balance in the region of 70 to 90 km. The results are in correspondence with direct wind measurements from ground-based observations at 95 km and 105 km altitude.

Synthesis of molecularly imprinted organic-inorganic hybrid azobenzene materials by sol-gel for radiation induced selective recognition of 2,4-dichlorophenoxyacetic acid

NASA Astrophysics Data System (ADS)

Shuai Jiang, Guang; An Zhong, Shi; Chen, Lan; Blakey, Idriss; Whitaker, Andrew

2011-02-01

A novel photoresponsive functional monomer bearing a siloxane polymerisable group and azobenzene moieties was synthesized. This monomer was then used to prepare photoresponsive molecularly imprinted polymers (MIP), which have specific binding sites for 2,4-dichlorophenoxyacetic acid (2,4-D) through hydrogen bonding moieties. The binding affinity of the imprinted recognition sites was switchable by alternate irradiations with ultraviolet and visible light, suggesting that azobenzene groups located inside the binding sites could be used as chemical sensors and the trans-cis isomerization could regulate the affinity for the 2,4-D. In addition, the concentration of the 2,4-D was able to be quantified by monitoring the trans-to-cis photoisomerization rate constant.

( sup 3 H)-DOB(4-bromo-2,5-dimethoxyphenylisopropylamine) and ( sup 3 H) ketanserin label two affinity states of the cloned human 5-hydroxytryptamine2 receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Branchek, T.; Adham, N.; Macchi, M.

1990-11-01

The binding properties of the 5-hydroxytryptamine2 (5-HT2) receptor have been the subject of much interest and debate in recent years. The hallucinogenic amphetamine derivative 4-bromo-2,5-dimethoxyphenylisopropylamine (DOB) has been shown to bind to a small number of binding sites with properties very similar to (3H)ketanserin-labeled 5-HT2 receptors, but with much higher agonist affinities. Some researchers have interpreted this as evidence for the existence of a new subtype of 5-HT2 receptor (termed 5-HT2A), whereas others have interpreted these data as indicative of agonist high affinity and agonist low affinity states for the 5-HT2 receptor. In this investigation, a cDNA clone encoding themore » serotonin 5-HT2 receptor was transiently transfected into monkey kidney Cos-7 cells and stably transfected into mouse fibroblast L-M(TK-) cells. In both systems, expression of this single serotonin receptor cDNA led to the appearance of both (3H)DOB and (3H)ketanserin binding sites with properties that matched their binding characteristics in mammalian brain homogenates. Addition of guanosine 5'-(beta, gamma-imido) triphosphate (Gpp(NH)p) to this system caused a rightward shift and steepening of agonist competition curves for (3H) ketanserin binding, converting a two-site binding curve to a single low affinity binding state. Gpp(NH)p addition also caused a 50% decrease in the number of high affinity (3H)DOB binding sites, with no change in the dissociation constant of the remaining high affinity states. These data on a single human 5-HT2 receptor cDNA expressed in two different transfection host cells indicate that (3H)DOB and (3H)ketanserin binding reside on the same gene product, apparently interacting with agonist and antagonist conformations of a single human 5-HT2 receptor protein.« less

Induction of the high-affinity Na(+)-dependent glutamate transport system XAG- by hypertonic stress in the renal epithelial cell line NBL-1.

PubMed Central

Ferrer-Martinez, A; Felipe, A; Nicholson, B; Casado, J; Pastor-Anglada, M; McGivan, J

1995-01-01

The high-affinity Na(+)-dependent glutamate transport system XAG- is induced (threefold increase in Vmax. with no change in Km) by hypertonicity in the renal epithelial cell line NBL-1. This effect is dependent on protein synthesis and glycosylation and is accompanied by an increase in EAAC1 mRNA levels. Other Na(+)-dependent transport systems in this cell line do not respond to hypertonic stress. In contrast to recent findings [Ruiz-Montasell, Gomez-Angelats, Casado, Felipe, McGivan and Pastor-Anglada (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 9569-9573] showing that increased system A activity after hyperosmotic shock results from induction of a regulatory protein, this is the first demonstration that hypertonicity may increase the expression of the gene for an amino acid transport protein itself. Images Figure 4 PMID:7654212

Nonlinear force propagation, anisotropic stiffening and non-affine relaxation in a model cytoskeleton

NASA Astrophysics Data System (ADS)

Mizuno, Daisuke; Head, David; Ikebe, Emi; Nakamasu, Akiko; Kinoshita, Suguru; Peijuan, Zhang; Ando, Shoji

2013-03-01

Forces are generated heterogeneously in living cells and transmitted through cytoskeletal networks that respond highly non-linearly. Here, we carry out high-bandwidth passive microrheology on vimentin networks reconstituted in vitro, and observe the nonlinear mechanical response due to forces propagating from a local source applied by an optical tweezer. Since the applied force is constant, the gel becomes equilibrated and the fluctuation-dissipation theorem can be employed to deduce the viscoelasticity of the local environment from the thermal fluctuations of colloidal probes. Our experiments unequivocally demonstrate the anisotropic stiffening of the cytoskeletal network behind the applied force, with greater stiffening in the parallel direction. Quantitative agreement with an affine continuum model is obtained, but only for the response at certain frequency ~ 10-1000 Hz which separates the high-frequency power law and low-frequency elastic behavior of the network. We argue that the failure of the model at lower frequencies is due to the presence of non-affinity, and observe that zero-frequency changes in particle separation can be fitted when an independently-measured, empirical nonaffinity factor is applied.

Paracetamol and cytarabine binding competition in high affinity binding sites of transporting protein

NASA Astrophysics Data System (ADS)

Sułkowska, A.; Bojko, B.; Równicka, J.; Sułkowski, W. W.

2006-07-01

Paracetamol (acetaminophen, AA) the most popular analgesic drug is commonly used in the treatment of pain in patients suffering from cancer. In our studies, we evaluated the competition in binding with serum albumin between paracetamol (AA) and cytarabine, antyleukemic drug (araC). The presence of one drug can alter the binding affinity of albumin towards the second one. Such interaction can result in changing of the free fraction of the one of these drugs in blood. Two spectroscopic methods were used to determine high affinity binding sites and the competition of the drugs. Basing on the change of the serum albumin fluorescence in the presence of either of the drugs the quenching ( KQ) constants for the araC-BSA and AA-BSA systems were calculated. Analysis of UV difference spectra allowed us to describe the changes in drug-protein complexes (araC-albumin and AA-albumin) induced by the presence of the second drug (AA and araC, respectively). The mechanism of competition between araC and AA has been proposed.

Insensitivity of cerebral oxygen transport to oxygen affinity of hemoglobin-based oxygen carriers

PubMed Central

Koehler, Raymond C.; Fronticelli, Clara; Bucci, Enrico

2008-01-01

The cerebrovascular effects of exchange transfusion of various cell-free hemoglobins that possess different oxygen affinities are reviewed. Reducing hematocrit by transfusion of a non-oxygen-carrying solution dilates pial arterioles on the brain surface and increases cerebral blood flow to maintain a constant bulk oxygen transport to the brain. In contrast, transfusion of hemoglobins with P50 of 4–34 Torr causes constriction of pial arterioles that offsets the decrease in blood viscosity to maintain cerebral blood flow and oxygen transport. The autoregulatory constriction is dependent on synthesis of 20-HETE from arachidonic acid. This oxygen-dependent reaction is apparently enhanced by facilitated oxygen diffusion from the red cell to the endothelium arising from increased plasma oxygen solubility in the presence of low or high-affinity hemoglobin. Exchange transfusion of recombinant hemoglobin polymers with P50 of 3 and 18 Torr reduces infarct volume from experimental stroke. Cell-free hemoglobins do not require a P50 as high as red blood cell hemoglobin to facilitate oxygen delivery. PMID:18230370

Robust estimation-free prescribed performance back-stepping control of air-breathing hypersonic vehicles without affine models

NASA Astrophysics Data System (ADS)

Bu, Xiangwei; Wu, Xiaoyan; Huang, Jiaqi; Wei, Daozhi

2016-11-01

This paper investigates the design of a novel estimation-free prescribed performance non-affine control strategy for the longitudinal dynamics of an air-breathing hypersonic vehicle (AHV) via back-stepping. The proposed control scheme is capable of guaranteeing tracking errors of velocity, altitude, flight-path angle, pitch angle and pitch rate with prescribed performance. By prescribed performance, we mean that the tracking error is limited to a predefined arbitrarily small residual set, with convergence rate no less than a certain constant, exhibiting maximum overshoot less than a given value. Unlike traditional back-stepping designs, there is no need of an affine model in this paper. Moreover, both the tedious analytic and numerical computations of time derivatives of virtual control laws are completely avoided. In contrast to estimation-based strategies, the presented estimation-free controller possesses much lower computational costs, while successfully eliminating the potential problem of parameter drifting. Owing to its independence on an accurate AHV model, the studied methodology exhibits excellent robustness against system uncertainties. Finally, simulation results from a fully nonlinear model clarify and verify the design.

Generation and characterization of high affinity humanized fab against hepatitis B surface antigen.

PubMed

Tiwari, Ashutosh; Dutta, Durgashree; Khanna, Navin; Acharya, Subrat K; Sinha, Subrata

2009-09-01

5S is a mouse monoclonal IgG1 that binds to the 'a' epitope of the Hepatitis B surface antigen (HBsAg) and tested positive in an in vitro test for virus neutralization. We have earlier reported the generation of humanized single chain variable fragment (scFv) from the same. In this article we report the generation of a recombinant Fab molecule by fusing humanized variable domains of 5S with the constant domains of human IgG1. The humanized Fab expressed in E. coli and subsequently purified, retained a high binding affinity (K(D) = 3.63 nmol/L) to HBsAg and bound to the same epitope of HBsAg as the parent molecule. The humanized Fab also maintained antigen binding in the presence of various destabilizing agents like 3 M NaCl, 30% DMSO, 8 M urea, and extreme pH. This high affinity humanized Fab provides a basis for the development of therapeutic molecules that can be safely utilized for the prophylaxis and treatment for Hepatitis B infection.

High-affinity receptors for bombesin-like peptides in normal guinea pig lung membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lach, E.; Trifilieff, A.; Landry, Y.

1991-01-01

The binding of the radiolabeled bombesin analogue ({sup 125}I-Tyr{sup 4})bombesin to guinea-pig lung membranes was investigated. Binding of ({sup 125}I-Tyr{sup 4})bombesin was specific, saturable, reversible and linearly related to the protein concentration. Scatchard analysis of equilibrium binding data at 25C indicated the presence of a single class of non-interacting binding sites for bombesin (B{sub max} = 7.7 fmol/mg protein). The value of the equilibrium dissociation constant (K{sub D} = 90 pM) agrees with a high-affinity binding site. Bombesin and structurally related peptides such as ({sup 125}I-Tyr{sup 4})bombesin, neuromedin B and neuromedin C inhibited the binding of ({sup 125}I-Tyr{sup 4})bombesin inmore » an order of potencies as follows: ({sup 125}I-Tyr{sup 4})bombesin {gt} bombesin {ge} neuromedin C {much gt} neuromedin B. These results indicate that guinea-pig lung membranes possess a single class of bombesin receptors with a high affinity for bombesin and a lower one for neuromedin B.« less

Kinetic studies on strand displacement in de novo designed parallel heterodimeric coiled coils.

PubMed

Groth, Mike C; Rink, W Mathis; Meyer, Nils F; Thomas, Franziska

2018-05-14

Among the protein folding motifs, which are accessible by de novo design, the parallel heterodimeric coiled coil is most frequently used in bioinspired applications and chemical biology in general. This is due to the straightforward sequence-to-structure relationships, which it has in common with all coiled-coil motifs, and the heterospecificity, which allows control of association. Whereas much focus was laid on designing orthogonal coiled coils, systematic studies on controlling association, for instance by strand displacement, are rare. As a contribution to the design of dynamic coiled-coil-based systems, we studied the strand-displacement mechanism in obligate heterodimeric coiled coils to investigate the suitability of the dissociation constants ( K D ) as parameters for the prediction of the outcome of strand-displacement reactions. We use two sets of heterodimeric coiled coils, the previously reported N-A x B y and the newly characterized C-A x B y . Both comprise K D values in the μM to sub-nM regime. Strand displacement is explored by CD titration and a FRET-based kinetic assay and is proved to be an equilibrium reaction with half-lifes from a few seconds up to minutes. We could fit the displacement data by a competitive binding model, giving rate constants and overall affinities of the underlying association and dissociation reactions. The overall affinities correlate well with the ratios of K D values determined by CD-thermal denaturation experiments and, hence, support the dissociative mechanism of strand displacement in heterodimeric coiled coils. From the results of more than 100 different displacement reactions we are able to classify three categories of overall affinities, which allow for easy prediction of the equilibrium of strand displacement in two competing heterodimeric coiled coils.

Kinetic studies on strand displacement in de novo designed parallel heterodimeric coiled coils† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc05342h

PubMed Central

Groth, Mike C.; Rink, W. Mathis; Meyer, Nils F.

2018-01-01

Among the protein folding motifs, which are accessible by de novo design, the parallel heterodimeric coiled coil is most frequently used in bioinspired applications and chemical biology in general. This is due to the straightforward sequence-to-structure relationships, which it has in common with all coiled-coil motifs, and the heterospecificity, which allows control of association. Whereas much focus was laid on designing orthogonal coiled coils, systematic studies on controlling association, for instance by strand displacement, are rare. As a contribution to the design of dynamic coiled-coil-based systems, we studied the strand-displacement mechanism in obligate heterodimeric coiled coils to investigate the suitability of the dissociation constants (KD) as parameters for the prediction of the outcome of strand-displacement reactions. We use two sets of heterodimeric coiled coils, the previously reported N-AxBy and the newly characterized C-AxBy. Both comprise KD values in the μM to sub-nM regime. Strand displacement is explored by CD titration and a FRET-based kinetic assay and is proved to be an equilibrium reaction with half-lifes from a few seconds up to minutes. We could fit the displacement data by a competitive binding model, giving rate constants and overall affinities of the underlying association and dissociation reactions. The overall affinities correlate well with the ratios of KD values determined by CD-thermal denaturation experiments and, hence, support the dissociative mechanism of strand displacement in heterodimeric coiled coils. From the results of more than 100 different displacement reactions we are able to classify three categories of overall affinities, which allow for easy prediction of the equilibrium of strand displacement in two competing heterodimeric coiled coils. PMID:29780562

Physiologic regulation of atrial natriuretic peptide receptors in rat renal glomeruli.

PubMed Central

Ballermann, B J; Hoover, R L; Karnovsky, M J; Brenner, B M

1985-01-01

Isolated rat renal glomeruli and cultured glomerular mesangial and epithelial cells were examined for atrial natriuretic peptide (ANP) receptors, and for ANP-stimulated cyclic guanosine monophosphate (cGMP) generation. In glomeruli from normal rats, human (1-28) 125I-ANP bound to a single population of high affinity receptors with a mean equilibrium dissociation constant of 0.46 nM. Human (1-28) ANP markedly stimulated cGMP generation, but not cAMP generation in normal rat glomeruli. Analogues of ANP that bound to the glomerular ANP receptor with high affinity stimulated cGMP accumulation, whereas the (13-28) ANP fragment, which failed to bind to the receptor, was devoid of functional activity. Cell surface receptors for ANP were expressed on cultured glomerular mesangial but not epithelial cells, and appreciable ANP-stimulated cGMP accumulation was elicited only in mesangial cells. Approximately 12,000 ANP receptor sites were present per mesangial cell, with an average value for the equilibrium dissociation constant of 0.22 nM. Feeding of a low-salt diet to rats for 2 wk resulted in marked up regulation of the glomerular ANP receptor density to a mean of 426 fmol/mg protein, compared with 116 fmol/mg in rats given a high-salt diet. A modest reduction in the affinity of glomerular ANP receptors was also observed in rats fed the low-salt diet. ANP-stimulated cGMP generation in glomeruli did not change with alterations in salt intake. We conclude that high salt feeding in the rat results in reduced glomerular ANP receptor density relative to values in salt restricted rats. Furthermore, the mesangial cell is a principal target for ANP binding in the glomerulus. Images PMID:3001139

A spectroscopic study of phenylbutazone and aspirin bound to serum albumin in rheumatoid diseases

NASA Astrophysics Data System (ADS)

Maciążek-Jurczyk, M.; Sułkowska, A.; Bojko, B.; Równicka-Zubik, J.; Sułkowski, W. W.

2011-11-01

Interaction of phenylbutazone (PBZ) and aspirin (ASA), two drugs recommended in rheumatoid diseases (RDs), when binding to human (HSA) and bovine (BSA) serum albumins, has been studied by quenching of fluorescence and proton nuclear magnetic resonance ( 1HNMR) techniques. On the basis of spectrofluorescence measurements high affinity binding sites of PBZ and ASA on albumin as well as their interaction within the binding sites were described. A low affinity binding site has been studied by proton nuclear magnetic resonance spectroscopy. Using fluorescence spectroscopy the location of binding site in serum albumin (SA) for PBZ and ASA was found. Association constants Ka were determined for binary (i.e. PBZ-SA and ASA-SA) and ternary complexes (i.e. PBZ-[ASA]-SA and ASA-[PBZ]-SA). PBZ and ASA change the affinity of each other to the binding site in serum albumin (SA). The presence of ASA causes the increase of association constants KaI of PBZ-SA complex. Similarly, PBZ influences KaI of ASA-SA complex. This phenomenon shows that the strength of binding and the stability of the complexes increase in the presence of the second drug. The decrease of KaII values suggests that the competition between PBZ and ASA in binding to serum albumin in the second class of binding sites occurs. The analysis of 1HNMR spectral parameters i.e. changes of chemical shifts and relaxation times of the drug indicate that the presence of ASA weakens the interaction of PBZ with albumin. Similarly PBZ weakens the interaction of ASA with albumin. This conclusion points to the necessity of using a monitoring therapy owning to the possible increase of uncontrolled toxic effects.

9-Benzoyl 9-deazaguanines as potent xanthine oxidase inhibitors.

PubMed

Rodrigues, Marili V N; Barbosa, Alexandre F; da Silva, Júlia F; dos Santos, Deborah A; Vanzolini, Kenia L; de Moraes, Marcela C; Corrêa, Arlene G; Cass, Quezia B

2016-01-15

A novel potent xanthine oxidase inhibitor, 3-nitrobenzoyl 9-deazaguanine (LSPN451), was selected from a series of 10 synthetic derivatives. The enzymatic assays were carried out using an on-flow bidimensional liquid chromatography (2D LC) system, which allowed the screening¸ the measurement of the kinetic inhibition constant and the characterization of the inhibition mode. This compound showed a non-competitive inhibition mechanism with more affinity for the enzyme-substrate complex than for the free enzyme, and inhibition constant of 55.1±9.80 nM, about thirty times more potent than allopurinol. Further details of synthesis and enzymatic studies are presented herein. Copyright © 2015 Elsevier Ltd. All rights reserved.

The effects of pH and temperature on fluorescent calcium indicators as determined with Chelex-100 and EDTA buffer systems.

PubMed

Lattanzio, F A

1990-08-31

A novel method of determining the apparent dissociation constants of fluorescent calcium indicators is described which utilizes Chelex-100 ion exchange resin and 45Ca. The affinity for calcium of indicators fluo-3, fura-2 and indo-1 measured at either 22 degrees or 37 degrees C decreases as pH is decreased from 7.4 to 5.5. These measurements agree with determinations made using EDTA-calcium buffers. The 1:1 calcium:indicator complex is maintained under all conditions. The necessity to correct dissociation constants during intracellular acidification to properly interpret fluorescence measurements is illustrated by indo-1 measurements in the ischemic rat heart.

On the simultaneous action of two competitive antagonists

PubMed Central

Ginsborg, B.L.; Stephenson, R.P.

1974-01-01

1 A hypothesis is outlined predicting the conditions in which the addition of a second competitive antagonist will increase rather than reduce the response to an agonist. 2 Experiments were performed with the guinea-pig ileum as the test tissue, hexyltrimethyl ammonium as the agonist, benzilyltropine methiodide as the `slow' antagonist and pentyltriethyl ammonium as the `fast' antagonist. 3 The results are consistent with the hypothesis, if the affinity constant for hexyltrimethyl ammonium is between 2.7 and 3.7 × 104 M-1, if the dissociation time constant for the slow antagonist is greater than 10 min and if that for the fast antagonist is less than 10 seconds. PMID:4451745

Surface Plasmon Resonance Biosensor Method for Palytoxin Detection Based on Na+,K+-ATPase Affinity

PubMed Central

Alfonso, Amparo; Pazos, María-José; Fernández-Araujo, Andrea; Tobio, Araceli; Alfonso, Carmen; Vieytes, Mercedes R.; Botana, Luis M.

2013-01-01

Palytoxin (PLTX), produced by dinoflagellates from the genus Ostreopsis was first discovered, isolated, and purified from zoanthids belonging to the genus Palythoa. The detection of this toxin in contaminated shellfish is essential for human health preservation. A broad range of studies indicate that mammalian Na+,K+-ATPase is a high affinity cellular receptor for PLTX. The toxin converts the pump into an open channel that stimulates sodium influx and potassium efflux. In this work we develop a detection method for PLTX based on its binding to the Na+,K+-ATPase. The method was developed by using the phenomenon of surface plasmon resonance (SPR) to monitor biomolecular reactions. This technique does not require any labeling of components. The interaction of PLTX over immobilized Na+,K+-ATPase is quantified by injecting different concentrations of toxin in the biosensor and checking the binding rate constant (kobs). From the representation of kobs versus PLTX concentration, the kinetic equilibrium dissociation constant (KD) for the PLTX-Na+,K+-ATPase association can be calculated. The value of this constant is KD = 6.38 × 10−7 ± 6.67 × 10−8 M PLTX. In this way the PLTX-Na+,K+-ATPase association was used as a suitable method for determination of the toxin concentration in a sample. This method represents a new and useful approach to easily detect the presence of PLTX-like compounds in marine products using the mechanism of action of these toxins and in this way reduce the use of other more expensive and animal based methods. PMID:24379088

Role of κ→λ light-chain constant-domain switch in the structure and functionality of A17 reactibody

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ponomarenko, Natalia; Chatziefthimiou, Spyros D.; Kurkova, Inna

2014-03-01

Catalytic antibody variants with κ and λ light-chain constant domains show differences in their crystal structures which lead to subtle changes in catalytic efficiency and thermodynamic parameters as well as in their affinity for peptide substrates. The engineering of catalytic function in antibodies requires precise information on their structure. Here, results are presented that show how the antibody domain structure affects its functionality. The previously designed organophosphate-metabolizing reactibody A17 has been re-engineered by replacing its constant κ light chain by the λ chain (A17λ), and the X-ray structure of A17λ has been determined at 1.95 Å resolution. It was foundmore » that compared with A17κ the active centre of A17λ is displaced, stabilized and made more rigid owing to interdomain interactions involving the CDR loops from the V{sub L} and V{sub H} domains. These V{sub L}/V{sub H} domains also have lower mobility, as deduced from the atomic displacement parameters of the crystal structure. The antibody elbow angle is decreased to 126° compared with 138° in A17κ. These structural differences account for the subtle changes in catalytic efficiency and thermodynamic parameters determined with two organophosphate ligands, as well as in the affinity for peptide substrates selected from a combinatorial cyclic peptide library, between the A17κ and A17λ variants. The data presented will be of interest and relevance to researchers dealing with the design of antibodies with tailor-made functions.« less

Surface plasmon resonance biosensor method for palytoxin detection based on Na+,K+-ATPase affinity.

PubMed

Alfonso, Amparo; Pazos, María-José; Fernández-Araujo, Andrea; Tobio, Araceli; Alfonso, Carmen; Vieytes, Mercedes R; Botana, Luis M

2013-12-27

Palytoxin (PLTX), produced by dinoflagellates from the genus Ostreopsis was first discovered, isolated, and purified from zoanthids belonging to the genus Palythoa. The detection of this toxin in contaminated shellfish is essential for human health preservation. A broad range of studies indicate that mammalian Na+,K+-ATPase is a high affinity cellular receptor for PLTX. The toxin converts the pump into an open channel that stimulates sodium influx and potassium efflux. In this work we develop a detection method for PLTX based on its binding to the Na+,K+-ATPase. The method was developed by using the phenomenon of surface plasmon resonance (SPR) to monitor biomolecular reactions. This technique does not require any labeling of components. The interaction of PLTX over immobilized Na+,K+-ATPase is quantified by injecting different concentrations of toxin in the biosensor and checking the binding rate constant (Kobs). From the representation of Kobs versus PLTX concentration, the kinetic equilibrium dissociation constant (K(D)) for the PLTX-Na+,K+-ATPase association can be calculated. The value of this constant is K(D) = 6.38 × 10-7 ± 6.67 × 10-8 M PLTX. In this way the PLTX-Na+,K+-ATPase association was used as a suitable method for determination of the toxin concentration in a sample. This method represents a new and useful approach to easily detect the presence of PLTX-like compounds in marine products using the mechanism of action of these toxins and in this way reduce the use of other more expensive and animal based methods.

Evaluation of Movement Restriction Zone Sizes in Controlling Classical Swine Fever Outbreaks

PubMed Central

Yadav, Shankar; Olynk Widmar, Nicole; Lay, Donald C.; Croney, Candace; Weng, Hsin-Yi

2017-01-01

The objective of this study was to compare the impacts of movement restriction zone sizes of 3, 5, 9, and 11 km with that of 7 km (the recommended zone size in the United States) in controlling a classical swine fever (CSF) outbreak. In addition to zone size, different compliance assumptions and outbreak types (single site and multiple site) were incorporated in the study. Three assumptions of compliance level were simulated: baseline, baseline ± 10%, and baseline ± 15%. The compliance level was held constant across all zone sizes in the baseline simulation. In the baseline ± 10% and baseline ± 15% simulations, the compliance level was increased for 3 and 5 km and decreased for 9 and 11 km from the baseline by the indicated percentages. The compliance level remained constant in all simulations for the 7-km zone size. Four single-site (i.e., with one index premises at the onset of outbreak) and four multiple-site (i.e., with more than one index premises at the onset of outbreak) CSF outbreak scenarios in Indiana were simulated incorporating various zone sizes and compliance assumptions using a stochastic between-premises disease spread model to estimate epidemic duration, percentage of infected, and preemptively culled swine premises. Furthermore, a risk assessment model that incorporated the results from the disease spread model was developed to estimate the number of swine premises under movement restrictions that would experience animal welfare outcomes of overcrowding or feed interruption during a CSF outbreak in Indiana. Compared with the 7-km zone size, the 3-km zone size resulted in a longer median epidemic duration, larger percentages of infected premises, and preemptively culled premises (P’s < 0.001) across all compliance assumptions and outbreak types. With the assumption of a higher compliance level, the 5-km zone size significantly (P < 0.001) reduced the epidemic duration and percentage of swine premises that would experience animal welfare outcomes in both outbreak types, whereas assumption of a lower compliance level for 9- and 11-km zone sizes significantly (P < 0.001) increased the epidemic duration and percentage of swine premises with animal welfare outcomes compared with the 7-km zone size. The magnitude of impact due to a zone size varied across the outbreak types (single site and multiple site). Overall, the 7-km zone size was found to be most effective in controlling CSF outbreaks, whereas the 5-km zone size was comparable to the 7-km zone size in some circumstances. PMID:28119920

The Original Michaelis Constant: Translation of the 1913 Michaelis-Menten Paper

PubMed Central

Johnson, Kenneth A.; Goody, Roger S.

2011-01-01

Nearly 100 years ago Michaelis and Menten published their now classic paper (Michaelis, L., and Menten, M. L. (1913) Die Kinetik der Invertinwirkung, Biochemische Zeitschrift 49, 333–369), in which they show that the rate of an enzyme-catalyzed reaction is proportional to the concentration of enzyme-substrate complex predicted by the Michaelis-Menten equation. Because the original text was written in German, yet is often quoted by English speaking authors, we undertook a complete translation of the 1913 publication, which we provide as an online supplement (http://pubs.acs.org). Here we introduce the translation, describe the historical context of the work, and show a new analysis of the original data. In doing so, we uncovered several surprises that reveal an interesting glimpse into the early history of enzymology. In particular, our re-analysis of Michaelis and Menten’s data using modern computational methods revealed an unanticipated rigor and precision in the original publication and uncovered a sophisticated, comprehensive analysis that has been overlooked in the century since their work was published. Michaelis and Menten not only analyzed initial velocity measurements, but they also fit their full time course data to the integrated form of the rate equations, including product inhibition, and derived a single global constant to represent all of their data. That constant was not the Michaelis constant, but rather, Vmax/Km, the specificity constant times the enzyme concentration (kcat/Km*E0). PMID:21888353

ATP sulfurylase from higher plants: kinetic and structural characterization of the chloroplast and cytosol enzymes from spinach leaf.

PubMed

Renosto, F; Patel, H C; Martin, R L; Thomassian, C; Zimmerman, G; Segel, I H

1993-12-01

Two forms of ATP sulfurylase were purified from spinach leaf. The major (chloroplast) form accounts for 85 to 90% of the total leaf activity (0.03 +/- 0.01 adenosine-5'-phosphosulfate (APS) synthesis units x gram fresh weight-1). Both enzyme forms appear to be tetramers composed of 49- to 50-kDa subunits with the minor (cytosolic) form being slightly larger than the chloroplast form. The specific activities (units x milligram protein-1) of the chloroplast form at pH 8.0, 30 degrees C, were as follows: APS synthesis, 16; molybdolysis, 229; ATP synthesis, 267; selenolysis, 4.1; fluorophosphate activation, 11. Kinetic constants for the physiological reaction were as follows: KmA = 0.046 mM, K(ia) = 0.85 mM, KmB = 0.25 mM, KmQ = 0.37 microM, K(iq) = 64-85 nM, and KmP = 10 microM, where A = MgATP, B = SO4(2-), P = total PPi at 5 mM Mg2+, and Q = APS. The kinetic constants for molybdolysis were similar to those of the APS synthesis reaction. The kinetic constants of the minor (cytosol) form were similar to those of the major form with two exceptions: (a) The molybdolysis activity was 120 units x milligram protein-1, yielding a Vmax (ATP synthesis)/Vmax (molybdolysis) ratio close to 2 (compared to about unity for the chloroplast form) and (b) KmA was greater (0.24 and 0.15 mM for APS synthesis and molybdolysis, respectively). Initial velocity measurements (made over an extended range of MgATP and SO4(2-) concentrations), product inhibition studies (by initial velocity methods and by reaction progress curve analyses), dead end inhibition studies (with monovalent and divalent oxyanions), and kcat/Km comparisons (for SO4(2-) and MoO4(2-) support a random AB-ordered PQ kinetic mechanism in which MgATP and SO4(2-) bind in a highly synergistic manner. Equilibrium binding studies indicated the presence of one APS site per subunit. HPLC elution profiles of chymotryptic and tryptic peptides were essentially the same for both enzyme forms. The N-terminal sequence of residues 5-20 of the cytosol enzyme was identical to residues 1-16 of the chloroplast enzyme.

ARC-1989-A89-7005

NASA Image and Video Library

1989-08-17

August 17 to 19, 1989 Range : 11.5 million km (7.1 million mi.) to 7.9 million km (4.9 million mi.) Four black and white images of Neptune's largest satellite, Triton, show it's rotation between the first (upper left) image and the last (lower right). Resolution improves from about 200 km (124 miles) to 150 km (93 miles) per line pair. Triton's south pole lies in the dark area near the bottom of the disk. Dark spots, roughly 1,000 km (620 miles) across, occur near the equator, and show Triton rotation between images. The rotation appears to be synchronous with Triton's 5.88-day orbital period (i.e., Triton rotates on its axis in the same time it revolves around Neptune.) The spots' constant rotation rate and their visibility near the edge of the disk suggest the spots are surface features. Whatever atmosphere is present on Triton appears transparent enough that Voyager 2's cameras can see through it.

The effects of acute whole body vibration as a recovery modality following high-intensity interval training in well-trained, middle-aged runners.

PubMed

Edge, J; Mündel, T; Weir, K; Cochrane, D J

2009-02-01

The main purpose of the present study was to examine the effects of acute whole body vibration (WBV) on recovery following a 3 km time trial (3 km TT) and high-intensity interval training (HIIT) (8 x 400 m). Post-HIIT measures included 3 km time-trial performance, exercise metabolism and markers of muscle damage (creatine kinase, CK) and inflammation (c-reactive protein, CRP). A second purpose was to determine the effects of a 3 km TT and HIIT on performance and metabolism the following day. Nine well-trained, middle-aged, male runners [(mean +/- SD) age 45 +/- 6 years, body mass 75 +/- 7 kg, VO2peak 58 +/- 5 ml kg(-1 )min(-1)] performed a constant pace run at 60 and 80% velocity at VO2peak (v VO2peak) followed by a 3-km TT and a 8 x 400-m HIIT session on two occasions. Following one occasion, the athletes performed 2 x 15 min of low frequency (12 Hz) WBV, whilst the other occasion was a non-WBV control. Twenty-four hours after each HIIT session (day 2) participants performed the constant pace run (60 and 80% v VO2peak) and 3 km TT again. There was a significant decrease in 3 km TT performance (~10 s) 24 h after the HIIT session (P < 0.05); however, there were no differences between conditions (control vs. vibration, P > 0.05). Creatine kinase was significantly elevated on day 2, though there were no differences between conditions (P > 0.05). VO2peak and blood lactate were lower on day 2 (P < 0.05), again with no differences between conditions (P > 0.05). These results show no benefit of WBV on running performance recovery following a HIIT session. However, we have shown that there may be acute alterations in metabolism 24 h following such a running session in well-trained, middle-aged runners.

Temperature-induced conformational change at the catalytic site of Sulfolobus solfataricus alcohol dehydrogenase highlighted by Asn249Tyr substitution. A hydrogen/deuterium exchange, kinetic, and fluorescence quenching study.

PubMed

Secundo, Francesco; Russo, Consiglia; Giordano, Antonietta; Carrea, Giacomo; Rossi, Mosè; Raia, Carlo A

2005-08-23

A combination of hydrogen/deuterium exchange, fluorescence quenching, and kinetic studies was used to acquire experimental evidence for the crystallographically hypothesized increase in local flexibility which occurs in thermophilic NAD(+)-dependent Sulfolobus solfataricus alcohol dehydrogenase (SsADH) upon substitution Asn249Tyr. The substitution, located at the adenine-binding site, proved to decrease the affinity for both coenzyme and substrate, rendering the mutant enzyme 6-fold more active when compared to the wild-type enzyme [Esposito et al. (2003) FEBS Lett. 539, 14-18]. The amide H/D exchange data show that the wild-type and mutant enzymes have similar global flexibility at 22 and 60 degrees C. However, the temperature dependence of the Stern-Volmer constant determined by acrylamide quenching shows that the increase in temperature affects the local flexibility differently, since the K(SV) increment is significantly higher for the wild-type than for the mutant enzyme over the range 18-45 degrees C. Interestingly, the corresponding van't Hoff plot (log K(SV) vs 1/T) proves nonlinear for the apo and holo wild-type and apo mutant enzymes, with a break at approximately 45 degrees C in all three cases due to a conformational change affecting the tryptophan microenvironment experienced by the quencher molecules. The Arrhenius and van't Hoff plots derived from the k(cat) and K(M) thermodependence measured with cyclohexanol and NAD(+) at different temperatures display an abrupt change of slope at 45-50 degrees C. This proves more pronounced in the case of the mutant enzyme compared to the wild-type enzyme due to a conformational change in the structure rather than to an overlapping of two or more rate-limiting reaction steps with different temperature dependencies of their rate constants. Three-dimensional analysis indicates that the observed conformational change induced by temperature is associated with the flexible loops directly involved in the substrate and coenzyme binding.

Molecular Cloning and Functional Analysis of a Na+-Insensitive K+ Transporter of Capsicum chinense Jacq

PubMed Central

Ruiz-Lau, Nancy; Bojórquez-Quintal, Emanuel; Benito, Begoña; Echevarría-Machado, Ileana; Sánchez-Cach, Lucila A.; Medina-Lara, María de Fátima; Martínez-Estévez, Manuel

2016-01-01

High-affinity K+ (HAK) transporters are encoded by a large family of genes and are ubiquitous in the plant kingdom. These HAK-type transporters participate in low- and high-affinity potassium (K+) uptake and are crucial for the maintenance of K+ homeostasis under hostile conditions. In this study, the full-length cDNA of CcHAK1 gene was isolated from roots of the habanero pepper (Capsicum chinense). CcHAK1 expression was positively regulated by K+ starvation in roots and was not inhibited in the presence of NaCl. Phylogenetic analysis placed the CcHAK1 transporter in group I of the HAK K+ transporters, showing that it is closely related to Capsicum annuum CaHAK1 and Solanum lycopersicum LeHAK5. Characterization of the protein in a yeast mutant deficient in high-affinity K+ uptake (WΔ3) suggested that CcHAK1 function is associated with high-affinity K+ uptake, with Km and Vmax for Rb of 50 μM and 0.52 nmol mg−1 min−1, respectively. K+ uptake in yeast expressing the CcHAK1 transporter was inhibited by millimolar concentrations of the cations ammonium (NH4+) and cesium (Cs+) but not by sodium (Na+). The results presented in this study suggest that the CcHAK1 transporter may contribute to the maintenance of K+ homeostasis in root cells in C. chinense plants undergoing K+-deficiency and salt stress. PMID:28083010

Computational design of nanoparticle drug delivery systems for selective targeting

NASA Astrophysics Data System (ADS)

Duncan, Gregg A.; Bevan, Michael A.

2015-09-01

Ligand-functionalized nanoparticles capable of selectively binding to diseased versus healthy cell populations are attractive for improved efficacy of nanoparticle-based drug and gene therapies. However, nanoparticles functionalized with high affinity targeting ligands may lead to undesired off-target binding to healthy cells. In this work, Monte Carlo simulations were used to quantitatively determine net surface interactions, binding valency, and selectivity between targeted nanoparticles and cell surfaces. Dissociation constant, KD, and target membrane protein density, ρR, are explored over a range representative of healthy and cancerous cell surfaces. Our findings show highly selective binding to diseased cell surfaces can be achieved with multiple, weaker affinity targeting ligands that can be further optimized by varying the targeting ligand density, ρL. Using the approach developed in this work, nanomedicines can be optimally designed for exclusively targeting diseased cells and tissues.Ligand-functionalized nanoparticles capable of selectively binding to diseased versus healthy cell populations are attractive for improved efficacy of nanoparticle-based drug and gene therapies. However, nanoparticles functionalized with high affinity targeting ligands may lead to undesired off-target binding to healthy cells. In this work, Monte Carlo simulations were used to quantitatively determine net surface interactions, binding valency, and selectivity between targeted nanoparticles and cell surfaces. Dissociation constant, KD, and target membrane protein density, ρR, are explored over a range representative of healthy and cancerous cell surfaces. Our findings show highly selective binding to diseased cell surfaces can be achieved with multiple, weaker affinity targeting ligands that can be further optimized by varying the targeting ligand density, ρL. Using the approach developed in this work, nanomedicines can be optimally designed for exclusively targeting diseased cells and tissues. Electronic supplementary information (ESI) available: Movie showing simulation renderings of targeted (ρL = 1820/μm2, KD = 120 μM) nanoparticle selective binding to cancer (ρR = 256/μm2) vs. healthy (ρR = 64/μm2) cell surfaces. Target membrane proteins have linear color scale depending on binding energy ranging from white when unbound (URL = 0) to red when tightly bound (URL = UM). See DOI: 10.1039/c5nr03691g

Peptide-Based Protein Capture Agents with High Affinity, Selectivity, and Stability as Antibody Replacements in Biodetection Assays

DTIC Science & Technology

2014-08-01

still require months to produce, whereas alternatives such as aptamers [5, 6] require less time to discover, but are not as thermally stable. Smart...Bioanalytical Chemistry, 402(10), 3027-3038 (2012). [5] K.-M. Song, S. Lee, and C. Ban, " Aptamers and Their Biological Applications," Sensors, 12(1...612-631 (2012). [6] B. Strehlitz, N. Nikolaus, and R. Stoltenburg, "Protein Detection with Aptamer Biosensors," Sensors, 8(7), 4296- 4307 (2008). [7

Layered intrusions of the Duluth Complex, Minnesota, USA

USGS Publications Warehouse

Miller, J.D.; Ripley, E.M.; ,

1996-01-01

The Duluth Complex and associated subvolcanic intrusions comprise a large (5000 km2) intrusive complex in northeastern Minnesota that was emplaced into comagmatic volcanics during the development of the 1.1 Ga Midcontinent rift in North America. In addition to anorthositic and felsic intrusions, the Duluth Complex is composed of many individual mafic layered intrusions of tholeiitic affinity. The cumulate stratigraphies and cryptic variations of six of the better exposed and better studied intrusions are described here to demonstrate the variability in their cumulus mineral paragenesis.

Dispersal of potato cyst nematodes measured using historical and spatial statistical analyses.

PubMed

Banks, N C; Hodda, M; Singh, S K; Matveeva, E M

2012-06-01

Rates and modes of dispersal of potato cyst nematodes (PCNs) were investigated. Analysis of records from eight countries suggested that PCNs spread a mean distance of 5.3 km/year radially from the site of first detection, and spread 212 km over ≈40 years before detection. Data from four countries with more detailed histories of invasion were analyzed further, using distance from first detection, distance from previous detection, distance from nearest detection, straight line distance, and road distance. Linear distance from first detection was significantly related to the time since the first detection. Estimated rate of spread was 5.7 km/year, and did not differ statistically between countries. Time between the first detection and estimated introduction date varied between 0 and 20 years, and differed among countries. Road distances from nearest and first detection were statistically significantly related to time, and gave slightly higher estimates for rate of spread of 6.0 and 7.9 km/year, respectively. These results indicate that the original site of introduction of PCNs may act as a source for subsequent spread and that this may occur at a relatively constant rate over time regardless of whether this distance is measured by road or by a straight line. The implications of this constant radial rate of dispersal for biosecurity and pest management are discussed, along with the effects of control strategies.

Creatine kinase and mitochondrial respiration in hearts of trout, cod and freshwater turtle.

PubMed

Birkedal, R; Gesser, H

2003-08-01

The importance of the creatine kinase system in the cardiac muscle of ectothermic vertebrates is unclear. Mammalian cardiac muscle seems to be structurally organized in a manner that compartmentalizes the intracellular environment as evidenced by the substantially higher mitochondrial apparent Km for ADP in skinned fibres compared to isolated mitochondria. A mitochondrial fraction of creatine kinase is functionally coupled to the mitochondrial respiration, and the transport of phosphocreatine and creatine as energy equivalents of ATP and ADP, respectively, increases the mitochondrial apparent ADP affinity, i.e. lowers the Km. This function of creatine kinase seems to be absent in hearts of frog species. To find out whether this applies to hearts of ectothermic vertebrate species in general, we investigated the effect of creatine on the mitochondrial respiration of saponin-skinned fibres from the ventricle of rainbow trout, Atlantic cod and freshwater turtle. For all three species, the apparent Km for ADP appeared to be substantially higher than for isolated mitochondria. Creatine lowered this Km in trout and turtle, thus indicating a functional coupling between mitochondrial creatine kinase and respiration. However, creatine had no effect on Km in cod ventricle. In conclusion, the creatine kinase-system in trout and turtle hearts seems to fulfil the same functions as in the mammalian heart, i.e. facilitating energy transport and communication between cellular compartments. In cod heart, however, this does not seem to be the case.

Development of bacterial display peptides for use in biosensing applications

NASA Astrophysics Data System (ADS)

Stratis-Cullum, Dimitra N.; Kogot, Joshua M.; Sellers, Michael S.; Hurley, Margaret M.; Sarkes, Deborah A.; Pennington, Joseph M.; Val-Addo, Irene; Adams, Bryn L.; Warner, Candice R.; Carney, James P.; Brown, Rebecca L.; Pellegrino, Paul M.

2012-06-01

Recent advances in synthetic library engineering continue to show promise for the rapid production of reagent technology in response to biological threats. A synthetic library of peptide mutants built off a bacterial host offers a convenient means to link the peptide sequence, (i.e., identity of individual library members) with the desired molecular recognition traits, but also allows for a relatively simple protocol, amenable to automation. An improved understanding of the mechanisms of recognition and control of synthetic reagent isolation and evolution remain critical to success. In this paper, we describe our approach to development of peptide affinity reagents based on peptide bacterial display technology with improved control of binding interactions for stringent evolution of reagent candidates, and tailored performance capabilities. There are four key elements to the peptide affinity reagent program including: (1) the diverse bacterial library technology, (2) advanced reagent screening amenable to laboratory automation and control, (3) iterative characterization and feedback on both affinity and specificity of the molecular interactions, and (3) integrated multiscale computational prescreening of candidate peptide ligands including in silico prediction of improved binding performance. Specific results on peptides binders to Protective Antigen (PA) protein of Bacillus anthracis and Staphylococcal Enterotoxin B (SEB) will be presented. Recent highlights of on cell vs. off-cell affinity behavior and correlation of the results with advanced docking simulations on the protein-peptide system(s) are included. The potential of this technology and approach to enable rapid development of a new affinity reagent with unprecedented speed (less than one week) would allow for rapid response to new and constantly emerging threats.

Constraining the local variance of H {sub 0} from directional analyses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bengaly, C.A.P. Jr., E-mail: carlosap@on.br

We evaluate the local variance of the Hubble Constant H {sub 0} with low-z Type Ia Supernovae (SNe). Our analyses are performed using a hemispherical comparison method in order to test whether taking the bulk flow motion into account can reconcile the measurement of the Hubble Constant H {sub 0} from standard candles ( H {sub 0} = 73.8±2.4 km s{sup -1} Mpc {sup -1}) with that of the Planck's Cosmic Microwave Background data ( H {sub 0} = 67.8 ± 0.9km s{sup -1} Mpc{sup -1}). We obtain that H {sub 0} ranges from 68.9±0.5 km s{sup -1} Mpc{sup -1}more » to 71.2±0.7 km s{sup -1} Mpc{sup -1} through the celestial sphere (1 σ uncertainty), implying a Hubble Constant maximal variance of δ H {sub 0} = (2.30±0.86) km s{sup -1} Mpc{sup -1} towards the ( l,b ) = (315°,27°) direction. Interestingly, this result agrees with the bulk flow direction estimates found in the literature, as well as previous evaluations of the H {sub 0} variance due to the presence of nearby inhomogeneities. We assess the statistical significance of this result with different prescriptions of Monte Carlo simulations, obtaining moderate statistical significance, i.e., 68.7% confidence level (CL) for such variance. Furthermore, we test the hypothesis of a higher H {sub 0} value in the presence of a bulk flow velocity dipole, finding some evidence for this result which, however, cannot be claimed to be significant due to the current large uncertainty in the SNe distance modulus. Then, we conclude that the tension between different H {sub 0} determinations can plausibly be caused to the bulk flow motion of the local Universe, even though the current incompleteness of the SNe data set, both in terms of celestial coverage and distance uncertainties, does not allow a high statistical significance for these results or a definitive conclusion about this issue.« less

Cellular and Biophysical Pipeline for the Screening of Peroxisome Proliferator-Activated Receptor Beta/Delta Agonists: Avoiding False Positives

PubMed Central

Batista, Fernanda Aparecida Heleno

2018-01-01

Peroxisome proliferator-activated receptor beta/delta (PPARß/δ) is considered a therapeutic target for metabolic disorders, cancer, and cardiovascular diseases. Here, we developed one pipeline for the screening of PPARß/δ agonists, which reduces the cost, time, and false-positive hits. The first step is an optimized 3-day long cellular transactivation assay based on reporter-gene technology, which is supported by automated liquid-handlers. This primary screening is followed by a confirmatory transactivation assay and by two biophysical validation methods (thermal shift assay (TSA) and (ANS) fluorescence quenching), which allow the calculation of the affinity constant, giving more information about the selected hits. All of the assays were validated using well-known commercial agonists providing trustworthy data. Furthermore, to validate and test this pipeline, we screened a natural extract library (560 extracts), and we found one plant extract that might be interesting for PPARß/δ modulation. In conclusion, our results suggested that we developed a cheaper and more robust pipeline that goes beyond the single activation screening, as it also evaluates PPARß/δ tertiary structure stabilization and the ligand affinity constant, selecting only molecules that directly bind to the receptor. Moreover, this approach might improve the effectiveness of the screening for agonists that target PPARß/δ for drug development.

Actinomycin D binding mode reveals the basis for its potent HIV-1 and cancer activity

NASA Astrophysics Data System (ADS)

Paramanathan, Thayaparan; Vladescu, Ioana D.; McCauley, Micah J.; Rouzina, Ioulia; Williams, Mark C.

2011-03-01

Actinomycin D (ActD) is one of the most studied antibiotics, which has been used as an anti-cancer agent and also shown to inhibit HIV reverse transcription. Initial studies with ActD established that it intercalates double stranded DNA (dsDNA). However, recent studies have shown that ActD binds with even higher affinity to single stranded DNA (ssDNA). In our studies we use optical tweezers to stretch and hold single dsDNA molecule at constant force in the presence of varying ActD concentrations until the binding reaches equilibrium. The change in dsDNA length upon ActD binding measured as a function of time yields the rate of binding in addition to the equilibrium lengthening of DNA. The results suggest extremely slow kinetics, on the order of several minutes and 0.52 +/- 0.06 μ M binding affinity. Holding DNA at constant force while stretching and relaxing suggests that ActD binds to two single strands that are close to each other rather than to pure dsDNA or ssDNA. This suggests that biological activity of ActD that contributes towards the inhibition of cellular replication is due to its ability to bind at DNA bubbles during RNA transcription, thereby stalling the transcription process.

Mechanism of Metal Ion Activation of the Diphtheria Toxin Repressor DtxR

DOE Office of Scientific and Technical Information (OSTI.GOV)

D'Aquino,J.; Tetenbaum-Novatt, J.; White, A.

2005-01-01

The diphtheria toxin repressor (DtxR) is a metal ion-activated transcriptional regulator that has been linked to the virulence of Corynebacterium diphtheriae. Structure determination has shown that there are two metal ion binding sites per repressor monomer, and site-directed mutagenesis has demonstrated that binding site 2 (primary) is essential for recognition of the target DNA repressor, leaving the role of binding site 1 (ancillary) unclear. Calorimetric techniques have demonstrated that although binding site 1 (ancillary) has high affinity for metal ion with a binding constant of 2 x 10{sup -7}, binding site 2 (primary) is a low-affinity binding site with amore » binding constant of 6.3 x 10{sup -4}. These two binding sites act in an independent fashion, and their contribution can be easily dissected by traditional mutational analysis. Our results clearly demonstrate that binding site 1 (ancillary) is the first one to be occupied during metal ion activation, playing a critical role in stabilization of the repressor. In addition, structural data obtained for the mutants Ni-DtxR(H79A, C102D), reported here, and the previously reported DtxR(H79A) have allowed us to propose a mechanism of metal activation for DtxR.« less

Coordination Chemistry of Bifunctional Chemical Agents Designed for Applications in 64Cu PET Imaging for Alzheimer's Disease.

PubMed

Sharma, Anuj K; Schultz, Jason W; Prior, John T; Rath, Nigam P; Mirica, Liviu M

2017-11-20

Positron emission tomography (PET) is emerging as one of the most important diagnostic tools for brain imaging, yet the most commonly used radioisotopes in PET imaging, 11 C and 18 F, have short half-lives, and their usage is thus somewhat limited. By comparison, the 64 Cu radionuclide has a half-life of 12.7 h, which is ideal for administering and imaging purposes. In spite of appreciable research efforts, high-affinity copper chelators suitable for brain imaging applications are still lacking. Herein, we present the synthesis and characterization of a series of bifunctional compounds (BFCs) based on macrocyclic 1,4,7-triazacyclononane and 2,11-diaza[3.3](2,6)pyridinophane ligand frameworks that exhibit a high affinity for Cu 2+ ions. In addition, these BFCs contain a 2-phenylbenzothiazole fragment that is known to interact tightly with amyloid β fibrillar aggregates. Determination of the protonation constants (pK a values) and stability constants (log β values) of these BFCs, as well as characterization of the isolated copper complexes using X-ray crystallography, electron paramagnetic resonance spectroscopy, and electrochemical studies, suggests that these BFCs exhibit desirable properties for the development of novel 64 Cu PET imaging agents for Alzheimer's disease.

A new real-time method for investigation of affinity properties and binding kinetics of magnetic nanoparticles

NASA Astrophysics Data System (ADS)

Orlov, Alexey V.; Nikitin, Maxim P.; Bragina, Vera A.; Znoyko, Sergey L.; Zaikina, Marina N.; Ksenevich, Tatiana I.; Gorshkov, Boris G.; Nikitin, Petr I.

2015-04-01

A method for quantitative investigation of affinity constants of receptors immobilized on magnetic nanoparticles (MP) is developed based on spectral correlation interferometry (SCI). The SCI records with a picometer resolution the thickness changes of a layer of molecules or nanoparticles due to a biochemical reaction on a cover slip, averaged over the sensing area. The method is compatible with other types of sensing surfaces employed in biosensing. The measured values of kinetic association constants of magnetic nanoparticles are 4 orders of magnitude higher than those of molecular antibody association with antigen. The developed method also suggests highly sensitive detection of antigens in a wide dynamic range. The limit of detection of 92 pg/ml has been demonstrated for prostate-specific antigen (PSA) with 50-nm MP employed as labels, which produce 3-order amplification of the SCI signals. The calibration curve features high sensitivity (slope) of 3-fold signal raise per 10-fold increase of PSA concentration within 4-order dynamic range, which is an attractive compromise for precise quantitative and highly sensitive immunoassay. The proposed biosensing technique offers inexpensive disposable sensor chips of cover slips and represents an economically sound alternative to traditional immunoassays for disease diagnostics, detection of pathogens in food and environmental monitoring.

Size and shape of Saturn's moon Titan.

PubMed

Zebker, Howard A; Stiles, Bryan; Hensley, Scott; Lorenz, Ralph; Kirk, Randolph L; Lunine, Jonathan

2009-05-15

Cassini observations show that Saturn's moon Titan is slightly oblate. A fourth-order spherical harmonic expansion yields north polar, south polar, and mean equatorial radii of 2574.32 +/- 0.05 kilometers (km), 2574.36 +/- 0.03 km, and 2574.91 +/- 0.11 km, respectively; its mean radius is 2574.73 +/- 0.09 km. Titan's shape approximates a hydrostatic, synchronously rotating triaxial ellipsoid but is best fit by such a body orbiting closer to Saturn than Titan presently does. Titan's lack of high relief implies that most--but not all--of the surface features observed with the Cassini imaging subsystem and synthetic aperture radar are uncorrelated with topography and elevation. Titan's depressed polar radii suggest that a constant geopotential hydrocarbon table could explain the confinement of the hydrocarbon lakes to high latitudes.

Size and shape of Saturn's moon Titan

USGS Publications Warehouse

Zebker, Howard A.; Stiles, Bryan; Hensley, Scott; Lorenz, Ralph; Kirk, Randolph L.; Lunine, Jonathan

2009-01-01

Cassini observations show that Saturn's moon Titan is slightly oblate. A fourth-order spherical harmonic expansion yields north polar, south polar, and mean equatorial radii of 2574.32 ± 0.05 kilometers (km), 2574.36 ± 0.03 km, and 2574.91 ± 0.11 km, respectively; its mean radius is 2574.73 ± 0.09 km. Titan's shape approximates a hydrostatic, synchronously rotating triaxial ellipsoid but is best fit by such a body orbiting closer to Saturn than Titan presently does. Titan's lack of high relief implies that most—but not all—of the surface features observed with the Cassini imaging subsystem and synthetic aperture radar are uncorrelated with topography and elevation. Titan's depressed polar radii suggest that a constant geopotential hydrocarbon table could explain the confinement of the hydrocarbon lakes to high latitudes.

Lunar Seismology: the Internal Structure of the Moon. Ph.D. Thesis

NASA Technical Reports Server (NTRS)

Goins, N. R.

1978-01-01

The direct P and S wave arrival times are the primary data set that can be measured on the seismograms of natural lunar seismic events. Polarization filtering techniques allow the enhancement of secondary body wave arrivals and record curves to identify the secondary phases and deduce structural information. Finally, shear wave amplitude vs. distance curves yield information on the location and magnitude of seismic velocity gradients in the interior. The results of these analyses show that the moon appears to have a two-layer crust at all four seismic stations: a 20 km upper crust that seems to be constant at all sites and a lower crust that is 40 km thick at stations 12 and 14 (mare), 55 + or - 10 km at station 16 (highland), and tentatively either 40 km or 70 km at station 15. The lower mantle extends from 480 km to at least 1100 km depth which is the maximum depth of penetration of all but a few seismic waves used as data. No definitive evidence for or against a lunar core exists.

Molecular Interaction Between Salivary Proteins and Food Tannins.

PubMed

Silva, Mafalda Santos; García-Estévez, Ignacio; Brandão, Elsa; Mateus, Nuno; de Freitas, Victor; Soares, Susana

2017-08-09

Polyphenols interaction with salivary proteins (SP) has been related with organoleptic features such as astringency. The aim of this work was to study the interaction between some human SP and tannins through two spectroscopic techniques, fluorescence quenching, and saturation transfer difference-nuclear magnetic resonance (STD-NMR). Generally, the results showed a significant interaction between SP and both condensed tannins and ellagitannins. Herein, STD-NMR proved to be a useful tool to map tannins' epitopes of binding, while fluorescence quenching allowed one to discriminate binding affinities. Ellagitannins showed the greatest binding constants values (K SV from 20.1 to 94.1 mM -1 ; K A from 0.7 to 8.3 mM -1 ) in comparison with procyanidins (K SV from 5.4 to 40.0 mM -1 ; K A from 1.1 to 2.7 mM -1 ). In fact, punicalagin was the tannin that demonstrated the highest affinity for all three SP. Regarding SP, P-B peptide was the one with higher affinity for ellagitannins. On the other hand, cystatins showed in general the lower K SV and K A values. In the case of condensed tannins, statherin was the SP with the highest affinity, contrasting with the other two SP. Altogether, these results are evidence that the distinct SP present in the oral cavity have different abilities to interact with food tannins class.

Identification of Elf-1 and B61 as high affinity ligands for the receptor tyrosine kinase MDK1.

PubMed

Ciossek, T; Ullrich, A

1997-01-09

Mouse Developmental Kinase 1 (MDK1) is a receptor tyrosine kinase of the eck/eph subfamily expressed in a variety of tissues during early mouse embryogenesis. To obtain further insight into the function of MDK1, we determined identity and localisation of its physiological ligand(s). Staining whole embryos with fusion proteins between the extracellular domain of MDK1 and human secreted alkaline phosphatase revealed areas of high receptor binding in the caudal mesencephalon, the frontal neocortex and the limb buds. This staining was sensitive to treatment with phosphatidylinositol-specific phospholipase C. Using Scatchard analysis, high affinity binding of Elf-1 (1.7 x 10(-10) M) and B61 (2.2 x 10(-10) M) towards MDK1 could be demonstrated. However, the transmembrane ligand Lerk2 displayed no measurable affinity for MDK1. Elf-1 and B61 bind to the three full-length MDK1 isoforms with similar dissociation constants. Slightly lower affinities were observed for the two truncated receptors MDK1-Tl and MDK1-T2. The activation of MDK1 with Elf-1 or B61 leads to the rapid autophosphorylation of MDK1 as well as tyrosine phosphorylation of an unknown 62 kDa phosphoprotein in Rat1 cells. These findings implicate MDK1 in patterning processes during early mouse embryogenesis and suggest MDK1 involvement in early organogenesis and midbrain development.

Choline Uptake in Agrobacterium tumefaciens by the High-Affinity ChoXWV Transporter▿

PubMed Central

Aktas, Meriyem; Jost, Kathinka A.; Fritz, Christiane; Narberhaus, Franz

2011-01-01

Agrobacterium tumefaciens is a facultative phytopathogen that causes crown gall disease. For successful plant transformation A. tumefaciens requires the membrane lipid phosphatidylcholine (PC), which is produced via the methylation and the PC synthase (Pcs) pathways. The latter route is dependent on choline. Although choline uptake has been demonstrated in A. tumefaciens, the responsible transporter(s) remained elusive. In this study, we identified the first choline transport system in A. tumefaciens. The ABC-type choline transporter is encoded by the chromosomally located choXWV operon (ChoX, binding protein; ChoW, permease; and ChoV, ATPase). The Cho system is not critical for growth and PC synthesis. However, [14C]choline uptake is severely reduced in A. tumefaciens choX mutants. Recombinant ChoX is able to bind choline with high affinity (equilibrium dissociation constant [KD] of ≈2 μM). Since other quaternary amines are bound by ChoX with much lower affinities (acetylcholine, KD of ≈80 μM; betaine, KD of ≈470 μM), the ChoXWV system functions as a high-affinity transporter with a preference for choline. Two tryptophan residues (W40 and W87) located in the predicted ligand-binding pocket are essential for choline binding. The structural model of ChoX built on Sinorhizobium meliloti ChoX resembles the typical structure of substrate binding proteins with a so-called “Venus flytrap mechanism” of substrate binding. PMID:21803998

Enzyme activity assay of glycoprotein enzymes based on a boronate affinity molecularly imprinted 96-well microplate.

PubMed

Bi, Xiaodong; Liu, Zhen

2014-12-16

Enzyme activity assay is an important method in clinical diagnostics. However, conventional enzyme activity assay suffers from apparent interference from the sample matrix. Herein, we present a new format of enzyme activity assay that can effectively eliminate the effects of the sample matrix. The key is a 96-well microplate modified with molecularly imprinted polymer (MIP) prepared according to a newly proposed method called boronate affinity-based oriented surface imprinting. Alkaline phosphatase (ALP), a glycoprotein enzyme that has been routinely used as an indicator for several diseases in clinical tests, was taken as a representative target enzyme. The prepared MIP exhibited strong affinity toward the template enzyme (with a dissociation constant of 10(-10) M) as well as superb tolerance for interference. Thus, the enzyme molecules in a complicated sample matrix could be specifically captured and cleaned up for enzyme activity assay, which eliminated the interference from the sample matrix. On the other hand, because the boronate affinity MIP could well retain the enzymatic activity of glycoprotein enzymes, the enzyme captured by the MIP was directly used for activity assay. Thus, additional assay time and possible enzyme or activity loss due to an enzyme release step required by other methods were avoided. Assay of ALP in human serum was successfully demonstrated, suggesting a promising prospect of the proposed method in real-world applications.

Tryptophan tags and de novo designed complementary affinity ligands for the expression and purification of recombinant proteins.

PubMed

Pina, Ana Sofia; Carvalho, Sara; Dias, Ana Margarida G C; Guilherme, Márcia; Pereira, Alice S; Caraça, Luciana T; Coroadinha, Ana Sofia; Lowe, Christopher R; Roque, A Cecília A

2016-11-11

A common strategy for the production and purification of recombinant proteins is to fuse a tag to the protein terminal residues and employ a "tag-specific" ligand for fusion protein capture and purification. In this work, we explored the effect of two tryptophan-based tags, NWNWNW and WFWFWF, on the expression and purification of Green Fluorescence Protein (GFP) used as a model fusion protein. The titers obtained with the expression of these fusion proteins in soluble form were 0.11mgml -1 and 0.48mgml -1 for WFWFWF and NWNWNW, respectively. A combinatorial library comprising 64 ligands based on the Ugi reaction was prepared and screened for binding GFP-tagged and non-tagged proteins. Complementary ligands A2C2 and A3C1 were selected for the effective capture of NWNWNW and WFWFWF tagged proteins, respectively, in soluble forms. These affinity pairs displayed 10 6 M -1 affinity constants and Qmax values of 19.11±2.60ugg -1 and 79.39ugg -1 for the systems WFWFWF AND NWNWNW, respectively. GFP fused to the WFWFWF affinity tag was also produced as inclusion bodies, and a refolding-on column strategy was explored using the ligand A4C8, selected from the combinatorial library of ligands but in presence of denaturant agents. Copyright © 2016 Elsevier B.V. All rights reserved.

Rapid characterization of a novel taspine derivative-HMQ1611 binding to EGFR by a cell membrane chromatography method.

PubMed

Du, Hui; Lv, Nan; Wang, Sicen; He, Langchong

2013-05-01

A new high-expression endothelial growth factor receptor (EGFR) cell membrane chromatography (CMC) method was applied to recognize the ligands acting on EGFR specifically, and investigate the affinity of gefitinib/HMQ1611 to EGFR. In the self and direct competitive assay, gefitinib/HMQ1611 was used as a competitor in the mobile phase to evaluate the effect of the competitor's concentrations on the retention of the ligands, respectively, and the competition between gefitinib and HMQ1611 binding to EGFR was also been examined. The retention behavior indicated that gefitinib had one type of binding sites on the EGFR, and the equilibrium dissociation constant (K(D)) was (9.11 ± 1.89) × 10(-6) M; HMQ1611 had two major binding regions on the EGFR, and the K(D) values obtained from the model were (2.39 ± 0.33) × 10(-7) and (3.87 ± 0.93) × 10(-5) M for HMQ1611 at the high- and low-affinity sites, respectively. The competition between gefitinib and HMQ1611 occurred at the low-affinity sites on the EGFR. The low-affinity sites were of higher concentrations and contributed to a much larger part of retention of HMQ1611. The results suggested that gefitinib and HMQ1611 competed for the common binding sites on the EGFR, no matter the ligand was used as an analyte or a competitor.

Influence of pH and ionic strength on electrostatic properties of ferredoxin, FNR, and hydrogenase and the rate constants of their interaction

NASA Astrophysics Data System (ADS)

Diakonova, A. N.; Khrushchev, S. S.; Kovalenko, I. B.; Riznichenko, G. Yu; Rubin, A. B.

2016-10-01

Ferredoxin (Fd) protein transfers electrons from photosystem I (PSI) to ferredoxin:NADP+-reductase (FNR) in the photosynthetic electron transport chain, as well as other metabolic pathways. In some photosynthetic organisms including cyanobacteria and green unicellular algae under anaerobic conditions Fd transfers electrons not only to FNR but also to hydrogenase—an enzyme which catalyzes reduction of atomic hydrogen to H2. One of the questions posed by this competitive relationship between proteins is which characteristics of thylakoid stroma media allow switching of the electron flow between the linear path PSI-Fd-FNR-NADP+ and the path PSI-Fd-hydrogenase-H2. The study was conducted using direct multiparticle simulation approach. In this method protein molecules are considered as individual objects that experience Brownian motion and electrostatic interaction with the surrounding media and each other. Using the model we studied the effects of pH and ionic strength (I) upon complex formation between ferredoxin and FNR and ferredoxin and hydrogenase. We showed that the rate constant of Fd-FNR complex formation is constant in a wide range of physiologically significant pH values. Therefore it can be argued that regulation of FNR activity doesn’t involve pH changes in stroma. On the other hand, in the model rate constant of Fd-hydrogenase interaction dramatically depends upon pH: in the range 7-9 it increases threefold. It may seem that because hydrogenase reduces protons it should be more active when pH is acidic. Apparently, regulation of hydrogenase’s affinity to both her reaction partners (H+ and Fd) is carried out by changes in its electrostatic properties. In the dark, the protein is inactive and in the light it is activated and starts to interact with both Fd and H+. Therefore, we can conclude that in chloroplasts the rate of hydrogen production is regulated by pH through the changes in the affinity between hydrogenase and ferredoxin.

Emerging spatial curvature can resolve the tension between high-redshift CMB and low-redshift distance ladder measurements of the Hubble constant

NASA Astrophysics Data System (ADS)

Bolejko, Krzysztof

2018-05-01

The measurements of the Hubble constant reveal a tension between high-redshift (CMB) and low-redshift (distance ladder) constraints. So far neither observational systematics nor new physics has been successfully implemented to explain away this tension. This paper presents a new solution to the Hubble constant problem. The solution is based on the Simsilun simulation (relativistic simulation of the large scale structure of the Universe) with the ray-tracing algorithm implemented. The initial conditions for the Simsilun simulation were set up as perturbations around the Λ CDM model. However, unlike in the standard cosmological model (i.e., Λ CDM model +perturbations ), within the Simsilun simulation relativistic and nonlinear evolution of cosmic structures lead to the phenomenon of emerging spatial curvature, where the mean spatial curvature evolves from the spatial flatness of the early Universe towards the slightly curved present-day Universe. Consequently, the present-day expansion rate is slightly faster compared to the spatially flat Λ CDM model. The results of the ray-tracing analysis show that the Universe which starts with initial conditions consistent with the Planck constraints should have the Hubble constant H0=72.5 ±2.1 km s-1 Mpc-1 . When the Simsilun simulation was rerun with no inhomogeneities imposed, the Hubble constant inferred within such a homogeneous simulation was H0=68.1 ±2.0 km s-1 Mpc-1 . Thus, the inclusion of nonlinear relativistic evolution that leads to the emergence of the spatial curvature can explain why the low-redshift measurements favor higher values compared to the high-redshift constraints and alleviate the tension between the CMB and distance ladder measurements of the Hubble constant.

Detection of Volatile Organic Compounds by Self-assembled Monolayer Coated Sensor Array with Concentration-independent Fingerprints

PubMed Central

Chang, Ye; Tang, Ning; Qu, Hemi; Liu, Jing; Zhang, Daihua; Zhang, Hao; Pang, Wei; Duan, Xuexin

2016-01-01

In this paper, we have modeled and analyzed affinities and kinetics of volatile organic compounds (VOCs) adsorption (and desorption) on various surface chemical groups using multiple self-assembled monolayers (SAMs) functionalized film bulk acoustic resonator (FBAR) array. The high-frequency and micro-scale resonator provides improved sensitivity in the detections of VOCs at trace levels. With the study of affinities and kinetics, three concentration-independent intrinsic parameters (monolayer adsorption capacity, adsorption energy constant and desorption rate) of gas-surface interactions are obtained to contribute to a multi-parameter fingerprint library of VOC analytes. Effects of functional group’s properties on gas-surface interactions are also discussed. The proposed sensor array with concentration-independent fingerprint library shows potential as a portable electronic nose (e-nose) system for VOCs discrimination and gas-sensitive materials selections. PMID:27045012

(/sup 3/H)-(Thr4,Gly7)OT: a highly selective ligand for central and peripheral OT receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Elands, J.; Barberis, C.; Jard, S.

1988-01-01

Oxytocin receptors in rat hippocampal synaptic plasma membranes were compared with mammary gland and uterine oxytocin receptors. For this purpose, a highly specific oxytocic agonist (Thr4,Gly7)oxytocin was tritiated. We demonstrated that this ligand labels oxytocin receptors selectively. Scatchard analyses revealed a high affinity for all the oxytocin receptors investigated, with equilibrium dissociation constants between 1.0 and 2.0 nM. Binding appeared to take place at a single population of receptor sites. Competition experiments confirmed the high affinity of arginine vasopressin for hippocampal oxytocin receptors but also revealed that mammary gland and uterine oxytocin receptors do not discriminate more efficiently between oxytocinmore » and arginine vasopressin. This lack in specificity is not affected by applying different concentrations of Mg ions.« less

A central black hole in M32

NASA Technical Reports Server (NTRS)

Tonry, John L.

1987-01-01

Observations are presented of the stellar rotation and velocity dispersion in M32. The projected rotation curve has an unresolved cusp at the center, with an amplitude of at least 60 km/s. The stellar velocity dispersion is constant at 56 + or - 5 km/s to a radius of 20 arcsec; a central bump in the observed dispersion is an artifact due to the rotation. The form of the rotation is such that isophotes have constant angular rotation velocity. The three-dimensional rotation field is modeled and the internal mean rotation of the stars around the center of M32 must reach at least 90 km/s at a radius of 2 pc. Hydrostatic equilibrium then requires 3-10 x 10 to the 6th solar masses of dark mass within the central parsec of M32. The possibility that M32 is undergoing core collapse and that this dark mass consists of dark stellar remnants is discussed, but ultimately rejected because the time scale for core collapse of M32 should be 2000 Hubble times. A more likely explanation of this dark mass, especially because of the presence of an X-ray point source at the center of M32, is a massive black hole.

Kinetic properties of the human liver cytosolic aldehyde dehydrogenase for retinal isomers.

PubMed

Bhat, P V; Samaha, H

1999-01-15

Retinoic acid exerts pleiotropic effects by acting through two families of nuclear receptors, RAR and RXR. All-trans and 9-cis retinoic acid bind RARs, whereas 9-cis retinoic acid binds and activates only the RXRs. To understand the role of human liver cytosolic aldehyde dehydrogenase (ALDH1) in retinoic acid synthesis, we examined the ability of ALDH 1 to catalyze the oxidation of the naturally occurring retinal isomers. ALDH1 catalyzed the oxidation of all-trans, 9-cis, and 13-cis retinal with equal efficiency. However, the affinity to all-trans retinal (Km = 2.2 microM) was twofold higher than to 9-cis (Km = 5.5 microM) and 13-cis (Km = 4.6 microM) retinal. All-trans retinol was a potent inhibitor of ALDH1 activity, and inhibited all-trans retinal oxidation uncompetitively. Comparison of the kinetic properties of ALDH1 for retinal isomers with those of previously reported rat kidney retinal dehydrogenase showed distinct differences, suggesting that ALDH1 may play a different role in retinal metabolism in liver.

Variation in light intensity with height and time from subsequent lightning return strokes

NASA Technical Reports Server (NTRS)

Jordan, D. M.; Uman, M. A.

1983-01-01

Photographic measurements of relative light intensity as a function of height and time have been conducted for seven return strokes in two lightning flashes at 7.8 and 8.7 km ranges, using film which possesses an approximately constant spectral response in the 300-670 nm range. The amplitude of the initial light peak is noted to decrease exponentially with height, with a decay constant of 0.6-0.8 km. The logarithm of the peak light intensity near the ground is found to be approximately proportional to the initial peak electric field intensity, implying that the current decrease with height may be much slower than the light decrease. Absolute light intensity is presently estimated through the integration of the photographic signals from individual channel segments, in order to simulate the calibrated, all-sky photoelectric data of Guo and Krider (1982).

Kinetics of ATP hydrolysis catalyzed by isolated TF1 and reconstituted TF0F1 ATPase.

PubMed

Rögner, M; Gräber, P

1986-09-01

The rate of ATP hydrolysis catalyzed by isolated TF1 and reconstituted TF0F1 was measured as a function of the ATP concentration in the presence of inhibitors [ADP, Pi and 3'-O-(1-naphthoyl)ATP]. ATP hydrolysis can be described by Michaelis-Menten kinetics with Km(TF1) = 390 microM and Km (TF0F1) = 180 microM. The inhibition constants are for ADP Ki(TF1) = 20 microM and Ki(TF0F1) = 100 microM, for 3'-O-(1-naphthoyl)ATP Ki(TF1) = 150 microM and Ki(TF0F1) = 3 microM, and for Pi Ki(TF1) = 60 mM. From these results it is concluded that upon binding of TF0 to TF1 the mechanism of ATP hydrolysis catalyzed by TF1 is not changed qualitatively; however, the kinetic constants differ quantitatively.

Influence of cimetidine and diethyldithiocarbamate on the metabolism of halothane and methoxyflurane in vitro.

PubMed

Loesch, J; Siegers, C P; Younes, M

1987-06-01

The metabolism of halothane and methoxyflurane was measured in vitro by the vial equilibration method using the S-9-fraction from rat liver as source of enzymes. Kinetic values were measured for halothane: Vmax = 11.6 nmol/g.min, KM = 19.6 mumol/l and methoxyflurane: Vmax = 12.0 nmol/g.min, KM = 17.5 mumol/l. Dithiocarb showed strong inhibitory activity on halothane and methoxyflurane metabolism; inhibition constants were calculated as Ki = 0.051 mmol/l and Ki = 0.004 mmol/l, respectively. Cimetidine inhibited the metabolism of both anesthetics to a lesser extent. Inhibition constants were calculated as Ki = 16.2 mmol/l and Ki = 8.2 mmol/l for halothane and methoxyflurane, respectively. The observed inhibitory properties of dithiocarb and cimetidine on the metabolism of halothane and methoxyflurane may be of interest in connection with the problem of toxic liver and kidney injury after anesthesia with these agents.

Distribution of fine-scale mantle heterogeneity from observations of Pdiff coda

USGS Publications Warehouse

Earle, P.S.; Shearer, P.M.

2001-01-01

We present stacked record sections of Global Seismic Network data that image the average amplitude and polarization of the high-frequency Pdiff coda and investigate their implications on the depth extent of fine-scale (~10 km) mantle heterogeneity. The extended 1-Hz coda lasts for at least 150 sec and is observed to a distance of 130??. The coda's polarization angle is about the same as the main Pdiff arrival (4.4 sec/deg) and is nearly constant with time. Previous studies show that multiple scattering from heterogeneity restricted to the lowermost mantle generates an extended Pdiff coda with a constant polarization. Here we present an alternative model that satisfies our Pdiff observations. The model consists of single scattering from weak (~1%) fine-scale (~2 km) structures distributed throughout the mantle. Although this model is nonunique, it demonstrates that Pdiff coda observations do not preclude the existence of scattering contributions from the entire mantle.

Probing the active site of alpha-class rat liver glutathione S-transferases using affinity labeling by monobromobimane.

PubMed Central

Hu, L.; Borleske, B. L.; Colman, R. F.

1997-01-01

Monobromobimane (mBBr) is a substrate of both mu- and alpha-class rat liver glutathione S-transferases, with Km values of 0.63 microM and 4.9 microM for the mu-class isozymes 3-3 and 4-4, respectively, and 26 microM for the alpha-class isozymes 1-1 and 2-2. In the absence of substrate glutathione, mBBr acts as an affinity label of the 1-1 as well as mu-class isozymes, but not of the alpha-class 2-2 isozyme. Incubation of rat liver isozyme 1-1 with mBBr at pH 7.5 and 25 degrees C results in a time-dependent inactivation of the enzyme but at a slower (threefold) rate than for reactions with the mu-class isozyme 3-3 and 4-4. The rate of inactivation of 1-1 isozyme by mBBr is not decreased but, rather, is slightly enhanced by S-methyl glutathione. In contrast, 17 beta-estradiol-3,17-disulfate (500 microM) gives a 12.5-fold decrease in the observed rate constant of inactivation by 4 mM mBBr. When incubated for 60 min with 4 mM mBBr, the 1-1 isozyme loses 60% of its activity and incorporates 1.7 mol reagent/mol subunit. Peptide analysis after thermolysin digestion indicates that mBBr modification is equally distributed between two cysteine residues at positions 17 and 111. Modification at these two sites is reduced equally in the presence of the added protectant, 17 beta-estradiol-3,17-disulfate, suggesting that Cys 17 and Cys 111 reside within or near the enzyme's steroid binding sites. In contrast to the 1-1 isozyme, the other alpha-class isozyme (2-2) is not inactivated by mBBr at concentrations as high as 15 mM. The different reaction kinetics and modification sites by mBBr suggest that distinct binding site structures are responsible for the characteristic substrate specificities of glutathione S-transferase isozymes. PMID:9007975

Probing the active site of alpha-class rat liver glutathione S-transferases using affinity labeling by monobromobimane.

PubMed

Hu, L; Borleske, B L; Colman, R F

1997-01-01

Monobromobimane (mBBr) is a substrate of both mu- and alpha-class rat liver glutathione S-transferases, with Km values of 0.63 microM and 4.9 microM for the mu-class isozymes 3-3 and 4-4, respectively, and 26 microM for the alpha-class isozymes 1-1 and 2-2. In the absence of substrate glutathione, mBBr acts as an affinity label of the 1-1 as well as mu-class isozymes, but not of the alpha-class 2-2 isozyme. Incubation of rat liver isozyme 1-1 with mBBr at pH 7.5 and 25 degrees C results in a time-dependent inactivation of the enzyme but at a slower (threefold) rate than for reactions with the mu-class isozyme 3-3 and 4-4. The rate of inactivation of 1-1 isozyme by mBBr is not decreased but, rather, is slightly enhanced by S-methyl glutathione. In contrast, 17 beta-estradiol-3,17-disulfate (500 microM) gives a 12.5-fold decrease in the observed rate constant of inactivation by 4 mM mBBr. When incubated for 60 min with 4 mM mBBr, the 1-1 isozyme loses 60% of its activity and incorporates 1.7 mol reagent/mol subunit. Peptide analysis after thermolysin digestion indicates that mBBr modification is equally distributed between two cysteine residues at positions 17 and 111. Modification at these two sites is reduced equally in the presence of the added protectant, 17 beta-estradiol-3,17-disulfate, suggesting that Cys 17 and Cys 111 reside within or near the enzyme's steroid binding sites. In contrast to the 1-1 isozyme, the other alpha-class isozyme (2-2) is not inactivated by mBBr at concentrations as high as 15 mM. The different reaction kinetics and modification sites by mBBr suggest that distinct binding site structures are responsible for the characteristic substrate specificities of glutathione S-transferase isozymes.

Carbon dioxide /V2/ radiance results using a new nonequilibrium model

NASA Astrophysics Data System (ADS)

Sharma, R. D.; Nadile, R. M.

1981-01-01

It was observed during the SPIRE experiment (Spectral Infrared Rocket Experiment) that the 15 micron limb radiance stays constant from 95 to 110 km despite the fact that CO2 concentration over this altitude range decreases by a factor of 20. The results of a 15 micron CO2 radiance model are presented which explain the observed anomaly. It is shown that CO2 deactivation by oxygen is the predominant factor in 15 micron emission above 95 km.

A non-local thermodynamic equilibrium radiative transfer model for ingrared emissions in the atmosphere of Mars. 1: Theoretical basis and nighttime populations of vibrational levels

NASA Astrophysics Data System (ADS)

Lopez-Valverde, M. A.; Lopez-Puertas, M.

1994-06-01

A radiative transfer model to study the infrared (1-20 micrometer) emissions of the CO and CO2 molecules in the atmosphere of Mars has been developed. The model runs from the planet's surface up to 180 km and has been especially elaborated to study non-local thermodynamic equilibrium (non-LTE) situations. it includes the most important energy levels and vibration-rotation bands able to give a significant atmospheric emission or produce a significant cooling/heating rate. Exchanges of energy in thermal and nonthermal (vibrational-vibrational) collisions as well as by radiative processes have been included. An exhaustive review of the rate constants for vibrational-thermal and vibrational-vibrational collisional exchanges has been carried out. Radiative transfer processes have been treated by using a modified Curtis matrix formulation. The populations of the excited vibrational levels for nighttime conditions are presented along with a sensitivity study of their variations to the kinetic temperature profile and to collisional rate constants. The populations of the CO2(0, nu2, 0) levels follow LTE up to about 85 km with the radiative transfer processes playing a very important role in maintaining this situation above the tropopause. This result is pratically insensitive to plausible variations in the kinetic temperature of the troposphere. The uncertainties in the rate constants play an important role in determining the populations of the levels at thermospheric altitudes, but they are of little significance for the heights where they start departing from LTE. The CO2(0, 00, 1) level breaks down from LTE at about 60 km, the laser bands at 10 micrometers giving a significant contribution to its population in the Martian mesosphere. The CO(1) level stars departing around 50 km and is noticeably enhanced in the upper thermosphere by absorption of upwelling flux from the planets' surface.

Effect of a 5-min cold-water immersion recovery on exercise performance in the heat.

PubMed

Peiffer, J J; Abbiss, C R; Watson, G; Nosaka, K; Laursen, P B

2010-05-01

This study examined the effect of a 5-min cold-water immersion (14 degrees C) recovery intervention on repeated cycling performance in the heat. 10 male cyclists performed two bouts of a 25-min constant-paced (254 (22) W) cycling session followed by a 4-km time trial in hot conditions (35 degrees C, 40% relative humidity). The two bouts were separated by either 15 min of seated recovery in the heat (control) or the same condition with 5-min cold-water immersion (5th-10th minute), using a counterbalanced cross-over design (CP(1)TT(1) --> CWI or CON --> CP(2)TT(2)). Rectal temperature was measured immediately before and after both the constant-paced sessions and 4-km timed trials. Cycling economy and Vo(2) were measured during the constant-paced sessions, and the average power output and completion times were recorded for each time trial. Compared with control, rectal temperature was significantly lower (0.5 (0.4) degrees C) in cold-water immersion before CP(2) until the end of the second 4-km timed trial. However, the increase in rectal temperature (0.5 (0.2) degrees C) during CP(2) was not significantly different between conditions. During the second 4-km timed trial, power output was significantly greater in cold-water immersion (327.9 (55.7) W) compared with control (288.0 (58.8) W), leading to a faster completion time in cold-water immersion (6.1 (0.3) min) compared with control (6.4 (0.5) min). Economy and Vo(2) were not influenced by the cold-water immersion recovery intervention. 5-min cold-water immersion recovery significantly lowered rectal temperature and maintained endurance performance during subsequent high-intensity exercise. These data indicate that repeated exercise performance in heat may be improved when a short period of cold-water immersion is applied during the recovery period.

Evaluation of kinetic constants of biomolecular interaction on optical surface plasmon resonance sensor with Newton Iteration Method

NASA Astrophysics Data System (ADS)

Zhao, Yuanyuan; Jiang, Guoliang; Hu, Jiandong; Hu, Fengjiang; Wei, Jianguang; Shi, Liang

2010-10-01

In the immunology, there are two important types of biomolecular interaction: antigens-antibodies and receptors-ligands. Monitoring the response rate and affinity of biomolecular interaction can help analyze the protein function, drug discover, genomics and proteomics research. Moreover the association rate constant and dissociation rate constant of receptors-ligands are the important parameters for the study of signal transmission between cells. Recent advances in bioanalyzer instruments have greatly simplified the measurement of the kinetics of molecular interactions. Non-destructive and real-time monitoring the response to evaluate the parameters between antigens and antibodies can be performed by using optical surface plasmon resonance (SPR) biosensor technology. This technology provides a quantitative analysis that is carried out rapidly with label-free high-throughput detection using the binding curves of antigens-antibodies. Consequently, the kinetic parameters of interaction between antigens and antibodies can be obtained. This article presents a low cost integrated SPR-based bioanalyzer (HPSPR-6000) designed by ourselves. This bioanalyzer is mainly composed of a biosensor TSPR1K23, a touch-screen monitor, a microprocessor PIC24F128, a microflow cell with three channels, a clamp and a photoelectric conversion device. To obtain the kinetic parameters, sensorgrams may be modeled using one of several binding models provided with BIAevaluation software 3.0, SensiQ or Autolab. This allows calculation of the association rate constant (ka) and the dissociation rate constant (kd). The ratio of ka to kd can be used to estimate the equilibrium constant. Another kind is the analysis software OriginPro, which can process the obtained data by nonlinear fitting and then get some correlative parameters, but it can't be embedded into the bioanalyzer, so the bioanalyzer don't support the use of OriginPro. This paper proposes a novel method to evaluate the kinetic parameters of biomolecular interaction by using Newton Iteration Method and Least Squares Method. First, the pseudo first order kinetic model of biomolecular interaction was established. Then the data of molecular interaction of HBsAg and HBsAb was obtained by bioanalyzer. Finally, we used the optical SPR bioanalyzer software which was written by ourselves to make nonlinear fit about the association and dissociation curves. The correlation coefficient R-squared is 0.99229 and 0.99593, respectively. Furthermore, the kinetic parameters and affinity constants were evaluated using the obtained data from the fitting results.

Activation of muscle nicotinic acetylcholine receptor channels by nicotinic and muscarinic agonists

PubMed Central

Akk, Gustav; Auerbach, Anthony

1999-01-01

The dose-response parameters of recombinant mouse adult neuromuscular acetylcholine receptor channels (nAChR) activated by carbamylcholine, nicotine, muscarine and oxotremorine were measured. Rate constants for agonist association and dissociation, and channel opening and closing, were estimated from single-channel kinetic analysis.The dissociation equilibrium constants were (mM): ACh (0.16)carbamylcholine (5.1)>oxotremorine M (0.6)>nicotine (0.5)>muscarine (0.15).Rat neuronal α4β2 nAChR can be activated by all of the agonists. However, detailed kinetic analysis was impossible because the recordings lacked clusters representing the activity of a single receptor complex. Thus, the number of channels in the patch was unknown and the activation rate constants could not be determined.Considering both receptor affinity and agonist efficacy, muscarine and oxotremorine are significant agonists of muscle-type nAChR. The results are discussed in terms of structure-function relationships at the nAChR transmitter binding site. PMID:10602325

Kinetic rate constant prediction supports the conformational selection mechanism of protein binding.

PubMed

Moal, Iain H; Bates, Paul A

2012-01-01

The prediction of protein-protein kinetic rate constants provides a fundamental test of our understanding of molecular recognition, and will play an important role in the modeling of complex biological systems. In this paper, a feature selection and regression algorithm is applied to mine a large set of molecular descriptors and construct simple models for association and dissociation rate constants using empirical data. Using separate test data for validation, the predicted rate constants can be combined to calculate binding affinity with accuracy matching that of state of the art empirical free energy functions. The models show that the rate of association is linearly related to the proportion of unbound proteins in the bound conformational ensemble relative to the unbound conformational ensemble, indicating that the binding partners must adopt a geometry near to that of the bound prior to binding. Mirroring the conformational selection and population shift mechanism of protein binding, the models provide a strong separate line of evidence for the preponderance of this mechanism in protein-protein binding, complementing structural and theoretical studies.

Isolation and characterization of Dehalobacter sp. strain UNSWDHB capable of chloroform and chlorinated ethane respiration.

PubMed

Wong, Yie K; Holland, Sophie I; Ertan, Haluk; Manefield, Mike; Lee, Matthew

2016-09-01

Dehalobacter sp. strain UNSWDHB can dechlorinate up to 4 mM trichloromethane at a rate of 0.1 mM per day to dichloromethane and 1,1,2-trichloroethane (1 mM, 0.1 mM per day) with the unprecedented product profile of 1,2-dichloroethane and vinyl chloride. 1,1,1-trichloroethane and 1,1-dichloroethane were slowly utilized by strain UNSWDHB and were not completely removed, with minimum threshold concentrations of 0.12 mM and 0.07 mM respectively under growth conditions. Enzyme kinetic experiments confirmed strong substrate affinity for trichloromethane and 1,1,2-trichloroethane (Km  = 30 and 62 µM respectively) and poor substrate affinity for 1,1,1-trichloroethane and 1,1-dichloroethane (Km  = 238 and 837 µM respectively). Comparison of enzyme kinetic and growth data with other trichloromethane respiring organisms (Dehalobacter sp. strain CF and Desulfitobacterium sp. strain PR) suggests an adaptation of strain UNSWDHB to trichloromethane. The trichloromethane RDase (TmrA) expressed by strain UNSWDHB was identified by BN-PAGE and functionally characterized. Amino acid comparison of homologous RDases from all three organisms revealed only six significant amino acid substitutions/deletions, which are likely to be crucial for substrate specificity. Furthermore, strain UNSWDHB was shown to grow without exogenous supply of cobalamin confirming genomic-based predictions of a fully functional cobalamin synthetic pathway. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

Molecular genetics of the platelet serotonin system in first-degree relatives of patients with autism

PubMed Central

Cross, Sarah; Kim, Soo-Jeong; Weiss, Lauren A.; Delahanty, Ryan J.; Sutcliffe, James S.; Leventhal, Bennett L.; Cook, Edwin H.; Veenstra-VanderWeele, Jeremy

2009-01-01

Elevated platelet serotonin (5-HT) is found in a subset of children with autism and in some of their first-degree relatives. Indices of the platelet serotonin system, including whole blood serotonin (5-HT), 5-HT binding affinity for the serotonin transporter (Km), 5-HT uptake (Vmax), and lysergic acid diethylamide (LSD) receptor binding, were previously studied in twenty-four first-degree relatives of probands with autism, half of whom were selected for elevated whole blood 5-HT levels. All subjects were then genotyped for selected polymorphisms at the SLC6A4, HTR7, HTR2A, ITGB3, and TPH1 loci. Previous studies allowed an a priori prediction of SLC6A4 haplotypes that separated the subjects into three groups that showed significantly different 5-HT binding affinity (Km, p = 0.005) and 5-HT uptake rate (Vmax, p = 0.046). Genotypes at four individual polymorphisms in SLC6A4 were not associated with platelet 5-HT indices. Haplotypes at SLC6A4 and individual genotypes of polymorphisms at SLC6A4, HTR7, HTR2A, ITGB3, and TPH1 showed no significant association with whole blood 5-HT. Haplotype analysis of two polymorphisms in TPH1 revealed a nominally significant association with whole blood 5-HT (p = 0.046). These initial studies of indices of the 5-HT system with several SNPs at loci in this system generate hypotheses for testing in other samples. PMID:17406648

Plasmin substrate binding site cooperativity guides the design of potent peptide aldehyde inhibitors.

PubMed

Swedberg, Joakim E; Harris, Jonathan M

2011-10-04

Perioperative bleeding is a cause of major blood loss and is associated with increased rates of postoperative morbidity and mortality. To combat this, antifibrinolytic inhibitors of the serine protease plasmin are commonly used to reduce bleeding during surgery. The most effective and previously widely used of these is the broad range serine protease inhibitor aprotinin. However, adverse clinical outcomes have led to use of alternative serine lysine analogues to inhibit plasmin. These compounds suffer from low selectivity and binding affinity. Consequently, a concerted effort to discover potent and selective plasmin inhibitors has developed. This study used a noncombinatorial peptide library to define plasmin's extended substrate specificity and guide the design of potent transition state analogue inhibitors. The various substrate binding sites of plasmin were found to exhibit a higher degree of cooperativity than had previously been appreciated. Peptide sequences capitalizing on these features produced high-affinity inhibitors of plasmin. The most potent of these, Lys-Met(sulfone)-Tyr-Arg-H [KM(O(2))YR-H], inhibited plasmin with a K(i) of 3.1 nM while maintaining 25-fold selectivity over plasma kallikrein. Furthermore, 125 nM (0.16 μg/mL) KM(O(2))YR-H attenuated fibrinolysis in vitro with an efficacy similar to that of 15 nM (0.20 μg/mL) aprotinin. To date, this is the most potent peptide inhibitor of plasmin that exhibits selectivity against plasma kallikrein, making this compound an attractive candidate for further therapeutic development.

Trypanosomatidae produce acetate via a mitochondrial acetate:succinate CoA transferase

PubMed Central

Van Hellemond, Jaap J.; Opperdoes, Fred R.; Tielens, Aloysius G. M.

1998-01-01

Hydrogenosome-containing anaerobic protists, such as the trichomonads, produce large amounts of acetate by an acetate:succinate CoA transferase (ASCT)/succinyl CoA synthetase cycle. The notion that mitochondria and hydrogenosomes may have originated from the same α-proteobacterial endosymbiont has led us to look for the presence of a similar metabolic pathway in trypanosomatids because these are the earliest-branching mitochondriate eukaryotes and because they also are known to produce acetate. The mechanism of acetate production in these organisms, however, has remained unknown. Four different members of the trypanosomatid family: promastigotes of Leishmania mexicana mexicana, L. infantum and Phytomonas sp., and procyclics of Trypanosoma brucei were analyzed as well as the parasitic helminth Fasciola hepatica. They all use a mitochondrial ASCT for the production of acetate from acetyl CoA. The succinyl CoA that is produced during acetate formation by ASCT is recycled presumably to succinate by a mitochondrial succinyl CoA synthetase, concomitantly producing ATP from ADP. The ASCT of L. mexicana mexicana promastigotes was further characterized after partial purification of the enzyme. It has a high affinity for acetyl CoA (Km 0.26 mM) and a low affinity for succinate (Km 6.9 mM), which shows that significant acetate production can occur only when high mitochondrial succinate concentrations prevail. This study identifies a metabolic pathway common to mitochondria and hydrogenosomes, which strongly supports a common origin for these two organelles. PMID:9501211

A new estimate of the Hubble constant using the Virgo cluster distance

NASA Astrophysics Data System (ADS)

Visvanathan, N.

The Hubble constant, which defines the size and age of the universe, remains substantially uncertain. Attention is presently given to an improved distance to the Virgo Cluster obtained by means of the 1.05-micron luminosity-H I width relation of spirals. In order to improve the absolute calibration of the relation, accurate distances to the nearby SMC, LMC, N6822, SEX A and N300 galaxies have also been obtained, on the basis of the near-IR P-L relation of the Cepheids. A value for the global Hubble constant of 67 + or 4 km/sec per Mpc is obtained.

Constant envelope OFDM scheme for 6PolSK-QPSK

NASA Astrophysics Data System (ADS)

Li, Yupeng; Ding, Ding

2018-03-01

A constant envelope OFDM scheme with phase modulator (PM-CE-OFDM) for 6PolSK-QPSK modulation was demonstrated. Performance under large fiber launch power is measured to check its advantages in counteracting fiber nonlinear impairments. In our simulation, PM-CE-OFDM, RF-assisted constant envelope OFDM (RF-CE-OFDM) and conventional OFDM (Con-OFDM) are transmitted through 80 km standard single mode fiber (SSMF) single channel and WDM system. Simulation results confirm that PM-CE-OFDM has best performance in resisting fiber nonlinearity. In addition, benefiting from the simple system structure, the complexity and cost of PM-CE-OFDM system could be reduced effectively.

AFRRI Reports, Second Quarter 1994

DTIC Science & Technology

1994-08-01

the antrum wete immediately placed in sterile 0.9% NaCl, kept on ice, coded, and then prepared for culture, smears, and urease assay by homogeniza...high urease specific activity (>1 |J.mol- min-1 ■ mg protein-1) plus high-affinity substrate binding (Mi- chaelis constant [K^\\ < 1 mmol/L),27 in at...031, respectively), and the characteristic bacterial growth with high-activity product.on of a urease with tight substrate binding " was found in

Measuring Binding Affinity of Protein-Ligand Interaction Using Spectrophotometry: Binding of Neutral Red to Riboflavin-Binding Protein

ERIC Educational Resources Information Center

Chenprakhon, Pirom; Sucharitakul, Jeerus; Panijpan, Bhinyo; Chaiyen, Pimchai

2010-01-01

The dissociation constant, K[subscript d], of the binding of riboflavin-binding protein (RP) with neutral red (NR) can be determined by titrating RP to a fixed concentration of NR. Upon adding RP to the NR solution, the maximum absorption peak of NR shifts to 545 nm from 450 nm for the free NR. The change of the absorption can be used to determine…

DOE Office of Scientific and Technical Information (OSTI.GOV)

Olsen, Todd Andrew; Brandt, Craig C.; Brooks, Scott C.

Mercury (Hg) methylation and methylmercury (MMHg) demethylation activity of periphyton biofilms from East Fork Poplar Creek, Tennessee, USA (EFPC) were measured during 2014-2015 using stable Hg isotopic rate assays. 201Hg II and MM 202Hg were added to intact periphyton samples and the formation of MM 201Hg and loss of MM 202Hg were monitored over time and used to calculate first-order rate constants for methylation and demethylation, respectively. The influence of location, temperature/season, light exposure and biofilm structure on methylation and demethylation were examined. Between-site differences in net methylation for samples collected from an upstream versus downstream location were driven bymore » differences in the demethylation rate constant (k d). In contrast, the within-site seasonal difference in net methylation was driven by changes in the methylation rate constant (k m). Samples incubated in the dark had lower net methylation due to km values that were 60% less than those incubated in the light. Disrupting the biofilm structure decreased km by 50% and resulted in net demethylating conditions. Overall, the measured rates resulted in a net excess of MMHg generated which could account for 27-85% of the MMHg flux in EFPC and suggests intact, actively photosynthesizing periphyton biofilms harbor zones of MMHg production.« less

Novel Xylose Dehydrogenase in the Halophilic Archaeon Haloarcula marismortui†

PubMed Central

Johnsen, Ulrike; Schönheit, Peter

2004-01-01

During growth of the halophilic archaeon Haloarcula marismortui on d-xylose, a specific d-xylose dehydrogenase was induced. The enzyme was purified to homogeneity. It constitutes a homotetramer of about 175 kDa and catalyzed the oxidation of xylose with both NADP+ and NAD+ as cosubstrates with 10-fold higher affinity for NADP+. In addition to d-xylose, d-ribose was oxidized at similar kinetic constants, whereas d-glucose was used with about 70-fold lower catalytic efficiency (kcat/Km). With the N-terminal amino acid sequence of the subunit, an open reading frame (ORF)—coding for a 39.9-kDA protein—was identified in the partially sequenced genome of H. marismortui. The function of the ORF as the gene designated xdh and coding for xylose dehydrogenase was proven by its functional overexpression in Escherichia coli. The recombinant enzyme was reactivated from inclusion bodies following solubilization in urea and refolding in the presence of salts, reduced and oxidized glutathione, and substrates. Xylose dehydrogenase showed the highest sequence similarity to glucose-fructose oxidoreductase from Zymomonas mobilis and other putative bacterial and archaeal oxidoreductases. Activities of xylose isomerase and xylulose kinase, the initial reactions of xylose catabolism of most bacteria, could not be detected in xylose-grown cells of H. marismortui, and the genes that encode them, xylA and xylB, were not found in the genome of H. marismortui. Thus, we propose that this first characterized archaeal xylose dehydrogenase catalyzes the initial step in xylose degradation by H. marismortui. PMID:15342590

Functional characterization of the organic cation transporters (OCTs) in human airway pulmonary epithelial cells.

PubMed

Ingoglia, Filippo; Visigalli, Rossana; Rotoli, Bianca Maria; Barilli, Amelia; Riccardi, Benedetta; Puccini, Paola; Dall'Asta, Valeria

2015-07-01

Organic cation transporters (OCT1-3) mediate the transport of organic cations including inhaled drugs across the cell membrane, although their role in lung epithelium hasn't been well understood yet. We address here the expression and functional activity of OCT1-3 in human airway epithelial cells A549, Calu-3 and NCl-H441. Kinetic and inhibition analyses, employing [(3)H]1-methyl-4-phenylpyridinium (MPP+) as substrate, and the compounds quinidine, prostaglandine E2 (PGE2) and corticosterone as preferential inhibitors of OCT1, OCT2, and OCT3, respectively, have been performed. A549 cells present a robust MPP+ uptake mediated by one high-affinity component (Km~50μM) which is identifiable with OCT3. Corticosterone, indeed, completely inhibits MPP+ transport, while quinidine and PGE2 are inactive and SLC22A3/OCT3 silencing with siRNA markedly lowers MPP+ uptake. Conversely, Calu-3 exhibits both a high (Km<20μM) and a low affinity (Km>0.6mM) transport components, referable to OCT3 and OCT1, respectively, as demonstrated by the inhibition analysis performed at proper substrate concentrations and confirmed by the use of specific siRNA. These transporters are active also when cells are grown under air-liquid interface (ALI) conditions. Only a very modest saturable MPP+ uptake is measurable in NCl-H441 cells and the inhibitory effect of quinidine points to OCT1 as the subtype functionally involved in this model. Finally, the characterization of MPP+ transport in human bronchial BEAS-2B cells suggests that OCT1 and OCT3 are operative. These findings could help to identify in vitro models to be employed for studies concerning the specific involvement of each transporter in drug transportation. Copyright © 2015 Elsevier B.V. All rights reserved.

Multiple melting stages and refertilization as indicators for ridge to subduction formation: The New Caledonia ophiolite

NASA Astrophysics Data System (ADS)

Ulrich, Marc; Picard, Christian; Guillot, Stéphane; Chauvel, Catherine; Cluzel, Dominique; Meffre, Sébastien

2010-03-01

The origin of the New Caledonia ophiolite (South West Pacific), one of the largest in the world, is controversial. This nappe of ultramafic rocks (300 km long, 50 km wide and 2 km thick) is thrust upon a smaller nappe (Poya terrane) composed of basalts from mid-ocean ridges (MORB), back arc basins (BABB) and ocean islands (OIB). This nappe was tectonically accreted from the subducting plate prior and during the obduction of the ultramafic nappe. The bulk of the ophiolite is composed of highly depleted harzburgites (± dunites) with characteristic U-shaped bulk-rock rare-earth element (REE) patterns that are attributed to their formation in a forearc environment. In contrast, the origin of spoon-shaped REE patterns of lherzolites in the northernmost klippes was unclear. Our new major element and REE data on whole rocks, spinel and clinopyroxene establish the abyssal affinity of these lherzolites. Significant LREE enrichment in the lherzolites is best explained by partial melting in a spreading ridge, followed by near in-situ refertilization from deeper mantle melts. Using equilibrium melting equations, we show that melts extracted from these lherzolites are compositionally similar to the MORB of the Poya terrane. This is used to infer that the ultramafic nappe and the mafic Poya terrane represent oceanic lithosphere of a single marginal basin that formed during the late Cretaceous. In contrast, our spinel data highlights the strong forearc affinities of the most depleted harzburgites whose compositions are best modeled by hydrous melting of a source that had previously experienced depletion in a spreading ridge. The New Caledonian boninites probably formed during this second stage of partial melting. The two melting events in the New Caledonia ophiolite record the rapid transition from oceanic accretion to convergence in the South Loyalty Basin during the Late Paleocene, with initiation of a new subduction zone at or near the ridge axis.

40 CFR 86.403-78 - Abbreviations.

Code of Federal Regulations, 2012 CFR

2012-07-01

.... cm—Centimetre(s). CO—Carbon monoxide. CO2—Carbon dioxide. Conc—Concentration. cu.—Cubic. CVS—Constant...—Fahrenheit. h—hour. HC—Hydrocarbon(s). Hg—Mercury. H2O—Water. in.—Inch(es). K—Kelvin. kg—Kilogram(s). km...

40 CFR 86.403-78 - Abbreviations.

Code of Federal Regulations, 2013 CFR

2013-07-01

.... cm—Centimetre(s). CO—Carbon monoxide. CO2—Carbon dioxide. Conc—Concentration. cu.—Cubic. CVS—Constant...—Fahrenheit. h—hour. HC—Hydrocarbon(s). Hg—Mercury. H2O—Water. in.—Inch(es). K—Kelvin. kg—Kilogram(s). km...

40 CFR 86.403-78 - Abbreviations.

Code of Federal Regulations, 2011 CFR

2011-07-01

.... cm—Centimetre(s). CO—Carbon monoxide. CO2—Carbon dioxide. Conc—Concentration. cu.—Cubic. CVS—Constant...—Fahrenheit. h—hour. HC—Hydrocarbon(s). Hg—Mercury. H2O—Water. in.—Inch(es). K—Kelvin. kg—Kilogram(s). km...

40 CFR 86.403-78 - Abbreviations.

Code of Federal Regulations, 2010 CFR

2010-07-01

.... cm—Centimetre(s). CO—Carbon monoxide. CO2—Carbon dioxide. Conc—Concentration. cu.—Cubic. CVS—Constant...—Fahrenheit. h—hour. HC—Hydrocarbon(s). Hg—Mercury. H2O—Water. in.—Inch(es). K—Kelvin. kg—Kilogram(s). km...

40 CFR 86.403-78 - Abbreviations.

Code of Federal Regulations, 2014 CFR

2014-07-01

.... cm—Centimetre(s). CO—Carbon monoxide. CO2—Carbon dioxide. Conc—Concentration. cu.—Cubic. CVS—Constant...—Fahrenheit. h—hour. HC—Hydrocarbon(s). Hg—Mercury. H2O—Water. in.—Inch(es). K—Kelvin. kg—Kilogram(s). km...

Transport of EDTA into cells of the EDTA-degrading bacterial strain DSM 9103.

PubMed

Witschel, M; Egli, T; Zehnder, A J; Wehrli, E; Spycher, M

1999-04-01

In the bacterial strain DSM 9103, which is able to grow with the complexing agent EDTA as the sole source of carbon, nitrogen and energy, the transport of EDTA into whole cells was investigated. EDTA uptake was found to be dependent on speciation: free EDTA and metal-EDTA complexes with low stability constants were readily taken up, whereas those with stability constants higher than 1016 were not transported. In EDTA-grown cells, initial transport rates of CaEDTA showed substrate-saturation kinetics with a high apparent affinity for CaEDTA (affinity constant Kt= 0.39 microM). Several uncouplers had an inhibitory effect on CaEDTA transport. CaEDTA uptake was also significantly reduced in the presence of an inhibitor of ATPase and the ionophore nigericin, which dissipates the proton gradient. Valinomycin, however, which affects the electrical potential, had little effect on uptake, indicating that EDTA transport is probably driven by the proton gradient. Of various structurally related compounds tested only Ca2+-complexed diethylenetriaminepentaacetate (CaDTPA) competitively inhibited CaEDTA transport. Uptake in fumarate-grown cells was low compared to that measured in EDTA-grown bacteria. These results strongly suggest that the first step in EDTA degradation by strain DSM 9103 consists of transport by an inducible energy-dependent carrier. Uptake experiments with 45Ca2+ in the presence and absence of EDTA indicated that Ca2+ is transported together with EDTA into the cells. In addition, these transport studies and electron-dispersive X-ray analysis of electron-dense intracellular bodies present in EDTA-grown cells suggest that two mechanisms acting simultaneously allow the cells to cope with the large amounts of metal ions taken up together with EDTA. In one mechanism the metal ions are excreted, in the other they are inactivated intracellularly in polyphosphate granules.

Na(+) transport, and the E(1)P-E(2)P conformational transition of the Na(+)/K(+)-ATPase.

PubMed Central

Babes, A; Fendler, K

2000-01-01

We have used admittance analysis together with the black lipid membrane technique to analyze electrogenic reactions within the Na(+) branch of the reaction cycle of the Na(+)/K(+)-ATPase. ATP release by flash photolysis of caged ATP induced changes in the admittance of the compound membrane system that are associated with partial reactions of the Na(+)/K(+)-ATPase. Frequency spectra and the Na(+) dependence of the capacitive signal are consistent with an electrogenic or electroneutral E(1)P <--> E(2)P conformational transition which is rate limiting for a faster electrogenic Na(+) dissociation reaction. We determine the relaxation rate of the rate-limiting reaction and the equilibrium constants for both reactions at pH 6.2-8.5. The relaxation rate has a maximum value at pH 7.4 (approximately 320 s(-1)), which drops to acidic (approximately 190 s(-1)) and basic (approximately 110 s(-1)) pH. The E(1)P <--> E(2)P equilibrium is approximately at a midpoint position at pH 6.2 (equilibrium constant approximately 0.8) but moves more to the E(1)P side at basic pH 8.5 (equilibrium constant approximately 0.4). The Na(+) affinity at the extracellular binding site decreases from approximately 900 mM at pH 6.2 to approximately 200 mM at pH 8.5. The results suggest that during Na(+) transport the free energy supplied by the hydrolysis of ATP is mainly used for the generation of a low-affinity extracellular Na(+) discharge site. Ionic strength and lyotropic anions both decrease the relaxation rate. However, while ionic strength does not change the position of the conformational equilibrium E(1)P <--> E(2)P, lyotropic anions shift it to E(1)P. PMID:11053130

The β subunit of the high-conductance calcium-activated potassium channel contributes to the high-affinity receptor for charybdotoxin

PubMed Central

Hanner, Markus; Schmalhofer, William A.; Munujos, Petraki; Knaus, Hans-Günther; Kaczorowski, Gregory J.; Garcia, Maria L.

1997-01-01

Transient expression of either α or α+β subunits of the high-conductance Ca2+-activated K+ (maxi-K) channel has been achieved in COS-1 cells. Expression has been studied using charybdotoxin (ChTX), a peptidyl inhibitor that binds in the pore on the α subunit. Although some properties of monoiodotyrosine-ChTX (125I-ChTX) binding to membranes derived from each type of transfected cells appear to be identical, other parameters of the binding reaction are markedly different. Under low ionic strength conditions, the affinity constant for 125I-ChTX measured under equilibrium binding conditions is increased ca. 50-fold in the presence of the β subunit. The rate constant for 125I-ChTX association is enhanced ca. 5-fold, whereas the dissociation rate constant is decreased more than 7-fold when the β subunit is present. These data indicate that functional coassembly of maxi-K channel subunits can be obtained in a transient expression system, and that the β subunit has profound effects on 125I-ChTX binding. We postulate that certain negatively charged residues in the large extracellular loop of β attract the positively charged 125I-ChTX to its binding site on α through electrostatic interactions, and account for effects observed on ligand association kinetics. Moreover, another residue(s) in the loop of β must contribute to stabilization of the toxin-bound state, either by a direct interaction with toxin, or through an allosteric effect on the α subunit. Certain regions in the extracellular loop of the β subunit may be in close proximity to the pore of the channel, and could play an important role in maxi-K channel function. PMID:9096310

Application of the Nernst-Planck approach to lead ion exchange in Ca-loaded Pelvetia canaliculata.

PubMed

Costa, Joana F de Sá S; Vilar, Vítor J P; Botelho, Cidália M S; da Silva, Eduardo A B; Boaventura, Rui A R

2010-07-01

Ca-loaded Pelvetia canaliculata biomass was used to remove Pb(2+) in aqueous solution from batch and continuous systems. The physicochemical characterization of algae Pelvetia particles by potentiometric titration and FTIR analysis has shown a gel structure with two major binding groups - carboxylic (2.8 mmol g(-1)) and hydroxyl (0.8 mmol g(-1)), with an affinity constant distribution for hydrogen ions well described by a Quasi-Gaussian distribution. Equilibrium adsorption (pH 3 and 5) and desorption (eluents: HNO(3) and CaCl(2)) experiments were performed, showing that the biosorption mechanism was attributed to ion exchange among calcium, lead and hydrogen ions with stoichiometry 1:1 (Ca:Pb) and 1:2 (Ca:H and Pb:H). The uptake capacity of lead ions decreased with pH, suggesting that there is a competition between H(+) and Pb(2+) for the same binding sites. A mass action law for the ternary mixture was able to predict the equilibrium data, with the selectivity constants alpha(Ca)(H)=9+/-1 and alpha(Ca)(Pb)=44+/-5, revealing a higher affinity of the biomass towards lead ions. Adsorption (initial solution pH 4.5 and 2.5) and desorption (0.3M HNO(3)) kinetics were performed in batch and continuous systems. A mass transfer model using the Nernst-Planck approximation for the ionic flux of each counter-ion was used for the prediction of the ions profiles in batch systems and packed bed columns. The intraparticle effective diffusion constants were determined as 3.73x10(-7)cm(2)s(-1) for H(+), 7.56x10(-8)cm(2)s(-1) for Pb(2+) and 6.37x10(-8)cm(2)s(-1) for Ca(2+). Copyright 2010 Elsevier Ltd. All rights reserved.

Gas Phase Molecular Spectroscopy: Electronic Spectroscopy of Combustion Intermediates, Chlorine Azide kinetics, and Rovibrational Energy Transfer in Acetylene

NASA Astrophysics Data System (ADS)

Freel, Keith A.

This dissertation is composed of three sections. The first deals with the electronic spectroscopy of combustion intermediates that are related to the formation of polycyclic aromatic hydrocarbons. Absorption spectra for phenyl, phenoxy, benzyl, and phenyl peroxy radicals were recorded using the technique of cavity ring-down spectroscopy. When possible, molecular constants, vibrational frequencies, and excited state lifetimes for these radicals were derived from these data. The results were supported by theoretical predictions. The second section presents a study of electron attachment to chlorine azide (ClN3) using a flowing-afterglow Langmuir-probe apparatus. Electron attachment rates were measured to be 3.5x10-8 and 4.5x10-8 cm3s-1 at 298 and 400 K respectively. The reactions of ClN3 with eighteen cations and seventeen anions were characterized. Rate constants were measured using a selected ion flow tube. The ionization energy (>9.6eV), proton affinity (713+/-41 kJ mol-1), and electron affinity (2.48+/-0.2 eV) for ClN 3 were determined from these data. The third section demonstrates the use of double resonance spectroscopy to observe state-selected rovibrational energy transfer from the first overtone asymmetric stretch of acetylene. The total population removal rate constants from various rotational levels of the (1,0,1,00,00) vibrational state were determined to be in the range of (9-17) x 10 -10 cm3s-1. Rotational energy transfer accounted for approximately 90% of the total removal rate from each state. Therefore, the upper limit of vibrational energy transfer from the (1,0,1,0 0,00) state was 10%.

High-affinity binding of laurate to naturally occurring mutants of human serum albumin and proalbumin.

PubMed

Kragh-Hansen, U; Pedersen, A O; Galliano, M; Minchiotti, L; Brennan, S O; Tárnoky, A L; Franco, M H; Salzano, F M

1996-12-15

Binding of laurate (n-dodecanoate) to genetic variants of albumin or its proprotein and to normal albumin isolated from the same heterozygous carriers was studied by a kinetic dialysis technique at physiological pH. The first stoichiometric association constant for binding to proalbumin Lille (Arg-2-->His) and albumin (Alb) Roma (Glu321-->Lys) was increased to 126% and 136% respectively compared with that for binding to normal albumin, whereas the constant for Alb Maku (Lys541-->Glu) was decreased to 80%. In contrast, normal laurate-binding properties were found for as many as nine other albumin variants with single amino acid substitutions. Because the net charges of all these mutants were different from that of normal albumin, the results suggest that the examples of modified laurate binding are not caused by long-range electrostatic effects. Rather, the three positions mentioned are located close to different binding sites for the fatty acid anion. The most pronounced effect was observed for the glycosylated Alb Casebrook, the binding constant of which was decreased to 20%. Binding to the glycosylated Alb Redhill was also decreased, but to a smaller extent (68%). These decreases in binding are caused by partial or total blocking of the high-affinity site by the oligosaccharides, by the negative charges of the oligosaccharides, and/or by conformational changes induced by these bulky moieties. Laurate binding to two chain-termination mutants (Alb Catania and Alb Venezia) was normal, indicating that the C-terminus of albumin is not important for binding. By using different preparations of normal albumin as controls in the binding experiments, it was also possible to compare the effect of various methods for isolation and defatting on laurate binding.

VEGF-Trap: a VEGF blocker with potent antitumor effects.

PubMed

Holash, Jocelyn; Davis, Sam; Papadopoulos, Nick; Croll, Susan D; Ho, Lillian; Russell, Michelle; Boland, Patricia; Leidich, Ray; Hylton, Donna; Burova, Elena; Ioffe, Ella; Huang, Tammy; Radziejewski, Czeslaw; Bailey, Kevin; Fandl, James P; Daly, Tom; Wiegand, Stanley J; Yancopoulos, George D; Rudge, John S

2002-08-20

Vascular endothelial growth factor (VEGF) plays a critical role during normal embryonic angiogenesis and also in the pathological angiogenesis that occurs in a number of diseases, including cancer. Initial attempts to block VEGF by using a humanized monoclonal antibody are beginning to show promise in human cancer patients, underscoring the importance of optimizing VEGF blockade. Previous studies have found that one of the most effective ways to block the VEGF-signaling pathway is to prevent VEGF from binding to its normal receptors by administering decoy-soluble receptors. The highest-affinity VEGF blocker described to date is a soluble decoy receptor created by fusing the first three Ig domains of VEGF receptor 1 to an Ig constant region; however, this fusion protein has very poor in vivo pharmacokinetic properties. By determining the requirements to maintain high affinity while extending in vivo half life, we were able to engineer a very potent high-affinity VEGF blocker that has markedly enhanced pharmacokinetic properties. This VEGF-Trap effectively suppresses tumor growth and vascularization in vivo, resulting in stunted and almost completely avascular tumors. VEGF-Trap-mediated blockade may be superior to that achieved by other agents, such as monoclonal antibodies targeted against the VEGF receptor.

In silico designed nanoMIP based optical sensor for endotoxins monitoring.

PubMed

Abdin, M J; Altintas, Z; Tothill, I E

2015-05-15

Molecular modelling was used to select specific monomers suitable for the design of molecularly imprinted polymers (MIPs) with high affinity towards endotoxins. MIPs were synthesised using solid-phase photopolymerisation with endotoxins from Escherichia coli 0111:B4 as the template. This technique also allowed the endotoxin template to be reused successfully. Particle size of ~190-220 nm was achieved with low polydispersity index, which confirms the quality of the produced MIPs. For the development of the optical sensor, SPR-2 biosensor system was used by functionalising the gold sensor chip with the MIP nanoparticles using EDC/NHS coupling procedure. The affinity based-endotoxin assay can detect endotoxins in the concentration range of 15.6-500 ng mL(-1). MIP surfaces were regenerated showing stability of the method for subsequent analysis and dissociation constants were calculated as 3.24-5.24×10(-8) M. The developed SPR sensor with the novel endotoxins nanoMIP showed the potential of the technology for endotoxins capture, detection and risk management and also the importance of computational modelling to design the artificial affinity ligands. Copyright © 2014 Elsevier B.V. All rights reserved.

Evaluation of protein adsorption and preferred binding regions in multimodal chromatography using NMR

PubMed Central

Chung, Wai Keen; Freed, Alexander S.; Holstein, Melissa A.; McCallum, Scott A.; Cramer, Steven M.

2010-01-01

NMR titration experiments with labeled human ubiquitin were employed in concert with chromatographic data obtained with a library of ubiquitin mutants to study the nature of protein adsorption in multimodal (MM) chromatography. The elution order of the mutants on the MM resin was significantly different from that obtained by ion-exchange chromatography. Further, the chromatographic results with the protein library indicated that mutations in a defined region induced greater changes in protein affinity to the solid support. Chemical shift mapping and determination of dissociation constants from NMR titration experiments with the MM ligand and isotopically enriched ubiquitin were used to determine and rank the relative binding affinities of interaction sites on the protein surface. The results with NMR confirmed that the protein possessed a distinct preferred binding region for the MM ligand in agreement with the chromatographic results. Finally, coarse-grained ligand docking simulations were employed to study the modes of interaction between the MM ligand and ubiquitin. The use of NMR titration experiments in concert with chromatographic data obtained with protein libraries represents a previously undescribed approach for elucidating the structural basis of protein binding affinity in MM chromatographic systems. PMID:20837551

Thermochemistry of the specific binding of C12 surfactants to bovine serum albumin.

PubMed

Nielsen, A D; Borch, K; Westh, P

2000-06-15

The specific binding to bovine serum albumin (BSA) of anionic and non-ionic surfactants with C12 acyl chains has been studied by high sensitivity isothermal titration calorimetry. This method proved particularly effective in resolving the binding of anionic surfactants into separate classes of sites with different affinity. For sodium dodecylsulfate (SDS) the measured binding curves could be rationalized as association to two classes (high affinity/low affinity) of sites comprising, respectively, three and six similar (i.e. thermodynamically equivalent), independent sites. Changes in the thermodynamic functions enthalpy, standard free energy, standard entropy and heat capacity could be discerned for each class of binding site, as well as for micelle formation. These data suggest that binding to low affinity sites (in analogy with micelle formation) exhibits energetic parameters; in particular, a large negative change in heat capacity, which is characteristic of hydrophobic interactions. The thermodynamics of high affinity binding, on the other hand, is indicative of other dominant forces; most likely electrostatic interactions. Other anionic ligands investigated (laurate and dodecyl benzylsulfonate) showed a behavior similar to SDS, the most significant difference being the high affinity binding of the alkylbenzyl sulfonate. For this ligand, the thermodynamic data is indicative of a more loosely associated complex than for SDS and laurate. BSA was found to bind one or two of the non-ionic surfactants (NIS) hepta- or penta(ethylene glycol) monododecyl ether (C12EO7 and C12EO5) with binding constants about three orders of magnitude lower than for SDS. Hence, the free energy of the surfactant in the weakly bound BSA-NIS complex is only slightly favored over the micellar state. The binding process is characterized by very large exothermic enthalpy changes (larger than for the charged surfactants) and a large, positive increment in heat capacity. These observations cannot be reconciled with a molecular picture based on simple hydrophobic condensation onto non-polar patches on the protein surface.

Dissecting the Dynamic Pathways of Stereoselective DNA Threading Intercalation

PubMed Central

Almaqwashi, Ali A.; Andersson, Johanna; Lincoln, Per; Rouzina, Ioulia; Westerlund, Fredrik; Williams, Mark C.

2016-01-01

DNA intercalators that have high affinity and slow kinetics are developed for potential DNA-targeted therapeutics. Although many natural intercalators contain multiple chiral subunits, only intercalators with a single chiral unit have been quantitatively probed. Dumbbell-shaped DNA threading intercalators represent the next order of structural complexity relative to simple intercalators, and can provide significant insights into the stereoselectivity of DNA-ligand intercalation. We investigated DNA threading intercalation by binuclear ruthenium complex [μ-dppzip(phen)4Ru2]4+ (Piz). Four Piz stereoisomers are defined by the chirality of the intercalating subunit (Ru(phen)2dppz) and the distal subunit (Ru(phen)2ip), respectively, each of which can be either right-handed (Δ) or left-handed (Λ). We used optical tweezers to measure single DNA molecule elongation due to threading intercalation, revealing force-dependent DNA intercalation rates and equilibrium dissociation constants. The force spectroscopy analysis provided the zero-force DNA binding affinity, the equilibrium DNA-ligand elongation Δxeq, and the dynamic DNA structural deformations during ligand association xon and dissociation xoff. We found that Piz stereoisomers exhibit over 20-fold differences in DNA binding affinity, from a Kd of 27 ± 3 nM for (Δ,Λ)-Piz to a Kd of 622 ± 55 nM for (Λ,Δ)-Piz. The striking affinity decrease is correlated with increasing Δxeq from 0.30 ± 0.02 to 0.48 ± 0.02 nm and xon from 0.25 ± 0.01 to 0.46 ± 0.02 nm, but limited xoff changes. Notably, the affinity and threading kinetics is 10-fold enhanced for right-handed intercalating subunits, and 2- to 5-fold enhanced for left-handed distal subunits. These findings demonstrate sterically dispersed transition pathways and robust DNA structural recognition of chiral intercalators, which are critical for optimizing DNA binding affinity and kinetics. PMID:27028636

Molecular Determinants of Epidermal Growth Factor Binding: A Molecular Dynamics Study

PubMed Central

Sanders, Jeffrey M.; Wampole, Matthew E.; Thakur, Mathew L.; Wickstrom, Eric

2013-01-01

The epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase family that plays a role in multiple cellular processes. Activation of EGFR requires binding of a ligand on the extracellular domain to promote conformational changes leading to dimerization and transphosphorylation of intracellular kinase domains. Seven ligands are known to bind EGFR with affinities ranging from sub-nanomolar to near micromolar dissociation constants. In the case of EGFR, distinct conformational states assumed upon binding a ligand is thought to be a determining factor in activation of a downstream signaling network. Previous biochemical studies suggest the existence of both low affinity and high affinity EGFR ligands. While these studies have identified functional effects of ligand binding, high-resolution structural data are lacking. To gain a better understanding of the molecular basis of EGFR binding affinities, we docked each EGFR ligand to the putative active state extracellular domain dimer and 25.0 ns molecular dynamics simulations were performed. MM-PBSA/GBSA are efficient computational approaches to approximate free energies of protein-protein interactions and decompose the free energy at the amino acid level. We applied these methods to the last 6.0 ns of each ligand-receptor simulation. MM-PBSA calculations were able to successfully rank all seven of the EGFR ligands based on the two affinity classes: EGF>HB-EGF>TGF-α>BTC>EPR>EPG>AR. Results from energy decomposition identified several interactions that are common among binding ligands. These findings reveal that while several residues are conserved among the EGFR ligand family, no single set of residues determines the affinity class. Instead we found heterogeneous sets of interactions that were driven primarily by electrostatic and Van der Waals forces. These results not only illustrate the complexity of EGFR dynamics but also pave the way for structure-based design of therapeutics targeting EGF ligands or the receptor itself. PMID:23382875

Cerebral Blood Flow Responses to Aquatic Treadmill Exercise.

PubMed

Parfitt, Rhodri; Hensman, Marianne Y; Lucas, Samuel J E

2017-07-01

Aquatic treadmills are used as a rehabilitation method for conditions such as spinal cord injury, osteoarthritis, and stroke, and can facilitate an earlier return to exercise training for athletes. However, their effect on cerebral blood flow (CBF) responses has not been examined. We tested the hypothesis that aquatic treadmill exercise would augment CBF and lower HR compared with land-based treadmill exercise. Eleven participants completed incremental exercise (crossover design) starting from walking pace (4 km·h, immersed to iliac crest [aquatic], 6 km·h [land]) and increasing 1 km·h every 2 min up to 10 km·h for aquatic (maximum belt speed) or 12 km·h for land. After this, participants completed two 2-min bouts of exercise immersed to midthigh and midchest at constant submaximal speed (aquatic), or were ramped to exhaustion (land; increased gradient 2° every min). Middle cerebral artery blood flow velocity (MCAv) and HR were measured throughout, and the initial 10 min of each protocol and responses at each immersion level were compared. Compared with land-based treadmill, MCAvmean increased more from baseline for aquatic exercise (21% vs 12%, P < 0.001), while being associated with lower overall HR (pooled difference, 11 bpm; P < 0.001). MCAvmean increased similarly during aquatic walking compared with land-based moderate intensity running (~10 cm·s, P = 0.56). Greater water immersion lowered HR (139 vs 178 bpm for midchest vs midthigh), whereas MCAvmean remained constant (P = 0.37). Findings illustrate the potential for aquatic treadmill exercise to enhance exercise-induced elevations in CBF and thus optimize shear stress-mediated adaptation of the cerebrovasculature.

Performance comparison of two fuel cell hybrid buses with different powertrain and energy management strategies

NASA Astrophysics Data System (ADS)

Ouyang, Minggao; Xu, Liangfei; Li, Jianqiu; Lu, Languang; Gao, Dawei; Xie, Qicheng

In order to assess the influences of different powertrain structures and energy management strategies on the performance of hybrid fuel cell buses (FCB), two buses (FCB A and FCB B) were constructed with a "energy hybrid structure" and "power hybrid structure", respectively. Different energy management strategies were investigated based on analysis of the two systems. And the two buses were compared with each other in a bus cycle and constant speed testing. The Polymer Electrolyte Membrane Fuel Cell (PEMFC) in FCB A showed an advantage in fuel economy for it worked usually in the high efficient range of the PEMFC engine. The hydrogen consumption rate in the cycle testing was 7.9 kg/100 km and 9.8 kg/100 km for FCB A and FCB B, and in the 40 kmph constant speed testing it was 3.3 kg/100 km and 4.0 kg/100 km, respectively. The fuel economy could be improved when the hydrogen and air supply subsystems are optimized and controlled with an advanced algorithm. It could also benefit from a braking energy regeneration system. Compared with FCB A, the PEMFC in FCB B worked under unfavorable operation conditions because its working range was comparatively wide, and the power changing rate was relatively large from a statistical point of view, which resulted in performance recession of the PEMFC in FCB B. After a mileage of 7000 km, the output power of the PEMFC in FCB B was reduced by 10%, compared with 2.4% in FCB A. An advanced energy management strategy is necessary to split the power between the PEMFC and a battery suitable for long durability of a PEMFC.

Sulfated Metabolites of Polychlorinated Biphenyls Are High-Affinity Ligands for the Thyroid Hormone Transport Protein Transthyretin

PubMed Central

Grimm, Fabian A.; Lehmler, Hans-Joachim; He, Xianran; Robertson, Larry W.

2013-01-01

Background: The displacement of l-thyroxine (T4) from binding sites on transthyretin (TTR) is considered a significant contributing mechanism in polychlorinated biphenyl (PCB)-induced thyroid disruption. Previous research has discovered hydroxylated PCB metabolites (OH-PCBs) as high-affinity ligands for TTR, but the binding potential of conjugated PCB metabolites such as PCB sulfates has not been explored. Objectives: We evaluated the binding of five lower-chlorinated PCB sulfates to human TTR and compared their binding characteristics to those determined for their OH-PCB precursors and for T4. Methods: We used fluorescence probe displacement studies and molecular docking simulations to characterize the binding of PCB sulfates to TTR. The stability of PCB sulfates and the reversibility of these interactions were characterized by HPLC analysis of PCB sulfates after their binding to TTR. The ability of OH-PCBs to serve as substrates for human cytosolic sulfotransferase 1A1 (hSULT1A1) was assessed by OH-PCB–dependent formation of adenosine-3´,5´-diphosphate, an end product of the sulfation reaction. Results: All five PCB sulfates were able to bind to the high-affinity binding site of TTR with equilibrium dissociation constants (Kd values) in the low nanomolar range (4.8–16.8 nM), similar to that observed for T4 (4.7 nM). Docking simulations provided corroborating evidence for these binding interactions and indicated multiple high-affinity modes of binding. All OH-PCB precursors for these sulfates were found to be substrates for hSULT1A1. Conclusions: Our findings show that PCB sulfates are high-affinity ligands for human TTR and therefore indicate, for the first time, a potential relevance for these metabolites in PCB-induced thyroid disruption. PMID:23584369

Development and validation of an affinity chromatography step using a peptide ligand for cGMP production of factor VIII.

PubMed

Kelley, Brian D; Tannatt, Molly; Magnusson, Robert; Hagelberg, Sigrid; Booth, James

2004-08-05

An affinity chromatography step was developed for purification of recombinant B-Domain Deleted Factor VIII (BDDrFVIII) using a peptide ligand selected from a phage display library. The peptide library had variegated residues, contained both within a disulfide bond-constrained ring and flanking the ring. The peptide ligand binds to BDDrFVIII with a dissociation constant of approximately 1 microM both in free solution and when immobilized on a chromatographic resin. The peptide is chemically synthesized and the affinity resin is produced by coupling the peptide to an agarose matrix preactivated with N-hydroxysuccinimide. Coupling conditions were optimized to give consistent and complete ligand incorporation and validated with a robustness study that tested various combinations of processing limits. The peptide affinity chromatographic operation employs conditions very similar to an immunoaffinity chromatography step currently in use for BDDrFVIII manufacture. The process step provides excellent recovery of BDDrFVIII from a complex feed stream and reduces host cell protein and DNA by 3-4 logs. Process validation studies established resin reuse over 26 cycles without changes in product recovery or purity. A robustness study using a factorial design was performed and showed that the step was insensitive to small changes in process conditions that represent normal variation in commercial manufacturing. A scaled-down model of the process step was qualified and used for virus removal studies. A validation package addressing the safety of the leached peptide included leaching rate measurements under process conditions, testing of peptide levels in product pools, demonstration of robust removal downstream by spiking studies, end product testing, and toxicological profiling of the ligand. The peptide ligand affinity step was scaled up for cGMP production of BDDrFVIII for clinical trials.

Determination of the affinity of drugs toward serum albumin by measurement of the quenching of the intrinsic tryptophan fluorescence of the protein.

PubMed

Epps, D E; Raub, T J; Caiolfa, V; Chiari, A; Zamai, M

1999-01-01

Binding of new chemical entities to serum proteins is an issue confronting pharmaceutical companies during development of potential therapeutic agents. Most drugs bind to the most abundant plasma protein, human serum albumin (HSA), at two major binding sites. Excepting fluorescence spectroscopy, existing methods for assaying drug binding to serum albumin are insensitive to higher-affinity compounds and can be labour-intensive, time-consuming, and usually require compound-specific assays. This led us to examine alternative ways to measure drug-albumin interaction. One method described here uses fluorescence quenching of the single tryptophan (Trp) residue in HSA excited at 295 nm to measure drug-binding affinity. Unfortunately, many compounds absorb, fluoresce, or both, in this UV wavelength region of the spectrum. Several types of binding phenomenon and spectral interference were identified by use of six structurally unrelated compounds and the equations necessary to make corrections mathematically were derived and applied to calculate binding constants accurately. The general cases were: direct quenching of Trp fluorescence by optically transparent ligands with low or high affinities; binding of optically transparent, non-fluorescent ligands to two specific sites where both sites or only one site result in Trp fluorescence quenching; and chromophores whose absorption either overlaps the Trp emission and quenches by energy transfer or absorbs light at the Trp fluorescence excitation wavelength producing absorptive screening as well as fluorescence quenching. Unless identification of the site specificity of drug binding to serum albumin is desired, quenching of the Trp fluorescence of albumin by titration with ligand is a rapid and facile method for determining the binding affinities of drugs for serum albumin.

The serotonin transporter: Examination of the changes in transporter affinity induced by ligand binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Humphreys, C.J.

1989-01-01

The plasmalemmal serotonin transporter uses transmembrane gradients of Na{sup +}, Cl{sup {minus}} and K{sup +} to accumulate serotonin within blood platelets. Transport is competitively inhibited by the antidepressant imipramine. Like serotonin transport, imipramine binding requires Na{sup +}. Unlike serotonin, however, imipramine does not appear to be transported. To gain insight into the mechanism of serotonin transport the author have analyzed the influences of Na{sup +} and Cl{sup {minus}}, the two ions cotransported with serotonin, on both serotonin transport and the interaction of imipramine and other antidepressant drugs with the plasmalemmal serotonin transporter of human platelets. Additionally, the author have synthesized,more » purified and characterized the binding of 2-iodoimipramine to the serotonin transporter. Finally, the author have conducted a preliminary study of the inhibition of serotonin transport and imipramine binding produced by dicyclohexylcarbodiimide. My results reveal many instances of positive heterotropic cooperativity in ligand binding to the serotonin transporter. Na{sup +} binding enhances the transporters affinity for imipramine and several other antidepressant drugs, and also increases the affinity for Cl{sup {minus}}. Cl{sup {minus}} enhances the transporters affinity for imipramine, as well as for Na{sup +}. At concentrations in the range of its K{sub M} for transport serotonin is a competitive inhibitor of imipramine binding. At much higher concentrations, however, serotonin also inhibits imipramines dissociation rate constant. This latter effect which is Na{sup +}-independent and species specific, is apparently produced by serotonin binding at a second, low affinity site on, or near, the transporter complex. Iodoimipramine competitively inhibit both ({sup 3}H)imipramine binding and ({sup 3}H)serotonin transport.« less

Analysis of free drug fractions in serum by ultrafast affinity extraction and two-dimensional affinity chromatography using α1-acid glycoprotein microcolumns.

PubMed

Bi, Cong; Zheng, Xiwei; Hage, David S

2016-02-05

In the circulatory system, many drugs are reversibly bound to serum proteins such as human serum albumin (HSA) and alpha1-acid glycoprotein (AGP), resulting in both free and protein-bound fractions for these drugs. This report examined the use of microcolumns containing immobilized AGP for the measurement of free drug fractions by ultrafast affinity extraction and a two-dimensional affinity system. Several drugs known to bind AGP were used as models to develop and evaluate this approach. Factors considered during the creation of this method included the retention of the drugs on the microcolumns, the injection flow rate, the microcolumn size, and the times at which a second AGP column was placed on-line with the microcolumn. The final system had residence times of only 110-830ms during sample passage through the AGP microcolumns and allowed free drug fractions to be determined within 10-20min when using only 3-10μL of sample per injection. This method was used to measure the free fractions of the model drugs at typical therapeutic levels in serum, giving good agreement with the results obtained by ultrafiltration. This approach was also used to estimate the binding constants for each drug with AGP in serum, even for drugs that had significant interactions with both AGP and HSA in such samples. These results indicated that AGP microcolumns could be used with ultrafast affinity extraction to measure free drug fractions in a label-free manner and to study the binding of drugs with AGP in complex samples such as serum. Copyright © 2015 Elsevier B.V. All rights reserved.

3- and 4-O-sulfoconjugated and methylated dopamine: highly reduced binding affinity to dopamine D2 receptors in rat striatal membranes.

PubMed

Werle, E; Lenz, T; Strobel, G; Weicker, H

1988-07-01

The binding properties of 3- and 4-O-sulfo-conjugated dopamine (DA-3-O-S, DA-4-O-S) as well as 3-O-methylated dopamine (MT) to rat striatal dopamine D2 receptors were investigated. 3H-spiperone was used as a radioligand in the binding studies. In saturation binding experiments (+)butaclamol, which has been reported to bind to dopaminergic D2 and serotoninergic 5HT2 receptors, was used in conjunction with ketanserin and sulpiride, which preferentially label 5HT2 and D2 receptors, respectively, in order to discriminate between 3H-spiperone binding to D2 and to 5HT2 receptors. Under our particular membrane preparation and assay conditions, 3H-spiperone binds to D2 and 5HT2 receptors with a maximal binding capacity (Bmax) of 340 fmol/mg protein in proportions of about 75%:25% with similar dissociation constants KD (35 pmol/l; 43 pmol/l). This result was verified by the biphasic competition curve of ketanserin, which revealed about 20% high (KD = 24 nmol/l) and 80% low (KD = 420 nmol/l) affinity binding sites corresponding to 5HT2 and D2 receptors, respectively. Therefore, all further competition experiments at a tracer concentration of 50 pmol/l were performed in the presence of 0.1 mumol/l ketanserin to mask the 5HT2 receptors. DA competition curves were best fitted assuming two binding sites, with high (KH = 0.12 mumol/l) and low (KL = 18 mumol/l) affinity, present in a ratio of 3:1. The high affinity binding sites were interconvertible by 100 mumol/l guanyl-5-yl imidodiphosphate [Gpp(NH)p], resulting in a homogenous affinity state of DA receptors (KD = 2.8 mumol/l).2+ off

Isolation and characterization of an IgNAR variable domain specific for the human mitochondrial translocase receptor Tom70.

PubMed

Nuttall, Stewart D; Krishnan, Usha V; Doughty, Larissa; Pearson, Kylie; Ryan, Michael T; Hoogenraad, Nicholas J; Hattarki, Meghan; Carmichael, Jennifer A; Irving, Robert A; Hudson, Peter J

2003-09-01

The new antigen receptor (IgNAR) from sharks is a disulphide bonded dimer of two protein chains, each containing one variable and five constant domains, and functions as an antibody. In order to assess the antigen-binding capabilities of isolated IgNAR variable domains (VNAR), we have constructed an in vitro library incorporating synthetic CDR3 regions of 15-18 residues in length. Screening of this library against the 60 kDa cytosolic domain of the 70 kDa outer membrane translocase receptor from human mitochondria (Tom70) resulted in one dominant antigen-specific clone (VNAR 12F-11) after four rounds of in vitro selection. VNAR 12F-11 was expressed into the Escherichia coli periplasm and purified by anti-FLAG affinity chromatography at yields of 3 mg x L(-1). Purified protein eluted from gel filtration columns as a single monomeric protein and CD spectrum analysis indicated correct folding into the expected beta-sheet conformation. Specific binding to Tom70 was demonstrated by ELISA and BIAcore (Kd = 2.2 +/- 0.31 x 10(-9) m-1) indicating that these VNAR domains can be efficiently displayed as bacteriophage libraries, and selected against target antigens with an affinity and stability equivalent to that obtained for other single domain antibodies. As an initial step in producing 'intrabody' variants of 12F-11, the impact of modifying or removing the conserved immunoglobulin intradomain disulphide bond was assessed. High affinity binding was only retained in the wild-type protein, which combined with our inability to affinity mature 12F-11, suggests that this particular VNAR is critically dependent upon precise CDR loop conformations for its binding affinity.

Kinetics of the hydrolysis of N-benzoyl-l-serine methyl ester catalysed by bromelain and by papain. Analysis of modifier mechanisms by lattice nomography, computational methods of parameter evaluation for substrate-activated catalyses and consequences of postulated non-productive binding in bromelain- and papain-catalysed hydrolyses

PubMed Central

Wharton, Christopher W.; Cornish-Bowden, Athel; Brocklehurst, Keith; Crook, Eric M.

1974-01-01

1. N-Benzoyl-l-serine methyl ester was synthesized and evaluated as a substrate for bromelain (EC 3.4.22.4) and for papain (EC 3.4.22.2). 2. For the bromelain-catalysed hydrolysis at pH7.0, plots of [S0]/vi (initial substrate concn./initial velocity) versus [S0] are markedly curved, concave downwards. 3. Analysis by lattice nomography of a modifier kinetic mechanism in which the modifier is substrate reveals that concave-down [S0]/vi versus [S0] plots can arise when the ratio of the rate constants that characterize the breakdown of the binary (ES) and ternary (SES) complexes is either less than or greater than 1. In the latter case, there are severe restrictions on the values that may be taken by the ratio of the dissociation constants of the productive and non-productive binary complexes. 4. Concave-down [S0]/vi versus [S0] plots cannot arise from compulsory substrate activation. 5. Computational methods, based on function minimization, for determination of the apparent parameters that characterize a non-compulsory substrate-activated catalysis are described. 6. In an attempt to interpret the catalysis by bromelain of the hydrolysis of N-benzoyl-l-serine methyl ester in terms of substrate activation, the general substrate-activation model was simplified to one in which only one binary ES complex (that which gives rise directly to products) can form. 7. In terms of this model, the bromelain-catalysed hydrolysis of N-benzoyl-l-serine methyl ester at pH7.0, I=0.1 and 25°C is characterized by Km1 (the dissociation constant of ES)=1.22±0.73mm, k (the rate constant for the breakdown of ES to E+products, P)=1.57×10−2±0.32×10−2s−1, Ka2 (the dissociation constant that characterizes the breakdown of SES to ES and S)=0.38±0.06m, and k′ (the rate constant for the breakdown of SES to E+P+S)=0.45±0.04s−1. 8. These parameters are compared with those in the literature that characterize the bromelain-catalysed hydrolysis of α-N-benzoyl-l-arginine ethyl ester and of α-N-benzoyl-l-arginine amide; Km1 and k for the serine ester hydrolysis are somewhat similar to Km and kcat. for the arginine amide hydrolysis and Kas and k′ for the serine ester hydrolysis are somewhat similar to Km and kcat. for the arginine ester hydrolysis. 9. A previous interpretation of the inter-relationships of the values of kcat. and Km for the bromelain-catalysed hydrolysis of the arginine ester and amide substrates is discussed critically and an alternative interpretation involving substantial non-productive binding of the arginine amide substrate to bromelain is suggested. 10. The parameters for the bromelain-catalysed hydrolysis of the serine ester substrate are tentatively interpreted in terms of non-productive binding in the binary complex and a decrease of this type of binding by ternary complex-formation. 11. The Michaelis parameters for the papain-catalysed hydrolysis of the serine ester substrate (Km=52±4mm, kcat.=2.80±0.1s−1 at pH7.0, I=0.1, 25.0°C) are similar to those for the papain-catalysed hydrolysis of methyl hippurate. 12. Urea and guanidine hydrochloride at concentrations of 1m have only small effects on the kinetic parameters for the hydrolysis of the serine ester substrate catalysed by bromelain and by papain. PMID:4455211

The high affinity of small-molecule antioxidants for hemoglobin.

PubMed

Puscas, Cristina; Radu, Luana; Carrascoza, Francisco; Mot, Augustin C; Amariei, Diana; Lungu, Oana; Scurtu, Florina; Podea, Paula; Septelean, Raluca; Matei, Alina; Mic, Mihaela; Attia, Amr A; Silaghi-Dumitrescu, Radu

2018-06-18

Hemoglobin has previously been shown to display ascorbate peroxidase and urate peroxidase activity, with measurable Michaelis-Menten parameters that reveal a particularly low Km for ascorbate as well as for urate - lower than the respective in vivo concentrations of these antioxidants in blood. Also, direct detection of a hemoglobin-ascorbate interaction was possible by monitoring the 1H-NMR spectrum of ascorbate in the presence of hemoglobin. The relative difference in structures between ascorbate and urate may raise the question as to exactly what the defining structural features would be, for a substrate that binds to hemoglobin with high affinity. Reported here are Michaelis-Menten parameters for hemoglobin acting as peroxidase against a number of other substrates of varying structures - gallate, caffeate, rutin, 3-hydroxyflavone, 3,6-dihydroxyflavone, quercetin, epicatechin, luteolin - all with high affinities (some higher than those of physiologically-relevant redox partners of Hb - ascorbate and urate). Moreover, this high affinity appears general to animal hemoglobins. 1 H-NMR and 13 C-NMR spectra reveal a general pattern wherein small hydrophilic antioxidants appear to all have their signals affected, presumably due to binding to hemoglobin. Fluorescence and calorimetry measurements confirm these conclusions. Docking calculations confirm the existence of binding sites on hemoglobin and on myoglobin for ascorbate as well as for other antioxidants. Support is found for involvement of Tyr42 in binding of three out of the four substrates investigated in the case of hemoglobin (including ascorbate and urate, as blood-contained relevant substrates), but also for Tyr145 (with urate and caffeate) and Tyr35 (with gallate). Copyright © 2018 Elsevier Inc. All rights reserved.

Dental affinities of the C-group inhabitants of Hierakonpolis, Egypt: Nubian, Egyptian, or both?

PubMed

Irish, J D; Friedman, R

2010-04-01

By c. 2050 BC a small community of C-Group Nubians was present deep within Egyptian territory at the city of Hierakonpolis. Their descendants stayed for the next 400 years. Today, the site of Hierakonpolis, 113 km north of Aswan, is known for its Egyptian deposits; however, it also contains a C-Group cemetery, which documents the northernmost occurrence of this culture. Sixty skeletons were excavated. Tombs feature Nubian architecture and goods, including leather garments, although the use of Egyptian mortuary practices and artifacts increased through time. Dates range from the early 11th Dynasty into the Second Intermediate period. During this time the Egyptian empire occupied Lower Nubia, and their state ideology vilified Nubians. Yet, at least in death, the C-Group inhabitants of Hierakonpolis proudly displayed their cultural heritage. Beyond discerning the reason(s) for their presence at the site (e.g., mercenaries, leather-workers, entertainers?), the focus of this report is to estimate their biological affinity. Were they akin to other Nubians, Egyptians, or both? And, was increasing 'Egyptianization' evident in the mortuary ritual accompanied by concomitant genetic influence? To address these queries, up to 36 dental morphological traits in the recovered individuals were compared to those in 26 regional comparative samples. The most influential traits were identified and phenetic affinities were calculated using the mean measure of divergence and other multivariate analyses. Assuming phenetic similarity provides an estimate of genetic relatedness, these affinities suggest the individuals comprising the C-Group sample were, and remained Nubian during their tenure at Hierakonpolis. Copyright 2010 Elsevier GmbH. All rights reserved.

Determinants of the Differential Antizyme-Binding Affinity of Ornithine Decarboxylase

PubMed Central

Liu, Yen-Chin; Hsu, Den-Hua; Huang, Chi-Liang; Liu, Yi-Liang; Liu, Guang-Yaw; Hung, Hui-Chih

2011-01-01

Ornithine decarboxylase (ODC) is a ubiquitous enzyme that is conserved in all species from bacteria to humans. Mammalian ODC is degraded by the proteasome in a ubiquitin-independent manner by direct binding to the antizyme (AZ). In contrast, Trypanosoma brucei ODC has a low binding affinity toward AZ. In this study, we identified key amino acid residues that govern the differential AZ binding affinity of human and Trypanosoma brucei ODC. Multiple sequence alignments of the ODC putative AZ-binding site highlights several key amino acid residues that are different between the human and Trypanosoma brucei ODC protein sequences, including residue 119, 124,125, 129, 136, 137 and 140 (the numbers is for human ODC). We generated a septuple human ODC mutant protein where these seven bases were mutated to match the Trypanosoma brucei ODC protein sequence. The septuple mutant protein was much less sensitive to AZ inhibition compared to the WT protein, suggesting that these amino acid residues play a role in human ODC-AZ binding. Additional experiments with sextuple mutants suggest that residue 137 plays a direct role in AZ binding, and residues 119 and 140 play secondary roles in AZ binding. The dissociation constants were also calculated to quantify the affinity of the ODC-AZ binding interaction. The K d value for the wild type ODC protein-AZ heterodimer ([ODC_WT]-AZ) is approximately 0.22 μM, while the K d value for the septuple mutant-AZ heterodimer ([ODC_7M]-AZ) is approximately 12.4 μM. The greater than 50-fold increase in [ODC_7M]-AZ binding affinity shows that the ODC-7M enzyme has a much lower binding affinity toward AZ. For the mutant proteins ODC_7M(-Q119H) and ODC_7M(-V137D), the K d was 1.4 and 1.2 μM, respectively. These affinities are 6-fold higher than the WT_ODC K d, which suggests that residues 119 and 137 play a role in AZ binding. PMID:22073206

Subalkaline andesite from Valu Fa Ridge, a back-arc spreading center in southern Lau Basin: petrogenesis, comparative chemistry, and tectonic implications

USGS Publications Warehouse

Vallier, T.L.; Jenner, G.A.; Frey, F.A.; Gill, J.B.; Davis, A.S.; Volpe, A.M.; Hawkins, J.W.; Morris, J.D.; Cawood, Peter A.; Morton, J.L.; Scholl, D. W.; Rautenschlein, M.; White, W.M.; Williams, Ross W.; Stevenson, A.J.; White, L.D.

1991-01-01

Tholeiitic andesite was dredged from two sites on Valu Fa Ridge (VFR), a back-arc spreading center in Lau Basin. Valu Fa Ridge, at least 200 km long, is located 40-50 km west of the active Tofua Volcanic Arc (TVA) axis and lies about 150 km above the subducted oceanic plate. One or more magma chambers, traced discontinuously for about 100 km along the ridge axis, lie 3-4 km beneath the ridge. The mostly aphyric and glassy lavas had high volatile contents, as shown by the abundance and large sizes of vesicles. An extensive fractionation history is inferred from the high SiO2 contents and FeO* MgO ratios. Chemical data show that the VFR lavas have both volcanic arc and back-arc basin affinities. The volcanic arc characteristics are: (1) relatively high abundances of most alkali and alkaline earth elements; (2) low abundances of high field strength elements Nb and Ta; (3) high U/Th ratios; (4) similar radiogenic isotope ratios in VFR and TVA lavas, in particular the enrichment of 87Sr 86Sr relative to 206Pb 204Pb; (5) high 238U 230Th, 230Th 232Th, and 226Ra 230Th activity ratios; and (6) high ratios of Rb/Cs, Ba/Nb, and Ba/La. Other chemical characteristics suggest that the VFR lavas are related to MORB-type back-arc basin lavas. For example, VFR lavas have (1) lower 87Sr 86Sr ratios and higher 143Nd 144Nd ratios than most lavas from the TVA, except samples from Ata Island, and are similar to many Lau Basin lavas; (2) lower Sr/REE, Rb/Zr, and Ba/Zr ratios than in arc lavas; and (3) higher Ti, Fe, and V, and higher Ti/V ratios than arc lavas generally and TVA lavas specifically. Most characteristics of VFR lavas can be explained by mixing depleted mantle with either small amounts of sediment and fluids from the subducting slab and/or an older fragment of volcanic arc lithosphere. The eruption of subalkaline andesite with some arc affinities along a back-arc spreading ridge is not unique. Collision of the Louisville and Tonga ridges probably activated back-arc extension that ultimately led to the creation and growth of Valu Fa Ridge. Some ophiolitic fragments in circum-Pacific and circum-Tethyan allochthonous terranes, presently interpreted to have originated in volcanic arcs, may instead be fragments of lithosphere that formed during early stages of seafloor spreading in a back-arc basin. ?? 1991.

Reef corals of Johnston Atoll: one of the world's most isolated reefs

NASA Astrophysics Data System (ADS)

Maragos, James E.; Jokiel, Paul L.

1986-01-01

Johnston Atoll lies 800 km southwest of the nearest reefs of Hawaii and over 1,500 km from other shallow reefs to the south and west. Only 33 species and 16 genera and subgenera of shallow water stony corals have been reported from the atoll. Endemic species are absent despite Johnston's great age and favorable environment. With few exceptions, only species with broad geographic distribution are represented. Factors contributing to the low number of species are remoteness, the atoll's small size, lack of favorable currents to transport larvae from the southwest Pacific, lack of reef “stepping stones” in the region since the Cretaceous, possible defaunation during eustatic sea-level rise and fall, and possible drowning from tectonic subsidence or tilting. The species list shows strongest affinity with that of Hawaii, but some unexpected discontinuities occur. Despite low species diversity, coral coverage is extremely high in most environments.

Estuarine sediment transport by gravity-driven movement of the nepheloid layer, Long Island Sound

USGS Publications Warehouse

Poppe, L.J.; McMullen, K.Y.; Williams, S.J.; Crocker, J.M.; Doran, E.F.

2008-01-01

Interpretation of sidescan-sonar imagery provides evidence that down-slope gravity-driven movement of the nepheloid layer constitutes an important mode of transporting sediment into the basins of north-central Long Island Sound, a major US East Coast estuary. In the Western Basin, this transport mechanism has formed dendritic drainage systems characterized by branching patterns of low backscatter on the seafloor that exceed 7.4 km in length and progressively widen down-slope, reaching widths of over 0.6 km at their southern distal ends. Although much smaller, dendritic patterns of similar morphology are also present in the northwestern part of the Central Basin. Because many contaminants display affinities for adsorption onto fine-grained sediments, and because the Sound is affected by seasonal hypoxia, mechanisms and dispersal pathways by which inorganic and organic sediments are remobilized and transported impact the eventual fate of the contaminants and environmental health of the estuary. 

Ion channel regulation by phosphoinositides analyzed with VSPs—PI(4,5)P2 affinity, phosphoinositide selectivity, and PI(4,5)P2 pool accessibility

PubMed Central

Rjasanow, Alexandra; Leitner, Michael G.; Thallmair, Veronika; Halaszovich, Christian R.; Oliver, Dominik

2015-01-01

The activity of many proteins depends on the phosphoinositide (PI) content of the membrane. E.g., dynamic changes of the concentration of PI(4,5)P2 are cellular signals that regulate ion channels. The susceptibility of a channel to such dynamics depends on its affinity for PI(4,5)P2. Yet, measuring affinities for endogenous PIs has not been possible directly, but has relied largely on the response to soluble analogs, which may not quantitatively reflect binding to native lipids. Voltage-sensitive phosphatases (VSPs) turn over PI(4,5)P2 to PI(4)P when activated by depolarization. In combination with voltage-clamp electrophysiology VSPs are useful tools for rapid and reversible depletion of PI(4,5)P2. Because cellular PI(4,5)P2 is resynthesized rapidly, steady state PI(4,5)P2 changes with the degree of VSP activation and thus depends on membrane potential. Here we show that titration of endogenous PI(4,5)P2 with Ci-VSP allows for the quantification of relative PI(4,5)P2 affinities of ion channels. The sensitivity of inward rectifier and voltage-gated K+ channels to Ci-VSP allowed for comparison of PI(4,5)P2 affinities within and across channel subfamilies and detected changes of affinity in mutant channels. The results also reveal that VSPs are useful only for PI effectors with high binding specificity among PI isoforms, because PI(4,5)P2 depletion occurs at constant overall PI level. Thus, Kir6.2, a channel activated by PI(4,5)P2 and PI(4)P was insensitive to VSP. Surprisingly, despite comparable PI(4,5)P2 affinity as determined by Ci-VSP, the Kv7 and Kir channel families strongly differed in their sensitivity to receptor-mediated depletion of PI(4,5)P2. While Kv7 members were highly sensitive to activation of PLC by Gq-coupled receptors, Kir channels were insensitive even when PI(4,5)P2 affinity was lowered by mutation. We hypothesize that different channels may be associated with distinct pools of PI(4,5)P2 that differ in their accessibility to PLC and VSPs. PMID:26150791

Osmotically-induced tension and the binding of N-BAR protein to lipid vesicles.

PubMed

Hutchison, Jaime B; Karunanayake Mudiyanselage, Aruni P K K; Weis, Robert M; Dinsmore, Anthony D

2016-02-28

The binding affinity of a curvature-sensing protein domain (N-BAR) is measured as a function of applied osmotic stress while the membrane curvature is nearly constant. Varying the osmotic stress allows us to control membrane tension, which provides a probe of the mechanism of binding. We study the N-BAR domain of the Drosophila amphiphysin and monitor its binding on 50 nm-radius vesicles composed of 90 mol% DOPC and 10 mol% PIP. We find that the bound fraction of N-BAR is enhanced by a factor of approximately 6.5 when the tension increases from zero to 2.6 mN m(-1). This tension-induced response can be explained by the hydrophobic insertion mechanism. From the data we extract a hydrophobic domain area that is consistent with known structure. These results indicate that membrane stress and strain could play a major role in the previously reported curvature-affinity of N-BAR.

Selection and characterization of a DNA aptamer to crystal violet.

PubMed

Chen, Yang; Wang, Jine; Zhang, Yajie; Xu, Lijun; Gao, Tian; Wang, Bing; Pei, Renjun

2018-06-13

Aptamers are short single-stranded DNA or RNA, which can be selected in vitro by systematic evolution of ligands by exponential enrichment (SELEX). In order to develop novel light-up probes to substitute G-quadruplex (G4), we selected a DNA aptamer for crystal violet (CV), a triphenylmethane light-up dye, by a modified affinity chromatography-based SELEX. The ssDNA pool was first coupled on streptavidin-coated agarose beads through a biotin labeled complementary oligonucleotide, and then the aptamer sequences would be released from agarose beads by CV affinity. This method is simple, straightforward and effective. The aptamer sequence with a low micromolar dissociation constant (Kd) and good specificity was achieved after 11 rounds of selection. The light-up properties of the CV-aptamer were also investigated, and the CV showed dramatic fluorescence enhancement. The CV-aptamer pair could be further used as a novel light-up fluorescent probe to design biosensors.

A Variable Step-Size Proportionate Affine Projection Algorithm for Identification of Sparse Impulse Response

NASA Astrophysics Data System (ADS)

Liu, Ligang; Fukumoto, Masahiro; Saiki, Sachio; Zhang, Shiyong

2009-12-01

Proportionate adaptive algorithms have been proposed recently to accelerate convergence for the identification of sparse impulse response. When the excitation signal is colored, especially the speech, the convergence performance of proportionate NLMS algorithms demonstrate slow convergence speed. The proportionate affine projection algorithm (PAPA) is expected to solve this problem by using more information in the input signals. However, its steady-state performance is limited by the constant step-size parameter. In this article we propose a variable step-size PAPA by canceling the a posteriori estimation error. This can result in high convergence speed using a large step size when the identification error is large, and can then considerably decrease the steady-state misalignment using a small step size after the adaptive filter has converged. Simulation results show that the proposed approach can greatly improve the steady-state misalignment without sacrificing the fast convergence of PAPA.

Optimality of affine control system of several species in competition on a sequential batch reactor

NASA Astrophysics Data System (ADS)

Rodríguez, J. C.; Ramírez, H.; Gajardo, P.; Rapaport, A.

2014-09-01

In this paper, we analyse the optimality of affine control system of several species in competition for a single substrate on a sequential batch reactor, with the objective being to reach a given (low) level of the substrate. We allow controls to be bounded measurable functions of time plus possible impulses. A suitable modification of the dynamics leads to a slightly different optimal control problem, without impulsive controls, for which we apply different optimality conditions derived from Pontryagin principle and the Hamilton-Jacobi-Bellman equation. We thus characterise the singular trajectories of our problem as the extremal trajectories keeping the substrate at a constant level. We also establish conditions for which an immediate one impulse (IOI) strategy is optimal. Some numerical experiences are then included in order to illustrate our study and show that those conditions are also necessary to ensure the optimality of the IOI strategy.

Properties of thymidylate synthetase from Ehrlich ascites carcinoma cells. Effect of Mg2/ and MgATP2-.

PubMed

Jastreboff, M; Kedzierska, B; Rode, W

1982-01-15

Ehrlich ascites carcinoma thymidylate synthetase was purified to electrophoretic homogeneity by affinity chromatography on 10-formyl-5,8-dideazofolate-ethyl-Sepharose. Electrophoretic analysis of the formation of the enzyme-5-fluorodeoxyuridylate-5,10-methylenetetrahydrofolate complexes showed the presence of two binding sites for 5-fluorodeoxyuridylate on the enzyme molecule. Molecular weight of the native enzyme was found to be 78,5000, whereas that of its monomer was 38, 500. The apparent Michaelis constants for dUMP and (+/-)-L-5,10-methylenetetrahydrofolate were 1.3 +/- 0.4 and 32.2 +/- 0.7 micrometers respectively. Phosphate acted as a weak inhibitor, competitive toward dUMP. The enzyme reaction exhibited a temperature-dependent change of activation energy, reflected in the binding affinity of dUMP, with a transitional temperature of 35.8 degrees. Both Mg2+ and MgATP2- were strong activators of the enzyme, MgATP2- being more effective.

Isolation and characterization of high affinity aptamers against DNA polymerase iota.

PubMed

Lakhin, Andrei V; Kazakov, Andrei A; Makarova, Alena V; Pavlov, Yuri I; Efremova, Anna S; Shram, Stanislav I; Tarantul, Viacheslav Z; Gening, Leonid V

2012-02-01

Human DNA-polymerase iota (Pol ι) is an extremely error-prone enzyme and the fidelity depends on the sequence context of the template. Using the in vitro systematic evolution of ligands by exponential enrichment (SELEX) procedure, we obtained an oligoribonucleotide with a high affinity to human Pol ι, named aptamer IKL5. We determined its dissociation constant with homogenous preparation of Pol ι and predicted its putative secondary structure. The aptamer IKL5 specifically inhibits DNA-polymerase activity of the purified enzyme Pol ι, but did not inhibit the DNA-polymerase activities of human DNA polymerases beta and kappa. IKL5 suppressed the error-prone DNA-polymerase activity of Pol ι also in cellular extracts of the tumor cell line SKOV-3. The aptamer IKL5 is useful for studies of the biological role of Pol ι and as a potential drug to suppress the increase of the activity of this enzyme in malignant cells.

Synthesis, anticancer activity, and iron affinity of the Actinoplanes metabolite 7,8-dihydroxy-1-methylnaphtho[2,3-c]furan-4,9-dione.

PubMed

Breyer, Sandra; Effenberger-Neidnicht, Katharina; Knauer, Sebastian; Schobert, Rainer

2011-02-01

The first synthesis of 7,8-dihydroxy-1-methylnaphtho[2,3-c]furan-4,9-dione (1), an isofuranonaphthoquinone produced by an Actinoplanes strain is described. Lactone ring opening of 6-methylfuro[3,4-c]furan-1(3H)-one (4) with ortho-lithiated veratrole (3), oxidation of product alcohol 5, and Friedel-Crafts acylation of the resulting aroylcarboxylic acid 7 afforded the mono methyl ether 2 of the target compound. The latter was obtained by demethylation of 2 with BBr(3) in 14% overall yield. While mono ether 2 was distinctly more cytotoxic than catechol 1 against a panel of five cancer cell lines, only the latter showed a siderophore-like binding affinity for Fe(III) with a complex dissociation constant K(D) of approximately 10(-29) M(3) (pM = 25.9). Copyright © 2010 Elsevier Ltd. All rights reserved.

Structural and functional difference of pheromone binding proteins in discriminating chemicals in the gypsy moth, Lymantria dispar.

PubMed

Yu, Yanxue; Ma, Fei; Cao, Yixia; Zhang, Junhua; Zhang, Yongan; Duan, Shengnan; Wei, Yadong; Zhu, Shuifang; Chen, Naizhong

2012-01-01

Pheromone-binding proteins (PBPs) of the gypsy moth, Lymantria dispar L., play an important role in olfaction. Here structures of PBPs were first built by Homology Modeling, and each model of PBPs had seven α-helices and a large hydrophobic cavity including 25 residues for PBP1 and 30 residues for PBP2. Three potential semiochemicals were first screened by CDOCKER program based on the PBP models and chemical database. These chemicals were Palmitic acid n-butyl ester (Pal), Bis(3,4-epoxycyclohexylmethyl) adipate (Bis), L-trans-epoxysuccinyl-isoleucyl-proline methyl ester propylamide (CA-074). The analysis of chemicals docking the proteins showed one hydrogen bond was established between the residues Lys94 and (+)-Disparlure ((+)-D), and л-л interactions were present between Phe36 of PBP1 and (+)-D. The Lys94 of PBP1 formed two and three hydrogen bonds with Bis and CA-074, respectively. There was no residue of PBP2 interacting with these four chemicals except Bis forming one hydrogen bond with Lys121. After simulating the conformational changes of LdisPBPs at pH7.3 and 5.5 by constant pH molecular dynamics simulation in implicit solvent, the N-terminal sequences of PBPs was unfolded, only having five α-helices, and PBP2 had larger binding pocket at 7.3 than PBP1. To investigate the changes of α-helices at different pH, far-UV and near-UV circular dichroism showed PBPs consist of α-helices, and the tertiary structures of PBP1 and PBP2 were influenced at pH7.3 and 5.5. The fluorescence binding assay indicated that PBP1 and PBP2 have similarly binding affinity to (+)-D at pH 5.5 and 7.3, respectively. At pH 5.5, the dissociation constant of the complex between PBP1 and 2-decyl-1-oxaspiro [2.2] pentane (OXP1) was 0.68 ± 0.01 μM, for (+)-D was 5.32 ± 0.11 μM, while PBP2 with OXP1 and (+)-D were 1.88 ± 0.02 μM and 5.54 ± 0.04 μM, respectively. Three chemicals screened had higher affinity to PBP1 than (+)-D except Pal at pH5.5, and had lower affinity than (+)-D at pH7.3. To PBP2, these chemicals had lower affinity than the sex pheromone except Bis at pH 5.5 and pH 7.3. Only PBP1 had higher affinity with Sal than the sex pheromone at pH 5.5. Therefore, the structures of PBP1 and PBP2 had different changes at pH5.5 and 7.3, showing different affinity to chemicals. This study helps understanding the role of PBPs as well as in developing more efficient chemicals for pest control.

Structural and Functional Difference of Pheromone Binding Proteins in Discriminating Chemicals in the Gypsy Moth, Lymantria Dispar

PubMed Central

Yu, Yanxue; Ma, Fei; Cao, Yixia; Zhang, Junhua; Zhang, Yongan; Duan, Shengnan; Wei, Yadong; Zhu, Shuifang; Chen, Naizhong

2012-01-01

Pheromone-binding proteins (PBPs) of the gypsy moth, Lymantria dispar L., play an important role in olfaction. Here structures of PBPs were first built by Homology Modeling, and each model of PBPs had seven α-helices and a large hydrophobic cavity including 25 residues for PBP1 and 30 residues for PBP2. Three potential semiochemicals were first screened by CDOCKER program based on the PBP models and chemical database. These chemicals were Palmitic acid n-butyl ester (Pal), Bis(3,4-epoxycyclohexylmethyl) adipate (Bis), L-trans-epoxysuccinyl-isoleucyl-proline methyl ester propylamide (CA-074). The analysis of chemicals docking the proteins showed one hydrogen bond was established between the residues Lys94 and (+)-Disparlure ((+)-D), and л-л interactions were present between Phe36 of PBP1 and (+)-D. The Lys94 of PBP1 formed two and three hydrogen bonds with Bis and CA-074, respectively. There was no residue of PBP2 interacting with these four chemicals except Bis forming one hydrogen bond with Lys121. After simulating the conformational changes of LdisPBPs at pH7.3 and 5.5 by constant pH molecular dynamics simulation in implicit solvent, the N-terminal sequences of PBPs was unfolded, only having five α-helices, and PBP2 had larger binding pocket at 7.3 than PBP1. To investigate the changes of α-helices at different pH, far-UV and near-UV circular dichroism showed PBPs consist of α-helices, and the tertiary structures of PBP1 and PBP2 were influenced at pH7.3 and 5.5. The fluorescence binding assay indicated that PBP1 and PBP2 have similarly binding affinity to (+)-D at pH 5.5 and 7.3, respectively. At pH 5.5, the dissociation constant of the complex between PBP1 and 2-decyl-1-oxaspiro [2.2] pentane (OXP1) was 0.68±0.01μM, for (+)-D was 5.32±0.11μM, while PBP2 with OXP1 and (+)-D were 1.88±0.02μM and 5.54±0.04μM, respectively. Three chemicals screened had higher affinity to PBP1 than (+)-D except Pal at pH5.5, and had lower affinity than (+)-D at pH7.3. To PBP2, these chemicals had lower affinity than the sex pheromone except Bis at pH 5.5 and pH 7.3. Only PBP1 had higher affinity with Sal than the sex pheromone at pH 5.5. Therefore, the structures of PBP1 and PBP2 had different changes at pH5.5 and 7.3, showing different affinity to chemicals. This study helps understanding the role of PBPs as well as in developing more efficient chemicals for pest control. PMID:22904666

Monitoring binding affinity between drug and α1-acid glycoprotein in real time by Venturi easy ambient sonic-spray ionization mass spectrometry.

PubMed

Liu, Ning; Lu, Xin; Yang, YuHan; Yao, Chen Xi; Ning, BaoMing; He, Dacheng; He, Lan; Ouyang, Jin

2015-10-01

A new approach for monitoring the binding affinity between drugs and alpha 1-acid glycoprotein in real time was developed based on a combination of drug-protein reaction followed by Venturi easy ambient sonic-spray ionization mass spectrometry determination of the free drug concentrations. A known basic drug, propranolol was used to validate the new built method. Binding constant values calculated by venturi easy ambient sonic-spray ionization mass spectrometry was in good accordance with a traditional ultrafiltration combined with high performance liquid chromatography method. Then six types of basic drugs were used as the samples to conduct the real time analysis. Upon injection of alpha 1-acid glycoprotein to the drug mixture, the ion chromatograms were extracted to show the changes in the free drug concentrations in real time. By observing the drop-out of six types of drugs during the whole binding reaction, the binding affinities of different drugs were distinguished. A volume shift validating experiment and an injection delay correcting experiment were also performed to eliminate extraneous factors and verify the reliability of our experiment. Therefore, the features of Venturi easy ambient sonic-spray ionization mass spectrometry (V-EASI-MS) and the experimental results indicate that our technique is likely to become a powerful tool for monitoring drug-AGP binding affinity in real time. Copyright © 2015 Elsevier B.V. All rights reserved.

Affinity purification of seminalplasmin and characterization of its interaction with calmodulin.

PubMed Central

Comte, M; Malnoë, A; Cox, J A

1986-01-01

Bull seminalplasmin antagonizes with high potency and selectivity the activating effect of calmodulin on target enzymes [Gietzen & Galla (1985) Biochem. J. 230, 277-280]. In the present paper we establish that seminalplasmin forms a 1:1, Ca2+-dependent and urea-resistant complex with calmodulin. The dissociation constant equals 1.6 nM. In the absence of Ca2+ a low-affinity complex is formed that is disrupted by 4 M-urea. On the basis of these properties, a fast affinity purification of seminalplasmin was developed. The high specificity of seminalplasmin as a calmodulin antagonist was demonstrated for the multipathway-regulated adenylate cyclase of bovine cerebellum. Far-u.v. c.d. properties are consistent with a random form of seminalplasmin in aqueous solution; 23% alpha-helix is induced on interaction with calmodulin. The fluorescence properties of the single tryptophan residue of seminalplasmin are markedly changed on formation of the complex. These studies allowed us to locate tentatively the peptide segment that interacts with calmodulin, and to ascertain the structural homology between seminalplasmin and other calmodulin-binding peptides. Additional material, showing the inhibition of calmodulin-mediated activation of bovine brain phosphodiesterase by melittin and seminalplasmin and also the near-u.v. spectrum of affinity-purified seminalplasmin, has been deposited as supplement SUP 50135 (4 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1986) 233, 5. Images Fig. 2. PMID:3814096

The solvatochromic effects of side chain substitution on the binding interaction of novel tricarbocyanine dyes with human serum albumin.

PubMed

Beckford, Garfield; Owens, Eric; Henary, Maged; Patonay, Gabor

2012-04-15

The effects of solvatochromism on protein-ligand interactions have been studied by absorbance and near-infrared laser induced fluorescence (NIR-LIF) spectroscopy. The utility of three novel classes of cyanine dyes designed for this purpose illustrates that the affinity interactions of ligands at the hydrophobic binding pockets of Human Serum Albumin (HSA) are not only dependent on the overall hydrophobic characteristics of the molecules but are highly influenced by the size of the ligands as well. Whereas changes to the chromophore moiety exhibited slight to moderate changes to the hydrophobic nature of these molecules, substitution at the alkyl indolium side chain has enabled us to vary the binding affinity towards serum albumin. Substitution at the indolium side chain among an ethyl to butyl group results in improved binding characteristics and an almost three-fold increase in affinity constant. In addition, replacement of the ethyl side chain with a phenylpropyl group also yielded unique solvotachromic patterns such as increased hydrophobicity and subsequent biocompatibility with the HSA binding regions. Ligand interaction was however inhibited by steric hindrance associated with the bulky phenyl ring system thus affecting the increased binding that could be realized from the improved hydrophobic nature of the molecules. This characteristic change in binding affinity is of potential interest to developing a methodology which reveals information on the hydrophobic character and steric specificity of the binding cavities. Copyright © 2012 Elsevier B.V. All rights reserved.

The energy and work of a ligand-gated ion channel

PubMed Central

Auerbach, Anthony

2015-01-01

Ligand-gated ion channels are allosteric membrane proteins that isomerize between C(losed) and O(pen) conformations. A difference in affinity for ligands in the two shapes influences the C↔O ‘gating’ equilibrium constant. The energies associated with adult-type mouse neuromuscular nicotinic acetylcholine receptor-channel (AChR) gating have been measured by using single-channel electrophysiology. Without ligands the free energy, enthalpy and entropy of gating are ΔG0=+8.4, ΔH0=+10.9 and ΔS0=+2.4 kcal/mol (−100 mV, 23 °C). Many mutations throughout the protein change ΔG0, including natural ones that cause disease. Agonists and most mutations change approximately independently the ground state energy difference, so it is possible to forecast and engineer AChR responses simply by combining perturbations. The free energy of the low↔high affinity change for the neurotransmitter at each of two functionally-equivalent binding sites is ΔGBACh=−5.1 kcal/mol. ΔGBACh is set mainly by interactions of ACh with just three binding site aromatic groups. For a series of structurally-related agonists there is a correlation between the energies of low- and high-affinity binding, which implies that gating commences with the formation of the low affinity complex. Brief, intermediate states in binding and gating have been detected. Several proposals for the nature of the gating transition state energy landscape and the isomerization mechanism are discussed. PMID:23357172

Immobilized metal ion affinity electrophoresis. A study with several model proteins containing histidine.

PubMed

Goubran-Botros, H; Nanak, E; Abdul Nour, J; Birkenmeir, G; Vijayalakshmi, M A

1992-04-24

Immobilized metal ion affinity electrophoresis (IMA-Elec) is one among the many methods derived from the immobilized metal ion affinity chromatography. Two approaches for incorporating the metal ligand, were studied. One was in the form of insoluble particulate material based on Sepharose 6B and the other in the form of soluble polymer based on polyethylene glycol (PEG) 5000. Both the polymers coupled with iminodiacetate and metallized with copper or zinc were used as ligands, incorporated into soluble agarose as the electrophoretic gel. Several histidine-containing model proteins were studied with both the systems and their metal binding strengths were determined as the dissociation constants, Kd. The results clearly demonstrated that the mechanism of protein recognition by immobilized copper or zinc via the accessible histidyl residues was maintained in the IMA-Elec system. Proteins with increasing numbers of histidine residues showed increasing binding strength (lower Kd values). While this basic mechanism was conserved, the supporting polymers (Sepharose 6B and the PEG 5000) showed significant differences in the metal binding to the protein. The polysaccharide Sepharose 6B enhanced the binding strength compared with PEG 5000. The optimum electrophoretic parameters were determined to be current intensities up to 20 mA and pH ca. 7.0. At pH greater than 8.0, a significant decrease in the affinity was observed, this decrease being greater with PEG 5000 than Sepharose 6B as supporting material.

Quantitative dissection of hydrogen bond-mediated proton transfer in the ketosteroid isomerase active site

PubMed Central

Sigala, Paul A.; Fafarman, Aaron T.; Schwans, Jason P.; Fried, Stephen D.; Fenn, Timothy D.; Caaveiro, Jose M. M.; Pybus, Brandon; Ringe, Dagmar; Petsko, Gregory A.; Boxer, Steven G.; Herschlag, Daniel

2013-01-01

Hydrogen bond networks are key elements of protein structure and function but have been challenging to study within the complex protein environment. We have carried out in-depth interrogations of the proton transfer equilibrium within a hydrogen bond network formed to bound phenols in the active site of ketosteroid isomerase. We systematically varied the proton affinity of the phenol using differing electron-withdrawing substituents and incorporated site-specific NMR and IR probes to quantitatively map the proton and charge rearrangements within the network that accompany incremental increases in phenol proton affinity. The observed ionization changes were accurately described by a simple equilibrium proton transfer model that strongly suggests the intrinsic proton affinity of one of the Tyr residues in the network, Tyr16, does not remain constant but rather systematically increases due to weakening of the phenol–Tyr16 anion hydrogen bond with increasing phenol proton affinity. Using vibrational Stark spectroscopy, we quantified the electrostatic field changes within the surrounding active site that accompany these rearrangements within the network. We were able to model these changes accurately using continuum electrostatic calculations, suggesting a high degree of conformational restriction within the protein matrix. Our study affords direct insight into the physical and energetic properties of a hydrogen bond network within a protein interior and provides an example of a highly controlled system with minimal conformational rearrangements in which the observed physical changes can be accurately modeled by theoretical calculations. PMID:23798390

Binding specificity of the juvenile hormone carrier protein from the hemolymph of the tobacco hornworm Manduca sexta Johannson (Lepidoptera: Sphingidae).

PubMed

Peterson, R C; Reich, M F; Dunn, P E; Law, J H; Katzenellnbogen, J A

1977-05-17

A series of analogues of insect juvenile hormone (four geometric isomers of methyl epoxyfarnesenate, several para-substituted epoxygeranyl phenyl ethers, and epoxyfarnesol and its acetate and haloacetate derivatives) was prepared to investigate the binding specificity of the hemolymph juvenile hormone binding protein from the tobacco hornworm Manduct sexta. The relative binding affinities were determined by a competition assay against radiolabeled methyl (E,E)-3,11-dimethyl-7-ethyl-cis-10,11-epoxytrideca-2,6-dienoate (JH I). The ratio of dissociation constants was estimated by plotting competitor data according to a linear transformation of the dissociation equations describing competition of two ligands for a binding protein. The importance of the geometry of the sesquiterpene hydrocarbon chain is indicated by the fact that the binding affinity is decreased as Z (cis) double bonds are substituted for E (trans) double bonds in the methyl epoxyfarnesenate series; the unepoxidized analogues do not bind. A carboxylic ester function is important although its orientation can be reversed, as indicated by the good binding of epoxyfarnesyl acetate. In the monoterpene series, methyl epoxygeranoate shows no affinity for the binding protein, but substitution of a phenyl or p-carbomethoxyphenyl ether for the ester function imparts a low, but significant affinity. These data taken together with earlier results indicate that the binding site for juvenile hormone in the hemolymph binding protein is characterized by a sterically defined hydrophobic region with polar sites that recognize the epoxide and the ester functions.

High-aluminum-affinity silica is a nanoparticle that seeds secondary aluminosilicate formation.

PubMed

Jugdaohsingh, Ravin; Brown, Andy; Dietzel, Martin; Powell, Jonathan J

2013-01-01

Despite the importance and abundance of aluminosilicates throughout our natural surroundings, their formation at neutral pH is, surprisingly, a matter of considerable debate. From our experiments in dilute aluminum and silica containing solutions (pH ~ 7) we previously identified a silica polymer with an extraordinarily high affinity for aluminium ions (high-aluminum-affinity silica polymer, HSP). Here, further characterization shows that HSP is a colloid of approximately 2.4 nm in diameter with a mean specific surface area of about 1,000 m(2) g(-1) and it competes effectively with transferrin for Al(III) binding. Aluminum binding to HSP strongly inhibited its decomposition whilst the reaction rate constant for the formation of the β-silicomolybdic acid complex indicated a diameter between 3.6 and 4.1 nm for these aluminum-containing nanoparticles. Similarly, high resolution microscopic analysis of the air dried aluminum-containing silica colloid solution revealed 3.9 ± 1.3 nm sized crystalline Al-rich silica nanoparticles (ASP) with an estimated Al:Si ratio of between 2 and 3 which is close to the range of secondary aluminosilicates such as imogolite. Thus the high-aluminum-affinity silica polymer is a nanoparticle that seeds early aluminosilicate formation through highly competitive binding of Al(III) ions. In niche environments, especially in vivo, this may serve as an alternative mechanism to polyhydroxy Al(III) species binding monomeric silica to form early phase, non-toxic aluminosilicates.

High-Aluminum-Affinity Silica Is a Nanoparticle That Seeds Secondary Aluminosilicate Formation

PubMed Central

Jugdaohsingh, Ravin; Brown, Andy; Dietzel, Martin; Powell, Jonathan J.

2013-01-01

Despite the importance and abundance of aluminosilicates throughout our natural surroundings, their formation at neutral pH is, surprisingly, a matter of considerable debate. From our experiments in dilute aluminum and silica containing solutions (pH ~ 7) we previously identified a silica polymer with an extraordinarily high affinity for aluminium ions (high-aluminum-affinity silica polymer, HSP). Here, further characterization shows that HSP is a colloid of approximately 2.4 nm in diameter with a mean specific surface area of about 1,000 m2 g-1 and it competes effectively with transferrin for Al(III) binding. Aluminum binding to HSP strongly inhibited its decomposition whilst the reaction rate constant for the formation of the β-silicomolybdic acid complex indicated a diameter between 3.6 and 4.1 nm for these aluminum-containing nanoparticles. Similarly, high resolution microscopic analysis of the air dried aluminum-containing silica colloid solution revealed 3.9 ± 1.3 nm sized crystalline Al-rich silica nanoparticles (ASP) with an estimated Al:Si ratio of between 2 and 3 which is close to the range of secondary aluminosilicates such as imogolite. Thus the high-aluminum-affinity silica polymer is a nanoparticle that seeds early aluminosilicate formation through highly competitive binding of Al(III) ions. In niche environments, especially in vivo, this may serve as an alternative mechanism to polyhydroxy Al(III) species binding monomeric silica to form early phase, non-toxic aluminosilicates. PMID:24349573

Evolutionary and Functional Relationships in the Truncated Hemoglobin Family.

PubMed

Bustamante, Juan P; Radusky, Leandro; Boechi, Leonardo; Estrin, Darío A; Ten Have, Arjen; Martí, Marcelo A

2016-01-01

Predicting function from sequence is an important goal in current biological research, and although, broad functional assignment is possible when a protein is assigned to a family, predicting functional specificity with accuracy is not straightforward. If function is provided by key structural properties and the relevant properties can be computed using the sequence as the starting point, it should in principle be possible to predict function in detail. The truncated hemoglobin family presents an interesting benchmark study due to their ubiquity, sequence diversity in the context of a conserved fold and the number of characterized members. Their functions are tightly related to O2 affinity and reactivity, as determined by the association and dissociation rate constants, both of which can be predicted and analyzed using in-silico based tools. In the present work we have applied a strategy, which combines homology modeling with molecular based energy calculations, to predict and analyze function of all known truncated hemoglobins in an evolutionary context. Our results show that truncated hemoglobins present conserved family features, but that its structure is flexible enough to allow the switch from high to low affinity in a few evolutionary steps. Most proteins display moderate to high oxygen affinities and multiple ligand migration paths, which, besides some minor trends, show heterogeneous distributions throughout the phylogenetic tree, again suggesting fast functional adaptation. Our data not only deepens our comprehension of the structural basis governing ligand affinity, but they also highlight some interesting functional evolutionary trends.

Evolutionary and Functional Relationships in the Truncated Hemoglobin Family

PubMed Central

Bustamante, Juan P.; Radusky, Leandro; Boechi, Leonardo; Estrin, Darío A.; ten Have, Arjen; Martí, Marcelo A.

2016-01-01

Predicting function from sequence is an important goal in current biological research, and although, broad functional assignment is possible when a protein is assigned to a family, predicting functional specificity with accuracy is not straightforward. If function is provided by key structural properties and the relevant properties can be computed using the sequence as the starting point, it should in principle be possible to predict function in detail. The truncated hemoglobin family presents an interesting benchmark study due to their ubiquity, sequence diversity in the context of a conserved fold and the number of characterized members. Their functions are tightly related to O2 affinity and reactivity, as determined by the association and dissociation rate constants, both of which can be predicted and analyzed using in-silico based tools. In the present work we have applied a strategy, which combines homology modeling with molecular based energy calculations, to predict and analyze function of all known truncated hemoglobins in an evolutionary context. Our results show that truncated hemoglobins present conserved family features, but that its structure is flexible enough to allow the switch from high to low affinity in a few evolutionary steps. Most proteins display moderate to high oxygen affinities and multiple ligand migration paths, which, besides some minor trends, show heterogeneous distributions throughout the phylogenetic tree, again suggesting fast functional adaptation. Our data not only deepens our comprehension of the structural basis governing ligand affinity, but they also highlight some interesting functional evolutionary trends. PMID:26788940

Titre and affinity of propylthiouracil-induced anti-myeloperoxidase antibodies are closely associated with the development of clinical vasculitis.

PubMed

Ye, Hua; Gao, Ying; Guo, Xiao-Hui; Zhao, Ming-Hui

2005-10-01

Substantial evidences suggested that propylthiouracil (PTU) could induced anti-myeloperoxidase (MPO) antibodies in sera from patients with hyperthyroidism, however, only a subgroup of the PTU-induced anti-MPO antibody positive patients developed clinical evident vasculitis. The aim of this study is to compare the titres and affinities of PTU induced anti-MPO antibodies in sera from patients with hyperthyroidism with and without clinical vasculitis. Anti-MPO antibody positive sera from patients diagnosed hyperthyroidism with (n = 13) and without (n = 14) clinical evident vasculitis were collected. The titre was determined by MPO-ELISA and expressed as logarithm value (lgT). The affinity constant (aK) of anti-MPO IgG was measured by antigen inhibition assay. The titre and aK values were compared between patients with and without vasculitis. In patients with vasculitis, the mean lgT of anti-MPO antibodies was 3.62 +/- 0.66; the median aK was 4.47 x 10(7)M(-1). In patients without vasculitis, the mean lgT was 2.54 +/- 0.29; the median aK was 0.14 x 10(7)M(-1), and both were significant lower than those in patients with vasculitis (t = 5.464; P = 0.000 & z = -4.373; P = 0.000, respectively). We concluded that the titre and affinity of anti-MPO antibodies might be associated with the development of clinical vasculitis in patients with PTU-induced ANCA.

Prolonged Maltose-Limited Cultivation of Saccharomyces cerevisiae Selects for Cells with Improved Maltose Affinity and Hypersensitivity

PubMed Central

Jansen, Mickel L. A.; Daran-Lapujade, Pascale; de Winde, Johannes H.; Piper, Matthew D. W.; Pronk, Jack T.

2004-01-01

Prolonged cultivation (>25 generations) of Saccharomyces cerevisiae in aerobic, maltose-limited chemostat cultures led to profound physiological changes. Maltose hypersensitivity was observed when cells from prolonged cultivations were suddenly exposed to excess maltose. This substrate hypersensitivity was evident from massive cell lysis and loss of viability. During prolonged cultivation at a fixed specific growth rate, the affinity for the growth-limiting nutrient (i.e., maltose) increased, as evident from a decreasing residual maltose concentration. Furthermore, the capacity of maltose-dependent proton uptake increased up to 2.5-fold during prolonged cultivation. Genome-wide transcriptome analysis showed that the increased maltose transport capacity was not primarily due to increased transcript levels of maltose-permease genes upon prolonged cultivation. We propose that selection for improved substrate affinity (ratio of maximum substrate consumption rate and substrate saturation constant) in maltose-limited cultures leads to selection for cells with an increased capacity for maltose uptake. At the same time, the accumulative nature of maltose-proton symport in S. cerevisiae leads to unrestricted uptake when maltose-adapted cells are exposed to a substrate excess. These changes were retained after isolation of individual cell lines from the chemostat cultures and nonselective cultivation, indicating that mutations were involved. The observed trade-off between substrate affinity and substrate tolerance may be relevant for metabolic engineering and strain selection for utilization of substrates that are taken up by proton symport. PMID:15066785

Chemically bonded stationary phases that use synthetic hosts containing aromatic binding clefts: HPLC analysis of nitro-substituted polycyclic aromatic hydrocarbons.

PubMed Central

Zimmerman, S C; Saionz, K W; Zeng, Z

1993-01-01

The synthesis of hosts with improved binding affinities for nitroaromatic guests is described. Association constants for several host-guest complexes were measured in chloroform solution and ranged over three orders of magnitude. Two hosts were covalently linked to silica gel to produce chemically bonded stationary phases for HPLC. The use of these phases for HPLC analysis of nitro-substituted polycyclic aromatic hydrocarbons is discussed. PMID:8433981

Affinity study on bovine serum albumin's peptides to amphiphilic gold nanoparticles: A test of epitopes and non-epitopes

NASA Astrophysics Data System (ADS)

Yuan, Ming; Li, Wanrong; Yang, Mingming; Huang, Xiufeng; Bai, Zhijun; Liu, Yushuang; Cai, Weijun; Wang, Yuqin; Zhang, Feng

2017-09-01

It is an inevitable event that nanoparticles (NPs) will encounter proteins/peptides in nano-medicine, so it has been significant to know their interaction mechanism before in vivo applications. Previously, a 105-amino-acid sequence had been reported as the binding site between bovine serum albumin (BSA) and amphiphilic polymer coated gold nanoparticles (AP-AuNPs) along with a mortise-tenon joint hypothesis. This article tested the affinity difference between two epitope peptide sequences such as: LGEYGFQNALIVR (S1), DAFLGSFLYEYSR (S2) and one non-epitope peptide sequence as: FDEHVKLVNELTEF (S3). With the photoluminescent amino acid residues, the fluorescence quenching method based on the nanometal surface energy transfer (NSET) principle was able to study the thermodynamics of the current binding system. The binding constants (Ka) were determined and followed the order as: Ka-S1 > Ka-S2 >> Ka-S3. Moreover, Hill constants indicated that cooperativity only presented in the interactions of AP-AuNP with either S1 or S2, but not for S3. Moreover, gel electrophoresis, surface plasmon resonance, atomic force microscopy and three dimensional fluorescence microscopy were all also used to comprehensively analyse the binding interaction mechanism. These results further provided useful information to better understand the mortise-tenon joint, which might find applications to nanofabrication and biomedicine.

Volatile anesthetics compete for common binding sites on bovine serum albumin: a 19F-NMR study.

PubMed Central

Dubois, B W; Cherian, S F; Evers, A S

1993-01-01

There is controversy as to the molecular nature of volatile anesthetic target sites. One proposal is that volatile anesthetics bind directly to hydrophobic binding sites on certain sensitive target proteins. Consistent with this hypothesis, we have previously shown that a fluorinated volatile anesthetic, isoflurane, binds saturably [Kd (dissociation constant) = 1.4 +/- 0.2 mM, Bmax = 4.2 +/- 0.3 sites] to fatty acid-displaceable domains on serum albumin. In the current study, we used 19F-NMR T2 relaxation to examine whether other volatile anesthetics bind to the same sites on albumin and, if so, whether they vary in their affinity for these sites. We show that three other fluorinated volatile anesthetics bind with varying affinity to fatty acid-displaceable domains on serum albumin: halothane, Kd = 1.3 +/- 0.2 mM; methoxyflurane, Kd = 2.6 +/- 0.3 mM; and sevoflurane, Kd = 4.5 +/- 0.6 mM. These three anesthetics inhibit isoflurane binding in a competitive manner: halothane, K(i) (inhibition constant) = 1.3 +/- 0.2 mM; methoxyflurane, K(i) = 2.5 +/- 0.4 mM; and sevoflurane, K(i) = 5.4 +/- 0.7 mM--similar to each anesthetic's respective Kd of binding to fatty acid displaceable sites. These results illustrate that a variety of volatile anesthetics can compete for binding to specific sites on a protein. PMID:8341659

Studies on Changes of β-Adrenergic Receptors in Polymorphonuclear Cell and Mononuclear Cell with the Changes of Thyroid Function

PubMed Central

Lee, Jong Do; You, Myung Hee; Kim, Young Seol; Kim, Jin Woo; Kim, Kwang Won; Kim, Sun Woo; Choi, Young Kil

1986-01-01

Although it has been well established that thyroid hormones increase β-adrenergic receptors of various tissues in the animal studies, there are controversies about the β-adrenergic receptor changes of human mononuclear cells and polymorphonuclear cells. The present study was performed to analyze the change of β-adrenergic receptor of those cells according to the thyroid functional status and to evaluate their usefulness in assessment of sympathetic hyperactivity. We measured [3H]-dihydroalprenolol binding to circulating mononuclear and polymorphonuclear cells from 18 patients with hyperthyrodism, 7 with hypothyroidism, 8 with euthyroid goiter and 21 normal controls. Only with polymorphonuclear cells the receptor concentration was significantly higher (P<0.01) in hyperthyroidism (46.07±4.78 fmol/mg protein) than in the normal control (28.42±2.06 fmol/mg protein) and the affinity constants of both cells were comparable to normal control values. And serum concentrations of T3 were not correlated well with the changes of receptor concentrations in hyperthyroidism. The patients with hypothyroidism and euthyroid goiter showed no significant difference in the receptor concentration and the affinity constants with both cell binding assays. These results indicate that thyroid hormones increase the receptor concentration in polymorphonuclear cells which might be responsible for the symptoms of sympathetic hyperactivity and the polymorphornuclear cells are useful for β-adrenergic receptor assay. PMID:15759381

BPS Z{sub N} string tensions, sine law and Casimir scaling, and integrable field theories

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kneipp, Marco A. C.; International Centre for Theoretical Physics

We consider a Yang-Mills-Higgs theory with spontaneous symmetry breaking of the gauge group G{yields}U(1){sup r}{yields}C{sub G}, with C{sub G} being the center of G. We study two vacua solutions of the theory which produce this symmetry breaking. We show that for one of these vacua, the theory in the Coulomb phase has the mass spectrum of particles and monopoles which is exactly the same as the mass spectrum of particles and solitons of two-dimensional affine Toda field theory, for suitable coupling constants. That result holds also for N=4 super Yang-Mills theories. On the other hand, in the Higgs phase, wemore » show that for each of the two vacua the ratio of the tensions of the BPS Z{sub N} strings satisfy either the Casimir scaling or the sine law scaling for G=SU(N). These results are extended to other gauge groups: for the Casimir scaling, the ratios of the tensions are equal to the ratios of the quadratic Casimir constant of specific representations; for the sine law scaling, the tensions are proportional to the components of the left Perron-Frobenius eigenvector of Cartan matrix K{sub ij} and the ratios of tensions are equal to the ratios of the soliton masses of affine Toda field theories.« less

Scale criticality in estimating ecosystem carbon dynamics

USGS Publications Warehouse

Zhao, Shuqing; Liu, Shuguang

2014-01-01

Scaling is central to ecology and Earth system sciences. However, the importance of scale (i.e. resolution and extent) for understanding carbon dynamics across scales is poorly understood and quantified. We simulated carbon dynamics under a wide range of combinations of resolution (nine spatial resolutions of 250 m, 500 m, 1 km, 2 km, 5 km, 10 km, 20 km, 50 km, and 100 km) and extent (57 geospatial extents ranging from 108 to 1 247 034 km2) in the southeastern United States to explore the existence of scale dependence of the simulated regional carbon balance. Results clearly show the existence of a critical threshold resolution for estimating carbon sequestration within a given extent and an error limit. Furthermore, an invariant power law scaling relationship was found between the critical resolution and the spatial extent as the critical resolution is proportional to An (n is a constant, and A is the extent). Scale criticality and the power law relationship might be driven by the power law probability distributions of land surface and ecological quantities including disturbances at landscape to regional scales. The current overwhelming practices without considering scale criticality might have largely contributed to difficulties in balancing carbon budgets at regional and global scales.

Observational effects of varying speed of light in quadratic gravity cosmological models

NASA Astrophysics Data System (ADS)

Izadi, Azam; Shacker, Shadi Sajedi; Olmo, Gonzalo J.; Banerjee, Robi

We study different manifestations of the speed of light in theories of gravity where metric and connection are regarded as independent fields. We find that for a generic gravity theory in a frame with locally vanishing affine connection, the usual degeneracy between different manifestations of the speed of light is broken. In particular, the space-time causal structure constant (cST) may become variable in that local frame. For theories of the form f(ℛ,ℛμνℛ μν), this variation in cST has an impact on the definition of the luminosity distance (and distance modulus), which can be used to confront the predictions of particular models against Supernovae type Ia (SN Ia) data. We carry out this test for a quadratic gravity model without cosmological constant assuming (i) a constant speed of light and (ii) a varying speed of light (VSL), and find that the latter scenario is favored by the data.

Predicting Stability Constants for Uranyl Complexes Using Density Functional Theory

DOE PAGES

Vukovic, Sinisa; Hay, Benjamin P.; Bryantsev, Vyacheslav S.

2015-04-02

The ability to predict the equilibrium constants for the formation of 1:1 uranyl:ligand complexes (log K 1 values) provides the essential foundation for the rational design of ligands with enhanced uranyl affinity and selectivity. We also use density functional theory (B3LYP) and the IEFPCM continuum solvation model to compute aqueous stability constants for UO 2 2+ complexes with 18 donor ligands. Theoretical calculations permit reasonably good estimates of relative binding strengths, while the absolute log K 1 values are significantly overestimated. Accurate predictions of the absolute log K 1 values (root mean square deviation from experiment < 1.0 for logmore » K 1 values ranging from 0 to 16.8) can be obtained by fitting the experimental data for two groups of mono and divalent negative oxygen donor ligands. The utility of correlations is demonstrated for amidoxime and imide dioxime ligands, providing a useful means of screening for new ligands with strong chelate capability to uranyl.« less

Simulation of NO and O2 transport facilitated by polymerized hemoglobin solutions in an arteriole that takes into account wall shear stress-induced NO production.

PubMed

Zhou, Yipin; Cabrales, Pedro; Palmer, Andre F

2012-03-01

A mathematical model was developed to study nitric oxide (NO) and oxygen (O(2)) transport in an arteriole and surrounding tissues exposed to a mixture of red blood cells (RBCs) and hemoglobin (Hb)-based O(2) carriers (HBOCs). A unique feature of this model is the inclusion of blood vessel wall shear stress-induced production of endothelial-derived NO, which is very sensitive to the viscosity of the RBC and HBOC mixture traversing the blood vessel lumen. Therefore in this study, a series of polymerized bovine Hb (PolyHb) solutions with high viscosity, varying O(2) affinities, NO dioxygenation rate constants and O(2) dissociation rate constants that were previously synthesized and characterized by our group was evaluated via mathematical modeling, in order to investigate the effect of these biophysical properties on the transport of NO and O(2) in an arteriole and its surrounding tissues subjected to anemia with the commercial HBOC Oxyglobin® and cell-free bovine Hb (bHb) serving as appropriate controls. The computer simulation results indicated that transfusion of high viscosity PolyHb solutions promoted blood vessel wall shear stress dependent generation of the vasodilator NO, especially in the blood vessel wall and should transport enough NO inside the smooth muscle layer to activate vasodilation compared to the commercial HBOC Oxyglobin® and cell-free bHb. However, NO scavenging in the arteriole lumen was unavoidable due to the intrinsic high NO dioxygenation rate constant of the HBOCs being studied. This study also observed that all PolyHbs could potentially improve tissue oxygenation under hypoxic conditions, while low O(2) affinity PolyHbs were more effective in oxygenating tissues under normoxic conditions compared with high O(2) affinity PolyHbs. In addition, all ultrahigh molecular weight PolyHbs displayed higher O(2) transfer rates than the commercial HBOC Oxyglobin® and cell-free bHb. Therefore, these results suggest that ultrahigh molecular weight PolyHb solutions could be used as safe and efficacious O(2) carriers for use in transfusion medicine. It also suggests that future generations of PolyHb solutions should possess lower NO dioxygenation reaction rate constants in order to reduce NO scavenging, while maintaining high solution viscosity to take advantage of wall shear stress-induced NO production. Taken together, we suggest that this mathematical model can be used to predict the vasoactivity of HBOCs and help guide the design and optimization of the next generation of HBOCs for use in transfusion medicine. Copyright © 2011 Elsevier B.V. All rights reserved.

Further considerations of engine emissions from subsonic aircraft at cruise altitude

NASA Astrophysics Data System (ADS)

Lee, S. H.; Le Dilosquer, M.; Singh, R.; Rycroft, M. J.

The most significant man-made sources of pollution of the higher troposphere and lower stratosphere are exhaust emissions from civil subsonic aircraft at cruise altitude (8-12 km). This paper examines such issues by computational modelling of Boeing 747-400 flights during their cruise phase between selected city pairs, for example London to Tokyo. The engine performance, exhaust pollutant prediction, and detailed flight history analysis effects of different Mach numbers and of increasing the cruise altitude from 9.8 to 12.1 km during the flight rather than staying at a constant cruise altitude of 10.5 km are studied in detail. To minimise the overall effects of atmospheric pollution, a Mach number of 0.85 and increasing altitude is the favoured cruise technique.