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Sample records for active site labeling

  1. Denaturation studies of active-site labeled papain using electron paramagnetic resonance and fluorescence spectroscopy.

    PubMed Central

    Ping, Z A; Butterfiel, D A

    1991-01-01

    A spin-labeled p-chloromercuribenzoate (SL-PMB) and a fluorescence probe, 6-acryloyl-2-dimethylaminonaphthalene (Acrylodan), both of which bind to the single SH group located in the active site of papain, were used to investigate the interaction of papain (EC 3.4.22.2) with two protein denaturants. It was found that the active site of papain was highly stable in urea solution, but underwent a large conformational change in guanidine hydrochloride solution. Electron paramagnetic resonance and fluorescence results were in agreement and both paralleled enzymatic activity of papain with respect to both the variation in pH and denaturation. These results strongly suggest that SL-PMB and Acrylodan labels can be used to characterize the physical state of the active site of the enzyme. PMID:1657229

  2. Affinity labeling and characterization of the active site histidine of glucosephosphate isomerase

    SciTech Connect

    Gibson, D.R.; Gracy, R.W.; Hartman, F.C.

    1980-10-10

    N-bromoacetylethanolamine phosphate was found to act as a specific affinity label for the active center of glucosephosphate isomerase. The inactivation process followed pseudo-first order kinetics, was irreversible, and exhibited rate saturation kinetics with minimal half-lives of inactivation of 4.5 and 6.3 min for the enzyme isolated from human placenta and rabbit muscle, respectively. The pH dependence of the inactivation process closely paralleled the pH dependence of the overall catalytic process with pK/sub a/ values at pH 6.4 and 9.0. The stoichiometry of labeling of either enzyme, as determined with N-bromo(/sup 14/C/sub 2/)acetylethanolamine phosphate, was 1 eq of the affinity label/subunit of enzyme. After acid hydrolysis and amino acid analysis of the radioactive affinity-labeled human enzyme, only radioactive 3-carboxymethyl histidine was found. In the case of the rabbit enzyme, the only radioactive derivative obtained was 1-carboxymethyl histidine. Active site tryptic peptides were isolated by solvent extraction, thin layer peptide fingerprinting, and ion exchange chromatography before and after removal of the phosphate from the active site peptide. Amino acid analysis of the labeled peptides from the two species were very similar. Using high sensitivity methods for sequence analysis, the primary structure of the active site was established as Val-Leu-His-Ala-Glu-Asn-Val-Asp (Gly,Thr,Ser) Glu-Ile (Thr-Gly-His-Lys-Glx)-Tyr-Phe. Apparent sequence homology between the catalytic center of glucosephosphate isomerase and triosephosphate isomerase suggest that the two enzymes may have evolved from a common ancestral gene.

  3. Active site labeling of the RNA polymerases A, B, and C from yeast.

    PubMed

    Riva, M; Schäffner, A R; Sentenac, A; Hartmann, G R; Mustaev, A A; Zaychikov, E F; Grachev, M A

    1987-10-25

    RNA polymerases A, B, and C from yeast were modified by reaction with 4-formylphenyl-gamma-ester of ATP as priming nucleotide followed by reduction with NaBH4. Upon phosphodiester bond formation with [alpha-32P]UTP, only the second largest subunit, A135, B150, or C128, was labeled in a template-dependent reaction. This indicates that these polypeptide chains are functionally homologous. The product covalently bound to B150 subunit was found to consist of a mixture of ApU and a trinucleotide. Enzyme labeling exhibited the characteristic alpha-amanitin sensitivity reported for A and B RNA polymerases. Labeling of both large subunits of enzyme A and B but not of any of the smaller subunits was observed when the reduction step stabilizing the binding of the priming nucleotide was carried out after limited chain elongation. These results illustrate the conservative evolution of the active site of eukaryotic RNA polymerases.

  4. Conformational dynamics of the active site loop of S-adenosylmethionine synthetase illuminated by site-directed spin labeling.

    PubMed

    Taylor, John C; Markham, George D

    2003-07-15

    S-adenosylmethionine synthetase (ATP: L-methionine S-adenosyltransferase, methionine adenosyltransferase, a.k.a. MAT) is one of numerous enzymes that have a flexible polypeptide loop that moves to gate access to the active site in a motion that is closely coupled to catalysis. Crystallographic studies of this tetrameric enzyme have shown that the loop is closed in the absence of bound substrates. However, the loop must open to allow substrate binding and a variety of data indicate that the loop is closed during the catalytic steps. Previous kinetic studies indicate that during turnover loop motion occurs on a time scale of 10(-2)s, ca. 10-fold faster than chemical transformations and turnover. Site-directed spin labeling has been used to introduce nitroxide groups at two positions in the loop to illuminate how the motion of the loop is affected by substrate binding. The two loop mutants constructed, G105C and D107C, retain wild type levels of MAT activity; attachment of a methanethiosulfonate spin label to convert the cysteine to the "R1" residue reduced the k(cat) only for the labeled D107R1 form (7-fold). The K(m) value for methionine increased 2- to 4-fold for the cysteine mutants and 2- to 7-fold for the labeled proteins, whereas the K(m) for ATP was changed by at most 2-fold. EPR spectra for both labeled proteins are nearly identical and show the presence of two major spin label environments with rotational diffusion rates differing by approximately 10-fold; the slower rate is ca. 4-fold faster than the estimated protein rotational rate. The spectra are not altered by addition of substrates or products. At both positions the less mobile conformation constitutes ca. 65% of the total species, indicating an equilibrium that only slightly favors one form, that in which the label is more immobilized. The equilibrium constant that relates the two forms is comparable to the equilibrium constant of 1.5 for a conformational change that was previously deduced from the

  5. Determination of lysine residues affinity labeled in the active site of yeast RNA polymerase II(B) by mutagenesis.

    PubMed Central

    Treich, I; Carles, C; Sentenac, A; Riva, M

    1992-01-01

    In a previous study, yeast RNA polymerase II(B) was affinity labeled with two nucleotide derivatives (III and VIII) (1). In both cases, the labeled site was localized to the C-terminal part of the B150 subunit. The potential target lysyl residues of derivative III were mapped to the conserved domain H, between Asn946 and Met999. In the present work, we have mutagenized to arginine the five lysines present in domain H. Three lysines can be replaced, individually or simultaneously, without affecting cell growth, and each mutated enzyme can still be affinity labeled. Hence one or both of the other two lysyl residues, Lys979 and Lys987, is the target of the affinity reagent. These two lysines were each found to be essential for cell viability. Derivative VIII labeled another domain in addition to domain H. Supported by analogous results obtained for E. coli RNA polymerase using derivative VIII (2), we hypothesized that the second domain labeled by this derivative in the B150 subunit was domain I. Mutagenesis of the unique lysine present in domain I demonstrated that Lys 1102 was the target of derivative VIII. These results indicate that in both prokaryotic and eukaryotic RNA polymerases, domains H and I are in close proximity and participate to the active site. Images PMID:1408783

  6. The active centre of rabbit muscle triose phosphate isomerase. The site that is labelled by glycidol phosphate.

    PubMed

    Miller, J C; Waley, S G

    1971-06-01

    1. Glycidol (2,3-epoxypropanol) phosphate is a specific irreversible inhibitor of rabbit muscle triose phosphate isomerase (EC 5.3.1.1); the site of attachment has now been studied. 2. The labelled enzyme was digested with pepsin and a modified peptide isolated. The sequence of the peptide is: Ala-Tyr-Glu-Pro-Val-Trp. 3. It is the glutamic acid residue in this peptide that is labelled: the peptide is thus a gamma-glutamyl ester derived from glycerol phosphoric acid. The same site is labelled by a mixture of glycidol and inorganic phosphate. 4. Kinetic and stereochemical features of these reactions are discussed.

  7. Site directed spin labelling and pulsed dipolar electron paramagnetic resonance (double electron electron resonance) of force activation in muscle

    NASA Astrophysics Data System (ADS)

    Fajer, Piotr G.

    2005-05-01

    The recent development of site specific spin labelling and advances in pulsed electron paramagnetic resonance (EPR) have established spin labelling as a viable structural biology technique. Specific protein sites or whole domains can be selectively targeted for spin labelling by cysteine mutagenesis. The secondary structure of the proteins is determined from the trends in EPR signals of labels attached to consecutive residues. Solvent accessibility or label mobility display periodicities along the labelled polypeptide chain that are characteristic of β-strands (periodicity of 2 residues) or α-helices (3.6 residues). Low-resolution 3D structure of proteins is determined from the distance restraints. Two spin labels placed within 60-70 Å of each other create a local dipolar field experienced by the other spin labels. The strength of this field is related to the interspin distance, {\\propto } r^{-3 } . The dipolar field can be measured by the broadening of the EPR lines for the short distances (8-20 Å) or for the longer distances (17-70 Å) by the pulsed EPR methods, double electron-electron resonance (DEER) and double quantum coherence (DQC). A brief review of the methodology and its applications to the multisubunit muscle protein troponin is presented below.

  8. Recombinant human nerve growth factor is biologically active and labels novel high-affinity binding sites in rat brain

    SciTech Connect

    Altar, C.A.; Burton, L.E.; Bennett, G.L.; Dugich-Djordjevic, M. )

    1991-01-01

    Iodinated recombinant human nerve growth factor (125I-rhNGF) stimulated neurite formation in PC12 cell cultures with a half-maximal potency of 35-49 pg/ml, compared with 39-52 pg/ml for rhNGF. In quantitative ligand autoradiography, the in vitro equilibrium binding of 125I-rhNGF to brain sections showed a 10-fold regional variation in density and was saturable, reversible, and specifically displaced by up to 74% with rhNGF or murine NGF (muNGF). At equilibrium, 125I-rhNGF bound to these sites with high affinity and low capacity (Bmax less than or equal to 13.2 fmol/mg of protein). Calculation of 125I-rhNGF binding affinity by kinetic methods gave average Kd values of 24 and 31 pM. Computer-generated maps revealed binding in brain regions not identified previously with 125I-muNGF, including hippocampus; dentate gyrus; amygdala; paraventricular thalamus; frontal, parietal, occipital, and cingulate cortices; nucleus accumbens; olfactory tubercle; subiculum; pineal gland; and medial geniculate nucleus. NGF binding sites were distributed in a 2-fold increasing medial-lateral gradient in the caudate-putamen and a 2-fold lateral-medial gradient in the nucleus accumbens. 125I-rhNGF binding sites were also found in most areas labeled by 125I-muNGF, including the interpedunucular nucleus, cerebellum, forebrain cholinergic nuclei, caudoventral caudate-putamen, and trigeminal nerve nucleus. 125I-rhNGF binding sites were absent from areas replete with low-affinity NGF binding sites, including circumventricular organs, myelinated fiber bundles, and choroid plexus. The present analysis provides an anatomical differentiation of high-affinity 125I-rhNGF binding sites and greatly expands the number of brain structures that may respond to endogenous NGF or exogenously administered rhNGF.

  9. Site-specific labeling of RNA.

    PubMed

    Nilsen, Timothy W

    2013-03-01

    In this protocol, RNAs containing a specific internally labeled phosphate are generated. The entire transcript of interest is synthesized using in vitro transcription. It is then digested with RNase H in the presence of a complementary chimeric oligonucleotide of the sequence 5'-NNNddddNNNNNNN-3', where N is a 2'-O-methyl (2' OMe) ribonucleotide and d is a de-oxy-nu-cle-o-tide. RNase H specifically cleaves the RNA in the oligonucleotide-RNA hybrid at the phosphodiester bond opposite the 5'-most deoxynucleotide, creating 3'-hydroxyl and 5'-phosphate termini. After dephosphorylation of the 5' end of the 3' fragment with phosphatase, this terminus is labeled to high specific activity with [γ-(32)P]ATP. The fragments of the original RNA are then resealed with T4 DNA ligase in the presence of a splint (or bridge) oligonucleotide. These labeled RNAs can be used in subsequent procedures to probe RNA-protein or RNA-RNA interactions.

  10. Site-directed isotope labelling and FTIR spectroscopy of bacteriorhodopsin.

    PubMed

    Sonar, S; Lee, C P; Coleman, M; Patel, N; Liu, X; Marti, T; Khorana, H G; RajBhandary, U L; Rothschild, K J

    1994-08-01

    Insight into integral membrane proteins function is presently limited by the difficulty of producing three-dimensional crystals. In addition, X-ray structures of proteins normally do not provide information about the protonation state and structural changes of individual residues. We report here the first use of site-directed isotope labelling and Fourier transform infrared (FTIR) difference spectroscopy to detect structural changes at the level of single residues in an integral membrane protein. Two site-directed isotope labeled (SDIL) tyrosine analogues of bacteriorhodopsin were produced which exhibit normal activity. FTIR spectroscopy shows that out of 11 tyrosines, only Tyr 185 is structurally active during the early photocycle and may be part of a proton wire.

  11. Affinity labeling of the ribosomal P site in Drosophila melanogaster

    SciTech Connect

    North, D.

    1987-01-01

    Several recent studies have probed the peptidyl transferase region of the Drosophila ribosome via the use of reactive site specific analogues (affinity labels). P site proteins adjacent to the 3' end of the amino acid bearing tRNA strand were labeled with modified tRNA fragments. Drugs affecting the binding of these agents were used to further clarify the nature of the region. The nascent peptide region of the P site was not labeled in previous experiments. To label that region radioactive Bromoacetylphenylalanyl-tRNA (BrAcphe-tRNA) was synthesized. The alpha-bromoacetyl group of this analogue is potentially reactive with nucleophiles present in either proteins or RNAs. Charged tRNAs and tRNA analogues bearing a peptide bond on the N-terminus of their amino acid are recognized as having affinity for the ribosomal P site. Specific labeling of the P site by BrAcphe-tRNA was confirmed by its ability to radioactively label proteins indirectly. As many as 8 ribosomal proteins may be labeled under these conditions, however, the majority of the bound label is associated with 3 large subunit proteins and 2 small subunit proteins. Overlaps between the proteins labeled by BrAcphe-tRNA and those labeled by other affinity labels are examined and a model of the peptidyl transferase region of Drosophila ribosomes is presented.

  12. Pretargeted PET Imaging Using a Site-Specifically Labeled Immunoconjugate.

    PubMed

    Cook, Brendon E; Adumeau, Pierre; Membreno, Rosemery; Carnazza, Kathryn E; Brand, Christian; Reiner, Thomas; Agnew, Brian J; Lewis, Jason S; Zeglis, Brian M

    2016-08-17

    In recent years, both site-specific bioconjugation techniques and bioorthogonal pretargeting strategies have emerged as exciting technologies with the potential to improve the safety and efficacy of antibody-based nuclear imaging. In the work at hand, we have combined these two approaches to create a pretargeted PET imaging strategy based on the rapid and bioorthogonal inverse electron demand Diels-Alder reaction between a (64)Cu-labeled tetrazine radioligand ((64)Cu-Tz-SarAr) and a site-specifically modified huA33-trans-cyclooctene immunoconjugate ((ss)huA33-PEG12-TCO). A bioconjugation strategy that harnesses enzymatic transformations and strain-promoted azide-alkyne click chemistry was used to site-specifically append PEGylated TCO moieties to the heavy chain glycans of the colorectal cancer-targeting huA33 antibody. Preclinical in vivo validation studies were performed in athymic nude mice bearing A33 antigen-expressing SW1222 human colorectal carcinoma xenografts. To this end, mice were administered (ss)huA33-PEG12-TCO via tail vein injection and-following accumulation intervals of 24 or 48 h-(64)Cu-Tz-SarAr. PET imaging and biodistribution studies reveal that this strategy clearly delineates tumor tissue as early as 1 h post-injection (6.7 ± 1.7%ID/g at 1 h p.i.), producing images with excellent contrast and high tumor-to-background activity concentration ratios (tumor:muscle = 21.5 ± 5.6 at 24 h p.i.). Furthermore, dosimetric calculations illustrate that this pretargeting approach produces only a fraction of the overall effective dose (0.0214 mSv/MBq; 0.079 rem/mCi) of directly labeled radioimmunoconjugates. Ultimately, this method effectively facilitates the high contrast pretargeted PET imaging of colorectal carcinoma using a site-specifically modified immunoconjugate. PMID:27356886

  13. Site-directed nanoparticle labeling of cytochrome c

    PubMed Central

    Aubin-Tam, Marie-Eve; Hwang, Wonmuk; Hamad-Schifferli, Kimberly

    2009-01-01

    Although nanoparticle-protein conjugates have been synthesized for numerous applications, bioconjugation remains a challenge, often resulting in denaturation or loss of protein function. This is partly because the protein–nanoparticle interface is poorly understood, which impedes the use of nanoparticles in nanomedicine. Although the effects of nanoparticle ligand and material on protein structure have been explored, the choice of the labeling site on the protein has not yet been systematically studied. To address this issue, we label cytochrome c site-specifically with a negatively charged Au nanoparticle via a covalent thiol–Au bond. The attachment site is controlled by cysteine mutations of surface residues. The effect of labeling on protein structure is probed by circular dichroism. Protein unfolding is the most severe when the nanoparticle is attached to the N- and C-terminal foldon, the core motif of cytochrome c. Also, when the nanoparticle is attached in the vicinity of charged residues, the amount of structural damage is greater because of salt-dependent electrostatic interactions with charged ligand bis(p-sulfonatophenyl) phenylphosphine on the nanoparticle. Molecular dynamics simulations also elucidate local to global structural perturbation depending on labeling site. These results suggest that the labeling site must be considered as one of the main design criteria for nanoparticle–protein conjugates. PMID:19251670

  14. Site-specific protein labeling with SNAP-tags.

    PubMed

    Cole, Nelson B

    2013-09-24

    Site-specific labeling of cellular proteins with chemical probes is a powerful tool for studying protein function in living cells. A number of small peptide and protein tags have been developed that can be labeled with synthetic probes with high efficiencies and specificities and provide flexibility not available with fluorescent proteins. The SNAP-tag is a modified form of the DNA repair enzyme human O(6)-alkylguanine-DNA-alkyltransferase, and undergoes a self-labeling reaction to form a covalent bond with O(6)-benzylguanine (BG) derivatives. BG can be modified with a wide variety of fluorophores and other reporter compounds, generally without affecting the reaction with the SNAP-tag. In this unit, basic strategies for labeling SNAP-tag fusion proteins, both for live cell imaging and for in vitro analysis, are described. This includes a description of a releasable SNAP-tag probe that allows the user to chemically cleave the fluorophore from the labeled SNAP-tag fusion. In vitro labeling of purified SNAP-tag fusions is briefly described.

  15. Isolation and sequencing of an active-site peptide from Rhodospirillum rubrum ribulosebisphosphate carboxylase/oxygenase after affinity labeling with 2-((Bromoacetyl)amino)pentitol 1,5-bisphosphate

    SciTech Connect

    Fraij, B.; Hartman, F.C.

    1983-01-01

    2-((Bromoacetyl)amino)pentitol 1,5-bisphosphate was reported to be a highly selective affinity label for ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum. The enzyme has now been inactivated with a /sup 14/C-labeled reagent in order to identify the target residue at the sequence level. Subsequent to inactivation, the enzyme was carboxymethylated with iodoacetate and then digested with trypsin. The only radioactive peptide in the digest was obtained at a high degree of purity by successive chromatography on DEAE-cellulose, SP-Sephadex, and Sephadex G-25. On the basis of amino acid analysis of the purified peptide, the derivatized residue was a methionyl sulfonium salt. Automated Edman degradation confirmed the purity of the labeled peptide and established its sequence as Leu-Gln-Gly-Ala-Ser-Gly-Ile-His-Thr-Gly-Thr-Met-Gly-Phe-Gly-Lys-Met-Glu-Gly-Glu-Ser-Ser-Asp-Arg. Cleavage of this peptide with cyanogen bromide showed that the reagent moiety was covalently attached to the second methionyl residue. Sequence homology with the carboxylase/oxygenase from spinach indicates that the lysyl residue immediately preceding the alkylated methionine corresponds to Lys-334, a residue previously implicated at the active site. 31 references, 4 figures, 3 tables.

  16. 16 CFR 305.6 - Duty to provide labels on Web sites.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... accessible Web site in a manner that allows catalog sellers to hyperlink to the label or download it for use in Web sites or paper catalogs. The label for each specific model must remain on the Web site for...

  17. Technological advances in site-directed spin labeling of proteins.

    PubMed

    Hubbell, Wayne L; López, Carlos J; Altenbach, Christian; Yang, Zhongyu

    2013-10-01

    Molecular flexibility over a wide time range is of central importance to the function of many proteins, both soluble and membrane. Revealing the modes of flexibility, their amplitudes, and time scales under physiological conditions is the challenge for spectroscopic methods, one of which is site-directed spin labeling EPR (SDSL-EPR). Here we provide an overview of some recent technological advances in SDSL-EPR related to investigation of structure, structural heterogeneity, and dynamics of proteins. These include new classes of spin labels, advances in measurement of long range distances and distance distributions, methods for identifying backbone and conformational fluctuations, and new strategies for determining the kinetics of protein motion.

  18. Site-Specific Covalent Labeling of RNA by Enzymatic Transglycosylation.

    PubMed

    Alexander, Seth C; Busby, Kayla N; Cole, Christian M; Zhou, Cun Yu; Devaraj, Neal K

    2015-10-14

    We demonstrate the site-specific incorporation of nucleobase derivatives bearing fluorophores or affinity labels into a short RNA stem loop recognition motif by exchange of a guanine residue. The RNA-TAG (transglycosylation at guanosine) is carried out by a bacterial (E. coli) tRNA guanine transglycosylase (TGT), whose natural substrate is the nitrogenous base PreQ1. Remarkably, we have successfully incorporated large functional groups including biotin, BODIPY, thiazole orange, and Cy7 through a polyethylene glycol linker attached to the exocyclic amine of PreQ1. Larger RNAs, such as mRNA transcripts, can be site-specifically labeled if they possess the 17-nucleotide hairpin recognition motif. The RNA-TAG methodology could facilitate the detection and manipulation of RNA molecules by enabling the direct incorporation of functional artificial nucleobases using a simple hairpin recognition element. PMID:26393285

  19. Simultaneous Site-Specific Dual Protein Labeling Using Protein Prenyltransferases.

    PubMed

    Zhang, Yi; Blanden, Melanie J; Sudheer, Ch; Gangopadhyay, Soumyashree A; Rashidian, Mohammad; Hougland, James L; Distefano, Mark D

    2015-12-16

    Site-specific protein labeling is an important technique in protein chemistry and is used for diverse applications ranging from creating protein conjugates to protein immobilization. Enzymatic reactions, including protein prenylation, have been widely exploited as methods to accomplish site-specific labeling. Enzymatic prenylation is catalyzed by prenyltransferases, including protein farnesyltransferase (PFTase) and geranylgeranyltransferase type I (GGTase-I), both of which recognize C-terminal CaaX motifs with different specificities and transfer prenyl groups from isoprenoid diphosphates to their respective target proteins. A number of isoprenoid analogues containing bioorthogonal functional groups have been used to label proteins of interest via PFTase-catalyzed reaction. In this study, we sought to expand the scope of prenyltransferase-mediated protein labeling by exploring the utility of rat GGTase-I (rGGTase-I). First, the isoprenoid specificity of rGGTase-I was evaluated by screening eight different analogues and it was found that those with bulky moieties and longer backbone length were recognized by rGGTase-I more efficiently. Taking advantage of the different substrate specificities of rat PFTase (rPFTase) and rGGTase-I, we then developed a simultaneous dual labeling method to selectively label two different proteins by using isoprenoid analogue and CaaX substrate pairs that were specific to only one of the prenyltransferases. Using two model proteins, green fluorescent protein with a C-terminal CVLL sequence (GFP-CVLL) and red fluorescent protein with a C-terminal CVIA sequence (RFP-CVIA), we demonstrated that when incubated together with both prenyltransferases and the selected isoprenoid analogues, GFP-CVLL was specifically modified with a ketone-functionalized analogue by rGGTase-I and RFP-CVIA was selectively labeled with an alkyne-containing analogue by rPFTase. By switching the ketone-containing analogue to an azide-containing analogue, it was

  20. Site-Specific Biomolecule Labeling with Gold Clusters

    PubMed Central

    Ackerson, Christopher J.; Powell, Richard D.; Hainfeld, James F.

    2013-01-01

    Site-specific labeling of biomolecules in vitro with gold clusters can enhance the information content of electron cryomicroscopy experiments. This chapter provides a practical overview of well-established techniques for forming biomolecule/gold cluster conjugates. Three bioconjugation chemistries are covered: Linker-mediated bioconjugation, direct gold–biomolecule bonding, and coordination-mediated bonding of nickel(II) nitrilotriacetic acid (NTA)-derivatized gold clusters to polyhistidine (His)-tagged proteins. PMID:20887859

  1. 2'-modified nucleosides for site-specific labeling of oligonucleotides

    NASA Technical Reports Server (NTRS)

    Krider, Elizabeth S.; Miller, Jeremiah E.; Meade, Thomas J.

    2002-01-01

    We report the synthesis of 2'-modified nucleosides designed specifically for incorporating labels into oligonucleotides. Conversion of these nucleosides to phosphoramidite and solid support-bound derivatives proceeds in good yield. Large-scale synthesis of 11-mer oligonucleotides possessing the 2'-modified nucleosides is achieved using these derivatives. Thermal denaturation studies indicate that the presence of 2'-modified nucleosides in 11-mer duplexes has minimal destabilizing effects on the duplex structure when the nucleosides are placed at the duplex termini. The powerful combination of phosphoramidite and support-bound derivatives of 2'-modified nucleosides affords the large-scale preparation of an entirely new class of oligonucleotides. The ability to synthesize oligonucleotides containing label attachment sites at 3', intervening, and 5' locations of a duplex is a significant advance in the development of oligonucleotide conjugates.

  2. Hydrazide Reactive Peptide Tags for Site-Specific Protein Labeling

    PubMed Central

    Eldridge, Glenn M.; Weiss, Gregory A.

    2011-01-01

    New site-specific protein labeling (SSPL) reactions for targeting specific, short peptides could be useful for the real time detection of proteins inside of living cells. One SSPL approach matches bioorthogonal reagents with complementary peptides. Here, hydrazide reactive peptides were selected from phage-displayed libraries using reaction-based selections. Selection conditions included washes of varying pH and treatment with NaCNBH3 in order to specifically select reactive carbonyl containing peptides. Selected peptides were fused to T4 lysozyme or synthesized on filter paper for colorimetric assays of the peptide-hydrazide interaction. A peptide-lysozyme protein fusion demonstrated specific, covalent labeling by the Hydrazide Reactive (HyRe) peptides in crude bacterial cell lysates, sufficient for the specific detection of an over-expressed protein fusion. Chemical synthesis of a short HyRe tag variant and subsequent reaction with two structurally distinct hydrazide probes produced covalent adducts observable by MALDI-TOF MS and MS/MS. Rather than isolating reactive carbonyl-containing peptides, we observed reaction with the N–terminal His of HyRe tag 114, amino acid sequence HKSNHSSKNRE, which attacks the hydrazide carbonyl at neutral pH. However, at the pH used during selection wash steps (<6.0), an alternative imine-containing product is formed that can be reduced with sodium cyanoborohydride. MSMS further reveals that this low pH product forms an adduct on Ser6. Further optimization of the novel bimolecular reaction described here could provide a useful tool for in vivo protein labeling and bioconjugate synthesis. The reported selection and screening methods could be widely applicable to the identification of peptides capable of other site-specific protein labeling reactions with bioorthogonal reagents. PMID:21905743

  3. NMR studies of active-site properties of human carbonic anhydrase II by using (15) N-labeled 4-methylimidazole as a local probe and histidine hydrogen-bond correlations.

    PubMed

    Shenderovich, Ilya G; Lesnichin, Stepan B; Tu, Chingkuang; Silverman, David N; Tolstoy, Peter M; Denisov, Gleb S; Limbach, Hans-Heinrich

    2015-02-01

    By using a combination of liquid and solid-state NMR spectroscopy, (15) N-labeled 4-methylimidazole (4-MI) as a local probe of the environment has been studied: 1) in the polar, wet Freon CDF3 /CDF2 Cl down to 130 K, 2) in water at pH 12, and 3) in solid samples of the mutant H64A of human carbonic anhydrase II (HCA II). In the latter, the active-site His64 residue is replaced by alanine; the catalytic activity is, however, rescued by the presence of 4-MI. For the Freon solution, it is demonstrated that addition of water molecules not only catalyzes proton tautomerism but also lifts its quasidegeneracy. The possible hydrogen-bond clusters formed and the mechanism of the tautomerism are discussed. Information about the imidazole hydrogen-bond geometries is obtained by establishing a correlation between published (1) H and (15) N chemical shifts of the imidazole rings of histidines in proteins. This correlation is useful to distinguish histidines embedded in the interior of proteins and those at the surface, embedded in water. Moreover, evidence is obtained that the hydrogen-bond geometries of His64 in the active site of HCA II and of 4-MI in H64A HCA II are similar. Finally, the degeneracy of the rapid tautomerism of the neutral imidazole ring His64 reported by Shimahara et al. (J. Biol. Chem.- 2007, 282, 9646) can be explained with a wet, polar, nonaqueous active-site conformation in the inward conformation, similar to the properties of 4-MI in the Freon solution. The biological implications for the enzyme mechanism are discussed. PMID:25521423

  4. NMR studies of active-site properties of human carbonic anhydrase II by using (15) N-labeled 4-methylimidazole as a local probe and histidine hydrogen-bond correlations.

    PubMed

    Shenderovich, Ilya G; Lesnichin, Stepan B; Tu, Chingkuang; Silverman, David N; Tolstoy, Peter M; Denisov, Gleb S; Limbach, Hans-Heinrich

    2015-02-01

    By using a combination of liquid and solid-state NMR spectroscopy, (15) N-labeled 4-methylimidazole (4-MI) as a local probe of the environment has been studied: 1) in the polar, wet Freon CDF3 /CDF2 Cl down to 130 K, 2) in water at pH 12, and 3) in solid samples of the mutant H64A of human carbonic anhydrase II (HCA II). In the latter, the active-site His64 residue is replaced by alanine; the catalytic activity is, however, rescued by the presence of 4-MI. For the Freon solution, it is demonstrated that addition of water molecules not only catalyzes proton tautomerism but also lifts its quasidegeneracy. The possible hydrogen-bond clusters formed and the mechanism of the tautomerism are discussed. Information about the imidazole hydrogen-bond geometries is obtained by establishing a correlation between published (1) H and (15) N chemical shifts of the imidazole rings of histidines in proteins. This correlation is useful to distinguish histidines embedded in the interior of proteins and those at the surface, embedded in water. Moreover, evidence is obtained that the hydrogen-bond geometries of His64 in the active site of HCA II and of 4-MI in H64A HCA II are similar. Finally, the degeneracy of the rapid tautomerism of the neutral imidazole ring His64 reported by Shimahara et al. (J. Biol. Chem.- 2007, 282, 9646) can be explained with a wet, polar, nonaqueous active-site conformation in the inward conformation, similar to the properties of 4-MI in the Freon solution. The biological implications for the enzyme mechanism are discussed.

  5. Site-specific fluorescent labeling to visualize membrane translocation of a myristoyl switch protein

    PubMed Central

    Yang, Sung-Tae; Lim, Sung In; Kiessling, Volker; Kwon, Inchan; Tamm, Lukas K.

    2016-01-01

    Fluorescence approaches have been widely used for elucidating the dynamics of protein-membrane interactions in cells and model systems. However, non-specific multi-site fluorescent labeling often results in a loss of native structure and function, and single cysteine labeling is not feasible when native cysteines are required to support a protein’s folding or catalytic activity. Here, we develop a method using genetic incorporation of non-natural amino acids and bio-orthogonal chemistry to site-specifically label with a single fluorescent small molecule or protein the myristoyl-switch protein recoverin, which is involved in rhodopsin-mediated signaling in mammalian visual sensory neurons. We demonstrate reversible Ca2+-responsive translocation of labeled recoverin to membranes and show that recoverin favors membranes with negative curvature and high lipid fluidity in complex heterogeneous membranes, which confers spatio-temporal control over down-stream signaling events. The site-specific orthogonal labeling technique is promising for structural, dynamical, and functional studies of many lipid-anchored membrane protein switches. PMID:27605302

  6. Site-specific fluorescent labeling to visualize membrane translocation of a myristoyl switch protein.

    PubMed

    Yang, Sung-Tae; Lim, Sung In; Kiessling, Volker; Kwon, Inchan; Tamm, Lukas K

    2016-01-01

    Fluorescence approaches have been widely used for elucidating the dynamics of protein-membrane interactions in cells and model systems. However, non-specific multi-site fluorescent labeling often results in a loss of native structure and function, and single cysteine labeling is not feasible when native cysteines are required to support a protein's folding or catalytic activity. Here, we develop a method using genetic incorporation of non-natural amino acids and bio-orthogonal chemistry to site-specifically label with a single fluorescent small molecule or protein the myristoyl-switch protein recoverin, which is involved in rhodopsin-mediated signaling in mammalian visual sensory neurons. We demonstrate reversible Ca(2+)-responsive translocation of labeled recoverin to membranes and show that recoverin favors membranes with negative curvature and high lipid fluidity in complex heterogeneous membranes, which confers spatio-temporal control over down-stream signaling events. The site-specific orthogonal labeling technique is promising for structural, dynamical, and functional studies of many lipid-anchored membrane protein switches. PMID:27605302

  7. 40 CFR 170.232 - Knowledge of labeling and site-specific information.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Knowledge of labeling and site... (CONTINUED) PESTICIDE PROGRAMS WORKER PROTECTION STANDARD Standard for Pesticide Handlers § 170.232 Knowledge of labeling and site-specific information. (a) Knowledge of labeling information. (1) The...

  8. 2-(4-bromoacetamido)anilino-2-deoxypentitol 1,5-bisphosphate, a new affinity label for ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum: determination of reaction parameters and characterization of an active site peptide

    SciTech Connect

    Herndon, C.S.; Hartman, F.C.

    1984-03-10

    Reductive amination of ribulose-P/sub 2/ with p-phenylenediamine in the presence of sodium cyanoborohydride yielded an epimeric mixture which was resolved by chromatography. Subsequent bromoacetylation of the isolated amino bisphosphates gave reagents A and B (ribo and arabino epimers of 2-(4-bromoacetamido)anilino-2-deoxypentitol 1.5-bisphosphate) which were competitive inhibitors of the carboxylase with K/sub i/ values of 705 and 104 ..mu..M, respectively. Reagent A exhibited no time-dependent effects on the carboxylase in either the deactivated or activated state. Incubation of the enzyme with reagent B in the presence of the essential activators CO/sub 2/ and Mg/sup 2 +/, however, resulted in an irreversible, time-dependent loss of activity, with a K/sub inact/ of 125 ..mu..M and a minimal half-time of 7.3 min. Covalent incorporation of (/sup 14/C)reagent B was directly proportional to the loss of activity, with total inactivation correlating with an incorporation of 1.1 mol of reagent/mol of subunit. Inclusion of the competitive inhibitor 2-carboxyribitol 1,5-bisphosphate protected against inactivation with a concomitant reduction in incorporation. Neither reagent affected the activity of spinach carboxylase. Fractionation of (/sup 14/C)reagent B-modified enzyme on DEAE-cellulose, subsequent to carboxymethylation and tryptic digestion, revealed two major radioactive peaks of approximately equal area. Digestion of each peak with alkaline phosphatase and rechromatography on DEAE-cellulose resulted in pure peptides I and II. The peptides were identical except in the site of labeling: peptide I contained a modified cysteinyl residue while peptide II contained a modified histidyl residue. 60 references, 7 figures, tables.

  9. Site-directed isotope labeling and ATR-FTIR difference spectroscopy of bacteriorhodopsin: the peptide carbonyl group of Tyr 185 is structurally active during the bR-->N transition.

    PubMed

    Ludlam, C F; Sonar, S; Lee, C P; Coleman, M; Herzfeld, J; RajBhandary, U L; Rothschild, K J

    1995-01-10

    The largest secondary structural change occurs in the bacteriorhodopsin (bR) photocycle during the M-->N transition. In this work site-directed isotope labeling (SDIL) and attenuated total reflection Fourier transform infrared (ATR-FTIR) difference spectroscopy were used to investigate this conformational change. L-Tyrosine containing a 13C isotope at the carbonyl carbon was selectively incorporated at Tyr 57, Tyr 147, and Tyr 185 by SDIL. This involves the cell-free expression of bR in the presence of Escherichia coli suppressor tRNA(CUATyr) aminoacylated with L-[1-13C]Tyr. ATR-FTIR difference spectroscopy reveals that of the 11 tyrosines, only the peptide carbonyl group of Tyr 185 undergoes a significant structural change during the bR-->N transition. Along with other spectroscopic evidence, this result suggests that the Tyr 185-Pro 186 region of the protein is structurally active and may function as a hinge which facilitates the tilt of the cytoplasmic portion of the F-helix in bacteriorhodopsin during the M-->N transition.

  10. Mode of action of exoglucanases from the cellulolytic fungus Trichoderma reesei: activity on reducing end-labeled cellooligosaccharides and topography of active sites of. beta. -glucosidase, cellobiohydrolase I(D), and cellobiohydrolase II

    SciTech Connect

    Chirico, W.J.

    1984-01-01

    The activity of purified ..beta..-glucosidase, cellobiohydrolase I(D) and cellobiohydrolase II from Trichoderma reesei was studied using (1-/sup 3/H)cellooligosaccharides. Reducing end-labeled cellooligosaccharides of high specific radioactivity were synthesized by reducing lactones of cellooligosaccharide aldonic acids with sodium boro(/sup 3/He)hydride. A thin-layer chromatographic method for separating (1-/sup 3/H)cellooligosaccharides with high resolution and quantitatively extracting them from silica gel was developed. The ..beta..-glucosidase was shown to bind (1-/sup 3/H)cellooligosaccharides in one productive mode and sequentially remove glucosyl residues from the nonreducing end. Cellobiohydrolase I(D) was shown to bind (1-/sup 3/H)cellooligosaccharides in more than one productive mode. Cellobiohydrolase II was also shown to bind (1-3H) cellooligosaccharides in more than one productive model; however, glycosidic bonds at the nonreducing end are preferentially hydrolyzed. The results are discussed in terms of the individual roles of exoglucanases during the saccharification of cellulose. A mechanism by which cellobiohydrolase I(D) and cellobiohydrolase II may synergistically degrade crystalline cellulose is proposed.

  11. 40 CFR 170.232 - Knowledge of labeling and site-specific information.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Knowledge of labeling and site-specific information. 170.232 Section 170.232 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS WORKER PROTECTION STANDARD Standard for Pesticide Handlers § 170.232 Knowledge of labeling and site-specific...

  12. Comparative conventional- and quantum dot-labeling strategies for LPS binding site detection in Arabidopsis thaliana mesophyll protoplasts

    PubMed Central

    Mgcina, Londiwe S.; Dubery, Ian A.; Piater, Lizelle A.

    2015-01-01

    Lipopolysaccharide (LPS) from Gram-negative bacteria is recognized as a microbe-associated molecular pattern (MAMP) and not only induces an innate immune response in plants, but also stimulates the development of characteristic defense responses. However, identification and characterization of a cell surface LPS-receptor/binding site, as described in mammals, remains elusive in plants. As an amphiphilic, macromolecular lipoglycan, intact LPS potentially contains three MAMP-active regions, represented by the O-polysaccharide chain, the core and the lipid A. Binding site studies with intact labeled LPS were conducted in Arabidopsis thaliana protoplasts and quantified using flow cytometry fluorescence changes. Quantum dots (Qdots), which allow non-covalent, hydrophobic labeling were used as a novel strategy in this study and compared to covalent, hydrophilic labeling with Alexa 488. Affinity for LPS-binding sites was clearly demonstrated by concentration-, temperature-, and time-dependent increases in protoplast fluorescence following treatment with the labeled LPS. Moreover, this induced fluorescence increase was convincingly reduced following pre-treatment with excess unlabeled LPS, thereby indicating reversibility of LPS binding. Inhibition of the binding process is also reported using endo- and exocytosis inhibitors. Here, we present evidence for the anticipated presence of LPS-specific binding sites in Arabidopsis protoplasts, and furthermore propose Qdots as a more sensitive LPS-labeling strategy in comparison to the conventional Alexa 488 hydrazide label for binding studies. PMID:26029233

  13. Analysis of a nucleotide-binding site of 5-lipoxygenase by affinity labelling: binding characteristics and amino acid sequences.

    PubMed Central

    Zhang, Y Y; Hammarberg, T; Radmark, O; Samuelsson, B; Ng, C F; Funk, C D; Loscalzo, J

    2000-01-01

    5-Lipoxygenase (5LO) catalyses the first two steps in the biosynthesis of leukotrienes, which are inflammatory mediators derived from arachidonic acid. 5LO activity is stimulated by ATP; however, a consensus ATP-binding site or nucleotide-binding site has not been found in its protein sequence. In the present study, affinity and photoaffinity labelling of 5LO with 5'-p-fluorosulphonylbenzoyladenosine (FSBA) and 2-azido-ATP showed that 5LO bound to the ATP analogues quantitatively and specifically and that the incorporation of either analogue inhibited ATP stimulation of 5LO activity. The stoichiometry of the labelling was 1.4 mol of FSBA/mol of 5LO (of which ATP competed with 1 mol/mol) or 0.94 mol of 2-azido-ATP/mol of 5LO (of which ATP competed with 0.77 mol/mol). Labelling with FSBA prevented further labelling with 2-azido-ATP, indicating that the same binding site was occupied by both analogues. Other nucleotides (ADP, AMP, GTP, CTP and UTP) also competed with 2-azido-ATP labelling, suggesting that the site was a general nucleotide-binding site rather than a strict ATP-binding site. Ca(2+), which also stimulates 5LO activity, had no effect on the labelling of the nucleotide-binding site. Digestion with trypsin and peptide sequencing showed that two fragments of 5LO were labelled by 2-azido-ATP. These fragments correspond to residues 73-83 (KYWLNDDWYLK, in single-letter amino acid code) and 193-209 (FMHMFQSSWNDFADFEK) in the 5LO sequence. Trp-75 and Trp-201 in these peptides were modified by the labelling, suggesting that they were immediately adjacent to the C-2 position of the adenine ring of ATP. Given the stoichiometry of the labelling, the two peptide sequences of 5LO were probably near each other in the enzyme's tertiary structure, composing or surrounding the ATP-binding site of 5LO. PMID:11042125

  14. Reactive sulfhydryl groups of sarcoplasmic reticulum ATPase. II. Site of labeling with iodoacetamide and its fluorescent derivative.

    PubMed

    Yamashita, T; Kawakita, M

    1987-02-01

    Iodoacetamide (IAA) and its fluorescent derivative, 5-(2-iodoacetamidoethyl) amino-naphthalene-1-sulfonate (IAEDANS) specifically bind to a site on the C-terminal half of sarcoplasmic reticulum (SR) Ca2+,Mg2+-ATPase. The location of this specific binding site was identified. SR membranes were treated with 150 microM [14C]IAA at pH 7.0 and 30 degrees C. One mole of IAA per mole of ATPase was bound in 6 h without affecting the Ca2+-transport activity. [14C]IAA-labeled SR membranes were cleaved with BrCN, and 14C-labeled peptide fragments were separated by Sephadex LH-60 chromatography and then digested further with trypsin. A radioactive peptide (Ala-Cys 674-Cys-Phe-Ala-Arg) was purified by Sephadex LH-20 chromatography and C18 reversed phase HPLC (Cys denotes the [14C]IAA-binding site). IAEDANS-labeling was carried out by reacting SR membranes with 50 microM IAEDANS for 5 h, at pH 7.0 and 30 degrees C. A fluorescent peptide was successfully purified by the same procedures as for the IAA-labeled peptide, and the amino acid sequence analysis of this peptide revealed that the IAEDANS labeling site was identical with the IAA binding site.

  15. Adenine nucleotide binding sites on beef heart F/sub 1/ ATPase: photoaffinity labeling of. beta. -subunit Tyr-368 at a noncatalytic site and. beta. Tyr-345 at a catalytic site

    SciTech Connect

    Cross, R.L.; Cunningham, D.; Miller, C.G.; Xue, Z.; Zhou, J.M.; Boyer, P.D.

    1987-08-01

    2-Azidoadenine (/sup 32/P)nucleotide was bound specifically at catalytic or noncatalytic nucleotide binding sites on beef heart mitochondrial F/sub 1/ ATPase. In both cases, photolysis resulted in nearly exclusive labeling of the ..beta.. subunit. The modified enzyme was digested with trypsin, and labeled peptides were purified by reversed-phase high-pressure liquid chromatography. Amino acid sequence analysis of the major /sup 32/P-labeled tryptic fragments showed ..beta..-subunit Tyr-368 to be present at noncatalytic sites and ..beta.. Tyr-345 to be present at catalytic sites. From the relationship between the degree of inhibition and extent of modification, it is estimated that one-third of the catalytic sites or two-thirds of the noncatalytic sites must be modified to give near-complete inhibition of catalytic activity.

  16. Effective and site-specific phosphoramidation reaction for universally labeling nucleic acids.

    PubMed

    Su, Yu-Chih; Chen, Hsing-Yin; Ko, Ni Chien; Hwang, Chi-Ching; Wu, Min Hui; Wang, Li-Fang; Wang, Yun-Ming; Chang, Sheng-Nan; Wang, Eng-Chi; Wang, Tzu-Pin

    2014-03-15

    Here we report efficient and selective postsynthesis labeling strategies, based on an advanced phosphoramidation reaction, for nucleic acids of either synthetic or enzyme-catalyzed origin. The reactions provided phosphorimidazolide intermediates of DNA or RNA which, whether reacted in one pot (one-step) or purified (two-step), were directly or indirectly phosphoramidated with label molecules. The acquired fluorophore-labeled nucleic acids, prepared from the phosphoramidation reactions, demonstrated labeling efficacy by their F/N ratio values (number of fluorophores per molecule of nucleic acid) of 0.02-1.2 which are comparable or better than conventional postsynthesis fluorescent labeling methods for DNA and RNA. Yet, PCR and UV melting studies of the one-step phosphoramidation-prepared FITC-labeled DNA indicated that the reaction might facilitate nonspecific hybridization in nucleic acids. Intrinsic hybridization specificity of nucleic acids was, however, conserved in the two-step phosphoramidation reaction. The reaction of site-specific labeling nucleic acids at the 5'-end was supported by fluorescence quenching and UV melting studies of fluorophore-labeled DNA. The two-step phosphoramidation-based, effective, and site-specific labeling method has the potential to expedite critical research including visualization, quantification, structural determination, localization, and distribution of nucleic acids in vivo and in vitro.

  17. Label-free catalytic and molecular beacon containing an abasic site for sensitive fluorescent detection of small inorganic and organic molecules.

    PubMed

    Song, Panshu; Xiang, Yu; Xing, Hang; Zhou, Zhaojuan; Tong, Aijun; Lu, Yi

    2012-03-20

    In this work, two methods with complementary features, catalytic and molecular beacon (CAMB) and label-free fluorescent sensors using an abasic site, have been combined into new label-free CAMB sensors that possess advantages of each method. The label-free method using a dSpacer-containing molecular beacon makes CAMB more cost-effective and less interfering with the catalytic activity, while CAMB allows the label-free method to use true catalytic turnovers for signal amplifications, resulting in a new label-free CAMB sensor for Pb(2+) ion, with a detection limit of 3.8 nM while maintaining the same selectivity. Furthermore, by using CAMB to overcome the label-free method's limitation of requiring excess enzyme strands, a new label-free CAMB sensor using aptazyme is also designed to detect adenosine down to 1.4 μM, with excellent selectivity over other nucleosides.

  18. REDOR NMR of stable-isotope-labeled protein binding sites

    SciTech Connect

    Schaefer, J.

    1994-12-01

    Rotational-echo, double resonance (REDOR) NMR, a new analytical spectroscopic technique for solids spinning at the magic angle, has been developed over the last 5 years. REDOR provides a direct measure of heteronuclear dipolar coupling between isolated pairs of labeled nuclei. In a solid with a {sup 13}C-{sup 15}N labeled pair, for example, the {sup 13}C rotational echoes that form each rotor period following a{sup 1}H-{sup 13}C cross-polarization transfer can be prevented from reaching full intensity by insertion of a {sup 15}N {pi} pulse each half rotor period. The REDOR difference (the difference between a {sup 13}C NMR spectrum obtained under these conditions and one obtained with no {sup 15}N {pi} pulses) has a strong dependence on the {sup 13}C-{sup 15}N dipolar coupling, and hence, the {sup 13}C-{sup 15}N internuclear distance. REDOR is described as double-resonance even though three radio frequencies (typically {sup 1}H, {sup 13}C, and {sup 15}N) are used because the protons are removed from the important evolution part of the experiment by resonant decoupling. The dephasing of magnetization in REDOR arises from a local dipolar {sup 13}C-{sup 15}N field gradient and involves no polarization transfer. REDOR has no dependence on {sup 13}C or {sup 15}N chemical-shift tensors and does not require resolution of a {sup 13}C-{sup 15}N coupling in the chemical-shift dimension.

  19. Site-specific labeling of proteins via sortase: protocols for the molecular biologist.

    PubMed

    Popp, Maximilian Wei-Lin

    2015-01-01

    Creation of site-specifically labeled protein bioconjugates is an important tool for the molecular biologist and cell biologist. Chemical labeling methods, while versatile with respect to the types of moieties that can be attached, suffer from lack of specificity, often targeting multiple positions within a protein. Here we describe protocols for the chemoenzymatic labeling of proteins at the C-terminus using the bacterial transpeptidase, sortase A. We detail a protocol for the purification of an improved pentamutant variant of the Staphylococcus aureus enzyme (SrtA 5(o)) that exhibits vastly improved kinetics relative to the wild-type enzyme. Importantly, a protocol for the construction of peptide probes compatible with sortase labeling using techniques that can be adapted to any cellular/molecular biology lab with no existing infrastructure for synthetic chemistry is described. Finally, we provide an example of how to optimize the labeling reaction using the improved SrtA 5(o) variant. PMID:25560076

  20. A versatile photoactivatable probe designed to label the diphosphate binding site of farnesyl diphosphate utilizing enzymes.

    PubMed

    Henry, Olivier; Lopez-Gallego, Fernando; Agger, Sean A; Schmidt-Dannert, Claudia; Sen, Stephanie; Shintani, David; Cornish, Katrina; Distefano, Mark D

    2009-07-01

    Farnesyl diphosphate (FPP) is a substrate for a diverse number of enzymes found in nature. Photoactive analogues of isoprenoid diphosphates containing either benzophenone, diazotrifluoropropionate or azide groups have been useful for studying both the enzymes that synthesize FPP as well as those that employ FPP as a substrate. Here we describe the synthesis and properties of a new class of FPP analogues that links an unmodified farnesyl group to a diphosphate mimic containing a photoactive benzophenone moiety; thus, importantly, these compounds are photoactive FPP analogues that contain no modifications of the isoprenoid portion of the molecule that may interfere with substrate binding in the active site of an FPP utilizing enzyme. Two isomeric compounds containing meta- and para-substituted benzophenones were prepared. These two analogues inhibit Saccharomyces cerevisiae protein farnesyltransferase (ScPFTase) with IC(50) values of 5.8 (meta isomer) and 3.0 microM (para isomer); the more potent analogue, the para isomer, was shown to be a competitive inhibitor of ScPFTase with respect to FPP with a K(I) of 0.46 microM. Radiolabeled forms of both analogues selectively labeled the beta-subunit of ScPFTase. The para isomer was also shown to label Escherichia coli farnesyl diphosphate synthase and Drosophila melanogaster farnesyl diphosphate synthase. Finally, the para isomer was shown to be an alternative substrate for a sesquiterpene synthase from Nostoc sp. strain PCC7120, a cyanobacterial source; the compound also labeled the purified enzyme upon photolysis. Taken together, these results using a number of enzymes demonstrate that this new class of probes should be useful for a plethora of studies of FPP-utilizing enzymes. PMID:19447628

  1. Indium-111-labeled leukocyte scintigraphy in hemodialysis access-site infection

    SciTech Connect

    Palestro, C.J.; Vega, A.; Kim, C.K.; Vallabhajosula, S.; Goldsmith, S.J. )

    1990-03-01

    Bacterial sepsis, a significant complication of chronic hemodialysis, is generally the result of infection at the vascular access site. We retrospectively reviewed the utility of indium-111-(111In) labeled autologous leukocyte scintigraphy in 26 patients (30 scans) with synthetic vascular grafts, on chronic hemodialysis, in whom hemodialysis access site infection was a diagnostic consideration. Leukocyte scintigraphy correctly identified all fifteen access-site infections; there was one false-positive study, for an overall sensitivity and specificity of 100% and 93%, respectively. Of particular significance is the fact that in nine (60%) of the fifteen access-site infections, physical examination was normal. Our data indicate that 111In-labeled leukocyte scintigraphy is a useful procedure for the diagnosis of hemodialysis access-site infection, and it is especially valuable when physical examination of the access site is normal.

  2. Site-specific DOTA/europium-labeling of recombinant human relaxin-3 for receptor-ligand interaction studies.

    PubMed

    Zhang, Wei-Jie; Luo, Xiao; Liu, Ya-Li; Shao, Xiao-Xia; Wade, John D; Bathgate, Ross A D; Guo, Zhan-Yun

    2012-08-01

    Relaxin-3 (also known as INSL7) is a recently identified neuropeptide belonging to the insulin/relaxin superfamily. It has putative roles in the regulation of stress responses, food intake, and reproduction by activation of its cognate G-protein-coupled receptor RXFP3. It also binds and activates the relaxin family peptide receptors RXFP1 and RXFP4 in vitro. To obtain a europium-labeled relaxin-3 as tracer for studying the interaction of these receptors with various ligands, in the present work we propose a novel site-specific labeling strategy for the recombinant human relaxin-3 that has been previously prepared in our laboratory. First, the N-terminal 6 × His-tag of the single-chain relaxin-3 precursor was removed by Aeromonas aminopeptidase and all of the primary amines of the resultant peptide were reversibly blocked by citroconic anhydride. Second, the A-chain N-terminus of the blocked peptide was released by endoproteinase Asp-N cleavage that removed the linker peptide between the B- and A-chains. Third, an alkyne moiety was introduced to the newly released A-chain N-terminus by reaction with the highly active primary amine-specific N-hydroxysuccinimide ester. Fourth, after removal of the reversible blockage under mild acidic condition, europium-loaded DOTA with an azide moiety was introduced to the two-chain relaxin-3 carrying the alkyne moiety through click chemistry. Using this site-specific labeling strategy, homogeneous monoeuropium-labeled human relaxin-3 could be obtained with good overall yield. In contrast, conventional random labeling resulted in a complex mixture that was poorly resolved because human relaxin-3 has four primary amine moieties that all react with the modification reagent. Both saturation and competition binding assays demonstrated that the DOTA/Eu(3+)-labeled relaxin-3 retained high binding affinity for human RXFP3, RXFP4, and RXFP1 and was therefore a suitable non-radioactive and stable tracer to study the interaction of various

  3. Rapid migration of /sup 111/indium-labeled granulocytes to sites of infection

    SciTech Connect

    Dutcher, J.P.; Schiffer, C.A.; Johnston, G.S.

    1981-03-05

    Results are reported of studies with homologous, ABO-matched, /sup 111/In-labeled granulocytes given to 14 granulocytopenic patients with specific, known sites of infection. In 13 of these patients, radioactivity was noted at the site of infection after only 30 minutes. Subsequent scans at four hours or 24 hours or both were all positive at the same site. This technique provides a method for rapid diagnosis of occult infection in this patient population.

  4. Site-Specific Chemoenzymatic Labeling of Aerolysin Enables the Identification of New Aerolysin Receptors

    PubMed Central

    Blom, Annet E. M.; Theile, Christopher S.; Li, Zeyang; Spooner, Eric; Ploegh, Hidde L.; Guimaraes, Carla P.

    2014-01-01

    Aerolysin is a secreted bacterial toxin that perforates the plasma membrane of a target cell with lethal consequences. Previously explored native and epitope-tagged forms of the toxin do not allow site-specific modification of the mature toxin with a probe of choice. We explore sortase-mediated transpeptidation reactions (sortagging) to install fluorophores and biotin at three distinct sites in aerolysin, without impairing binding of the toxin to the cell membrane and with minimal impact on toxicity. Using a version of aerolysin labeled with different fluorophores at two distinct sites we followed the fate of the C-terminal peptide independently from the N-terminal part of the toxin, and show its loss in the course of intoxication. Making use of the biotinylated version of aerolysin, we identify mesothelin, urokinase plasminogen activator surface receptor (uPAR, CD87), glypican-1, and CD59 glycoprotein as aerolysin receptors, all predicted or known to be modified with a glycosylphosphatidylinositol anchor. The sortase-mediated reactions reported here can be readily extended to other pore forming proteins. PMID:25275512

  5. Affinity Labeling of Hepatitis C Virus Replicase with a Nucleotide Analog: Identification of binding site

    PubMed Central

    Manvar, Dinesh; Singh, Kamlendra; Pandey, Virendra N.

    2013-01-01

    We have used an ATP analog, 5′-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA), to modify HCV replicase in order to identify ATP binding site in the enzyme. FSBA inactivates HCV replicase activity in a concentration dependent manner with a binding stoichiometry of 2 moles of FSBA per mole of enzyme. The enzyme activity is protected from FSBA in the presence of rNTP substrates or double stranded RNA template primers that do not support ATP as the incoming nucleotide but not in the presence of polyrU.rA26. The HPLC analysis of tryptic peptides of FSBA-modified enzyme revealed the presence of two distinct peptides eluted at 23 min and 36 min; these were absent in the control. Further we noted that both the peptides were protected from FSBA modification in the presence of Mg.ATP. The LC/MS/MS analysis of the affinity-labeled tryptic peptides purified from HPLC, identified two major modification sites at positions 382 (Tyr), and 491 (Lys) and a minor site at position 38 (Tyr). To validate the functional significance of Tyr38, Tyr382 and Lys491 in catalysis, we individually substituted these residues by alanine and examined their ability to catalyze RdRp activity. We found that both Y382A and K491A mutants were significantly affected in their ability to catalyze RdRp activity while Y38A remained unaffected. We further observed that both Y382A and K491A mutants were not affected in their ability to bind template primer but significantly affected in their ability to photo-crosslink ATP in the absence or presence of template primer. PMID:23268692

  6. Affinity labeling of hepatitis C virus replicase with a nucleotide analogue: identification of binding site.

    PubMed

    Manvar, Dinesh; Singh, Kamlendra; Pandey, Virendra N

    2013-01-15

    We have used an ATP analogue 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA) to modify HCV replicase in order to identify the ATP binding site in the enzyme. FSBA inactivates HCV replicase activity in a concentration-dependent manner with a binding stoichiometry of 2 moles of FSBA per mole of enzyme. The enzyme activity is protected from FSBA in the presence of rNTP substrates or double-stranded RNA template primers that do not support ATP as the incoming nucleotide but not in the presence of polyrU.rA(26). HPLC analysis of tryptic peptides of FSBA-modified enzyme revealed the presence of two distinct peptides eluted at 23 and 36 min; these were absent in the control. Further we noted that both peptides were protected from FSBA modification in the presence of Mg·ATP. The LC/MS/MS analysis of the affinity-labeled tryptic peptides purified from HPLC, identified two major modification sites at positions 382 (Tyr), and 491 (Lys) and a minor site at position 38 (Tyr). To validate the functional significance of Tyr38, Tyr382, and Lys491 in catalysis, we individually substituted these residues by alanine and examined their ability to catalyze RdRp activity. We found that both Y382A and K491A mutants were significantly affected in their ability to catalyze RdRp activity while Y38A remained unaffected. We further observed that both Y382A and K491A mutants were not affected in their ability to bind template primer but were significantly affected in their ability to photo-cross-link ATP in the absence or presence of template primer. PMID:23268692

  7. Design, Synthesis, and Functional Activity of Labeled CD1d Glycolipid Agonists

    PubMed Central

    2013-01-01

    Invariant natural killer T cells (iNKT cells) are restricted by CD1d molecules and activated upon CD1d-mediated presentation of glycolipids to T cell receptors (TCRs) located on the surface of the cell. Because the cytokine response profile is governed by the structure of the glycolipid, we sought a method for labeling various glycolipids to study their in vivo behavior. The prototypical CD1d agonist, α-galactosyl ceramide (α-GalCer) 1, instigates a powerful immune response and the generation of a wide range of cytokines when it is presented to iNKT cell TCRs by CD1d molecules. Analysis of crystal structures of the TCR−α-GalCer–CD1d ternary complex identified the α-methylene unit in the fatty acid side chain, and more specifically the pro-S hydrogen at this position, as a site for incorporating a label. We postulated that modifying the glycolipid in this way would exert a minimal impact on the TCR–glycolipid–CD1d ternary complex, allowing the labeled molecule to function as a good mimic for the CD1d agonist under investigation. To test this hypothesis, the synthesis of a biotinylated version of the CD1d agonist threitol ceramide (ThrCer) was targeted. Both diastereoisomers, epimeric at the label tethering site, were prepared, and functional experiments confirmed the importance of substituting the pro-S, and not the pro-R, hydrogen with the label for optimal activity. Significantly, functional experiments revealed that biotinylated ThrCer (S)-10 displayed behavior comparable to that of ThrCer 5 itself and also confirmed that the biotin residue is available for streptavidin and antibiotin antibody recognition. A second CD1d agonist, namely α-GalCer C20:2 4, was modified in a similar way, this time with a fluorescent label. The labeled α-GalCer C20:2 analogue (11) again displayed functional behavior comparable to that of its unlabeled substrate, supporting the notion that the α-methylene unit in the fatty acid amide chain should be a suitable site for

  8. Chemical tags for site-specific fluorescent labeling of biomolecules.

    PubMed

    Freidel, Christoph; Kaloyanova, Stefka; Peneva, Kalina

    2016-06-01

    This review focuses on the various approaches to covalently attach a chromophore to a biomolecule of interest in site-specific manner. Novel methods like inverse electron-demand Diels-Alder reaction, Pictet-Spengler ligation and enzyme tags like SNAP and Halo-tags are critically discussed and compared to established techniques like copper-free click reaction and native chemical ligation. Selected examples in which the tags have been exploited for in vitro or in vivo imaging are reviewed and evaluated. PMID:26969255

  9. Photoaffinity Labeling the Propofol Binding Site in GLIC†

    PubMed Central

    Chiara, David C.; Gill, Jonathan F.; Chen, Qiang; Tillman, Tommy; Dailey, William P.; Eckenhoff, Roderic G.; Xu, Yan; Tang, Pei; Cohen, Jonathan B.

    2014-01-01

    Propofol, an intravenous general anesthetic, produces many of its anesthetic effects in vivo by potentiating the responses of GABA type A receptors (GABAAR), members of the superfamily of pentameric ligand-gated ion channels (pLGICs) that contain anion-selective channels. Propofol also inhibits pLGICs containing cation-selective channels, including nicotinic acetylcholine receptors and GLIC, a prokaryotic proton-gated homolog from Gloeobacter violaceus. In the structure of GLIC co-crystallized with propofol at pH 4 (presumed open/desensitized states), propofol was localized to an intrasubunit pocket at the extracellular end of the transmembrane domain within the bundle of transmembrane α-helices [Nury, H, et. al. (2011) Nature 469, 428–431]. To identify propofol binding sites in GLIC in solution, we used a recently developed photoreactive propofol analog (2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol or AziPm) which acts as an anesthetic in vivo and potentiates GABAAR in vitro. For GLIC expressed in Xenopus oocytes, propofol and AziPm inhibited current responses at pH 5.5 (EC20) with IC50s of 20 and 50 μM, respectively. When [3H]AziPm (7 μM) was used to photolabel detergent-solubilized, affinity-purified GLIC at pH 4.4, protein microsequencing identified propofol-inhibitable photolabeling of three residues in the GLIC transmembrane domain: Met-205, Tyr-254, and Asn-307 in the M1, M3, and M4 transmembrane helices, respectively. Thus, in GLIC in solution, propofol and AziPm bind competitively to a site in proximity to these residues, which in the GLIC crystal structure are in contact with the propofol bound in the intrasubunit pocket. PMID:24341978

  10. Salt site performance assessment activities

    SciTech Connect

    Kircher, J.F.; Gupta, S.K.

    1983-01-01

    During this year the first selection of the tools (codes) for performance assessments of potential salt sites have been tentatively selected and documented; the emphasis has shifted from code development to applications. During this period prior to detailed characterization of a salt site, the focus is on bounding calculations, sensitivity and with the data available. The development and application of improved methods for sensitivity and uncertainty analysis is a focus for the coming years activities and the subject of a following paper in these proceedings. Although the assessments to date are preliminary and based on admittedly scant data, the results indicate that suitable salt sites can be identified and repository subsystems designed which will meet the established criteria for protecting the health and safety of the public. 36 references, 5 figures, 2 tables.

  11. Synthesis and evaluation of radioactive and fluorescent residualizing labels for identifying sites of plasma protein catabolism

    SciTech Connect

    Maxwell, J.L.; Baynes, J.W.; Thorpe, S.R.

    1986-05-01

    Inulin and lactose were each coupled to tyramine by reductive amination with NaBH/sub 3/CN and the tyramine then labeled with /sup 125/I. Dilactitol-/sup 125/I-tyramine (DLT) and inulin-/sup 125/I-tyramine (InTn) were coupled by reductive amination and cyanuric chloride, respectively, to asialofetuin (ASF), fetuin and rat serum albumin (RSA). Attachment of either label had no effect on the circulating half-lives of the proteins. Radioactivity from labeled ASF was recovered in rat liver (> 90%) by 1 h post-injection and remained in liver with half-lives of 2 and 6 days, respectively, for the DLT and InTn labels. Whole body recoveries of radioactivity from DLT- and InTn labels. Whole body recoveries of radioactivity from DLT- and InTn-labeled RSA were 5 and 6.5 days, respectively, again indicating that the larger glycoconjugate label residualized more efficiently in cells following protein degradation. (Lactitol)/sub 2/-N-CH/sub 2/-CH/sub 2/-NH-fluroescein (DLF) was also coupled to ASF by reductive amination and recovered quantitatively in liver at 1 h post-injection. Native ASF was an effective competitor for clearance of DLF-ASF from the circulation. Fluorescent degradation products were retained in liver with a half-life of 1.2 days. Residualizing fluorescent labels should be useful for identification and sorting of cells active in the degradation of plasma proteins.

  12. Direct photoaffinity labeling by nucleotides of the apparent catalytic site on the heavy chains of smooth muscle and Acanthamoeba myosins

    SciTech Connect

    Maruta, H.; Korn, E.D.

    1981-01-10

    The heavy chains of Acanthamoeba myosins, IA, IB and II, turkey gizzard myosin, and rabbit skeletal muscle myosin subfragment-1 were specifically labeled by radioactive ATP, ADP, and UTP, each of which is a substrate or product of myosin ATPase activity, when irradiated with uv light at 0/sup 0/C. With UTP, as much as 0.45 mol/mol of Acanthamoeba myosin IA heavy chain and 1 mol/mol of turkey gizzard myosin heavy chain was incorporated. Evidence that the ligands were associated with the catalytic site included the observations that reaction occurred only with nucleotides that are substrates or products of the ATPase activity; that the reaction was blocked by pyrophosphate which is an inhibitor of the ATPase activity; that ATP was bound as ADP; and that label was probably restricted to a single peptide following limited subtilisin proteolysis of labeled Acanthamoeba myosin IA heavy chain and extensive cleavage with CNBr and trypsin of labeled turkey gizzard myosin heavy chain.

  13. Site-directed spin labeling reveals pentameric ligand-gated ion channel gating motions.

    PubMed

    Dellisanti, Cosma D; Ghosh, Borna; Hanson, Susan M; Raspanti, James M; Grant, Valerie A; Diarra, Gaoussou M; Schuh, Abby M; Satyshur, Kenneth; Klug, Candice S; Czajkowski, Cynthia

    2013-11-01

    Pentameric ligand-gated ion channels (pLGICs) are neurotransmitter-activated receptors that mediate fast synaptic transmission. In pLGICs, binding of agonist to the extracellular domain triggers a structural rearrangement that leads to the opening of an ion-conducting pore in the transmembrane domain and, in the continued presence of neurotransmitter, the channels desensitize (close). The flexible loops in each subunit that connect the extracellular binding domain (loops 2, 7, and 9) to the transmembrane channel domain (M2-M3 loop) are essential for coupling ligand binding to channel gating. Comparing the crystal structures of two bacterial pLGIC homologues, ELIC and the proton-activated GLIC, suggests channel gating is associated with rearrangements in these loops, but whether these motions accurately predict the motions in functional lipid-embedded pLGICs is unknown. Here, using site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy and functional GLIC channels reconstituted into liposomes, we examined if, and how far, the loops at the ECD/TMD gating interface move during proton-dependent gating transitions from the resting to desensitized state. Loop 9 moves ∼9 Å inward toward the channel lumen in response to proton-induced desensitization. Loop 9 motions were not observed when GLIC was in detergent micelles, suggesting detergent solubilization traps the protein in a nonactivatable state and lipids are required for functional gating transitions. Proton-induced desensitization immobilizes loop 2 with little change in position. Proton-induced motion of the M2-M3 loop was not observed, suggesting its conformation is nearly identical in closed and desensitized states. Our experimentally derived distance measurements of spin-labeled GLIC suggest ELIC is not a good model for the functional resting state of GLIC, and that the crystal structure of GLIC does not correspond to a desensitized state. These findings advance our understanding

  14. Complementary-addressed site-directed spin labeling of long natural RNAs

    PubMed Central

    Babaylova, Elena S.; Malygin, Alexey A.; Lomzov, Alexander A.; Pyshnyi, Dmitrii V.; Yulikov, Maxim; Jeschke, Gunnar; Krumkacheva, Olesya A.; Fedin, Matvey V.; Karpova, Galina G.; Bagryanskaya, Elena G.

    2016-01-01

    Nanoscale distance measurements by pulse dipolar Electron paramagnetic resonance (EPR) spectroscopy allow new insights into the structure and dynamics of complex biopolymers. EPR detection requires site directed spin labeling (SDSL) of biomolecule(s), which remained challenging for long RNAs up-to-date. Here, we demonstrate that novel complementary-addressed SDSL approach allows efficient spin labeling and following structural EPR studies of long RNAs. We succeeded to spin-label Hepatitis C Virus RNA internal ribosome entry site consisting of ≈330 nucleotides and having a complicated spatial structure. Application of pulsed double electron–electron resonance provided spin–spin distance distribution, which agrees well with the results of molecular dynamics (MD) calculations. Thus, novel SDSL approach in conjunction with EPR and MD allows structural studies of long natural RNAs with nanometer resolution and can be applied to systems of biological and biomedical significance. PMID:27269581

  15. A readily synthesized cyclic pyrrolysine analogue for site-specific protein "click" labeling.

    PubMed

    Hao, Ziyang; Song, Yanqun; Lin, Shixian; Yang, Maiyun; Liang, Yujie; Wang, Jing; Chen, Peng R

    2011-04-21

    A concise route was developed for the facile synthesis of a cyclic pyrrolysine analogue bearing an azide handle. Directed evolution enabled the encoding of this non-natural amino acid in both prokaryotic and eukaryotic cells, which offers a highly efficient approach for the site-specific protein labeling using click chemistry.

  16. Protein-induced changes in DNA structure and dynamics observed with noncovalent site-directed spin labeling and PELDOR

    PubMed Central

    Reginsson, Gunnar W.; Shelke, Sandip A.; Rouillon, Christophe; White, Malcolm F.; Sigurdsson, Snorri Th.; Schiemann, Olav

    2013-01-01

    Site-directed spin labeling and pulsed electron–electron double resonance (PELDOR or DEER) have previously been applied successfully to study the structure and dynamics of nucleic acids. Spin labeling nucleic acids at specific sites requires the covalent attachment of spin labels, which involves rather complicated and laborious chemical synthesis. Here, we use a noncovalent label strategy that bypasses the covalent labeling chemistry and show that the binding specificity and efficiency are large enough to enable PELDOR or DEER measurements in DNA duplexes and a DNA duplex bound to the Lac repressor protein. In addition, the rigidity of the label not only allows resolution of the structure and dynamics of oligonucleotides but also the determination of label orientation and protein-induced conformational changes. The results prove that this labeling strategy in combination with PELDOR has a great potential for studying both structure and dynamics of oligonucleotides and their complexes with various ligands. PMID:22941643

  17. Active site of ribulosebisphosphate carboxylase/oxygenase

    SciTech Connect

    Hartman, F.C.; Stringer, C.D.; Milanez, S.; Lee, E.H.

    1985-01-01

    Previous affinity labeling studies and comparative sequence analyses have identified two different lysines at the active site of ribulosebisphosphate carboxylase/oxygenase and have suggested their essentiality to function. The essential lysines occupy positions 166 and 329 in the Rhodospirillum rubrum enzyme and positions 175 and 334 in the spinach enzyme. Based on the pH-dependencies of inactivations of the two enzymes by trinitrobenzene sulfonate, Lys-166 (R. rubrum enzyme) exhibits a pK/sub a/ of 7.9 and Lys-334 (spinach enzyme) exhibits a pK/sub a/ of 9.0. These low pK/sub a/ values as well as the enhanced nucleophilicities of the lysyl residues argue that both are important to catalysis rather than to substrate binding. Lys-166 may correspond to the essential base that initiates catalysis and that displays a pK/sub a/ of 7.5 in the pH-curve for V/sub max//K/sub m/. Cross-linking experiments with 4,4'-diisothiocyano-2,2'-disulfonate stilbene demonstrate that the two active-site lysines are within 12 A. 50 refs., 7 figs., 1 tab.

  18. Label-free optical activation of astrocyte in vivo

    NASA Astrophysics Data System (ADS)

    Choi, Myunghwan; Yoon, Jonghee; Ku, Taeyun; Choi, Kyungsun; Choi, Chulhee

    2011-07-01

    As the most abundant cell type in the central nervous system, astrocyte has been one of main research topics in neuroscience. Although various tools have been developed, at present, there is no tool that allows noninvasive activation of astrocyte in vivo without genetic or pharmacological perturbation. Here we report a noninvasive label-free optical method for physiological astrocyte activation in vivo using a femtosecond pulsed laser. We showed the laser stimulation robustly induced astrocytic calcium activation in vivo and further verified physiological relevance of the calcium increase by demonstrating astrocyte mediated vasodilation in the brain. This novel optical method will facilitate noninvasive physiological study on astrocyte function.

  19. Enhanced Aqueous Suzuki–Miyaura Coupling Allows Site-Specific Polypeptide 18F-Labeling

    PubMed Central

    2013-01-01

    The excesses of reagents used in protein chemistry are often incompatible with the reduced or even inverse stoichiometries used for efficient radiolabeling. Analysis and screening of aqueous Pd(0) ligand systems has revealed the importance of a guanidine core and the discovery of 1,1-dimethylguanidine as an enhanced ligand for aqueous Suzuki–Miyaura cross-coupling. This novel Pd catalyst system has now allowed the labeling of small molecules, peptides, and proteins with the fluorine-18 prosthetic [18F]4-fluorophenylboronic acid. These findings now enable site-specific protein 18F-labeling under biologically compatible conditions using a metal-triggered reaction. PMID:23991754

  20. Site-Specific Labeling of Protein Kinase CK2: Combining Surface Display and Click Chemistry for Drug Discovery Applications.

    PubMed

    Nienberg, Christian; Retterath, Anika; Becher, Kira-Sophie; Saenger, Thorsten; Mootz, Henning D; Jose, Joachim

    2016-01-01

    Human CK2 is a heterotetrameric constitutively active serine/threonine protein kinase and is an emerging target in current anti-cancer drug discovery. The kinase is composed of two catalytic CK2α subunits and two regulatory CK2β subunits. In order to establish an assay to identify protein-protein-interaction inhibitors (PPI) of the CK2α/CK2β interface, a bioorthogonal click reaction was used to modify the protein kinase α-subunit with a fluorophore. By expanding the genetic code, the unnatural amino acid para azidophenylalanine (pAzF) could be incorporated into CK2α. Performing the SPAAC click reaction (Strain-Promoted Azide-Alkyne Cycloaddition) by the use of a dibenzylcyclooctyne-fluorophore (DBCO-fluorophore) led to a specifically labeled human protein kinase CK2α. This site-specific labeling does not impair the phosphorylation activity of CK2, which was evaluated by capillary electrophoresis. Furthermore a dissociation constant (KD) of 631 ± 86.2 nM was determined for the substrate αS1-casein towards CK2α. This labeling strategy was also applied to CK2β subunit on Escherichia coli, indicating the site-specific modifications of proteins on the bacterial cell surface when displayed by Autodisplay. PMID:27355959

  1. Site-Specific Labeling of Protein Kinase CK2: Combining Surface Display and Click Chemistry for Drug Discovery Applications †

    PubMed Central

    Nienberg, Christian; Retterath, Anika; Becher, Kira-Sophie; Saenger, Thorsten; Mootz, Henning D.; Jose, Joachim

    2016-01-01

    Human CK2 is a heterotetrameric constitutively active serine/threonine protein kinase and is an emerging target in current anti-cancer drug discovery. The kinase is composed of two catalytic CK2α subunits and two regulatory CK2β subunits. In order to establish an assay to identify protein-protein-interaction inhibitors (PPI) of the CK2α/CK2β interface, a bioorthogonal click reaction was used to modify the protein kinase α-subunit with a fluorophore. By expanding the genetic code, the unnatural amino acid para azidophenylalanine (pAzF) could be incorporated into CK2α. Performing the SPAAC click reaction (Strain-Promoted Azide-Alkyne Cycloaddition) by the use of a dibenzylcyclooctyne-fluorophore (DBCO-fluorophore) led to a specifically labeled human protein kinase CK2α. This site-specific labeling does not impair the phosphorylation activity of CK2, which was evaluated by capillary electrophoresis. Furthermore a dissociation constant (KD) of 631 ± 86.2 nM was determined for the substrate αS1-casein towards CK2α. This labeling strategy was also applied to CK2β subunit on Escherichia coli, indicating the site-specific modifications of proteins on the bacterial cell surface when displayed by Autodisplay. PMID:27355959

  2. Site-Specific Labeling of Protein Kinase CK2: Combining Surface Display and Click Chemistry for Drug Discovery Applications.

    PubMed

    Nienberg, Christian; Retterath, Anika; Becher, Kira-Sophie; Saenger, Thorsten; Mootz, Henning D; Jose, Joachim

    2016-06-27

    Human CK2 is a heterotetrameric constitutively active serine/threonine protein kinase and is an emerging target in current anti-cancer drug discovery. The kinase is composed of two catalytic CK2α subunits and two regulatory CK2β subunits. In order to establish an assay to identify protein-protein-interaction inhibitors (PPI) of the CK2α/CK2β interface, a bioorthogonal click reaction was used to modify the protein kinase α-subunit with a fluorophore. By expanding the genetic code, the unnatural amino acid para azidophenylalanine (pAzF) could be incorporated into CK2α. Performing the SPAAC click reaction (Strain-Promoted Azide-Alkyne Cycloaddition) by the use of a dibenzylcyclooctyne-fluorophore (DBCO-fluorophore) led to a specifically labeled human protein kinase CK2α. This site-specific labeling does not impair the phosphorylation activity of CK2, which was evaluated by capillary electrophoresis. Furthermore a dissociation constant (KD) of 631 ± 86.2 nM was determined for the substrate αS1-casein towards CK2α. This labeling strategy was also applied to CK2β subunit on Escherichia coli, indicating the site-specific modifications of proteins on the bacterial cell surface when displayed by Autodisplay.

  3. Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction

    PubMed Central

    Torres-Kolbus, Jessica; Chou, Chungjung; Deiters, Alexander; Chin, Jason W.

    2013-01-01

    The site-specific incorporation of bioorthogonal groups via genetic code expansion provides a powerful general strategy for site-specifically labelling proteins with any probe. However, the slow reactivity of the bioorthogonal functional groups that can be encoded genetically limits the utility of this strategy. We demonstrate the genetic encoding of a norbornene amino acid using the pyrrolysyl tRNA synthetase/tRNACUA pair in Escherichia coli and mammalian cells. We developed a series of tetrazine-based probes that exhibit `turn-on' fluorescence on their rapid reaction with norbornenes. We demonstrate that the labelling of an encoded norbornene is specific with respect to the entire soluble E. coli proteome and thousands of times faster than established encodable bioorthogonal reactions. We show explicitly the advantages of this approach over state-of-the-art bioorthogonal reactions for protein labelling in vitro and on mammalian cells, and demonstrate the rapid bioorthogonal site-specific labelling of a protein on the mammalian cell surface. PMID:22437715

  4. Interaction of P-aminobenzoic acid with normal and sickel erythrocyte membrane: photoaffinity labelling of the binding sites

    SciTech Connect

    Premachandra, B.R.

    1986-03-05

    Electron microscopic studies revealed that P-Amino benzoic acid (PABA) could prevent eichinocytosis of red cells in vitro. Equilibrium binding studies with right side out membrane vesicles (ROV) revealed a similar number of binding sites (1.2-1.4 ..mu..mol/mg) and Kd (1.4-1.6 mM) values for both normal and sickle cell membranes. /sup 14/C-Azide analogue of PABA was synthesized as a photoaffinity label to probe its sites of interaction on the erythrocyte membranes. Competitive binding studies of PABA with its azide indicated that both the compounds share common binding sites on the membrane surface since a 20 fold excess of azide inhibited PABA binding in a linear fashion. The azide was covalently incorporated into the membrane components only upon irradiation (52-35% of the label found in the proteins and the rest in lipids). Electrophoretic analysis of photolabelled ROV revealed that the azide interacts chiefly with Band 3 protein. PABA inhibited both high and low affinity calcium (Ca) binding sites situated on either surface of the membrane in a non-competitive manner; however, Ca binding stimulated by Mg-ATP was not affected. Ca transport into inside out vesicles was inhibited by PABA; but it did not affect the calcium ATP-ase activity. The authors studies suggest that the mechanism of action of PABA is mediated by its interaction with Band 3 protein (anion channel), calcium channel and calcium binding sites of erythrocyte membrane.

  5. Affinity labeling of the carbohydrate binding site of the lectin discoidin I using a photoactivatable radioiodinated monosaccharide

    SciTech Connect

    Kohnken, R.E.; Berger, E.A.

    1987-12-29

    N-(4-Azidosalicyl) galactosamine (GalNASA), a photoactivatable, radioiodinatable analog of N-acetylgalactosamine (GalNAc), has been prepared and characterized. The authors have used this reagent for labeling of the carbohydrate binding site of discoidin I, an endogenous lectin produced by Dictyostelium discoideum. GalNASA behaved as a ligand for discoidin I, as judged by its ability to compete in an assay measuring the carbohydrate binding activity of discoidin I. In this assay, it exhibited a K/sub i,app/ of 800 ..mu..M, comparable to that of GalNAc. The K/sub i,app/ of GalNASA decreased to 40 ..mu..m upon prior photolysis with ultraviolet light. In contrast, N-(4-azidosalicyl) ethanolamine produced no inhibition of carbohydrate binding regardless of photolysis. Covalent labeling of discoidin I with /sup 125/I-GalNASA was entirely dependent upon ultraviolet light. A portion of labeling, representing 40-60% of the total, was sensitive to reagents which were known to inhibit carbohydrate binding by discoidin I, including GalNAc, asialofetuin, and ethyl-enediaminetetraacetic acid. The carbohydrate-sensitive fraction of discoidin I photolabeling with /sup 125/I-GalNASA exhibited a K/sub d/ of 15-40 ..mu..M, in agreement with the K/sub i,app/ of prephotolyzed GalNASA observed in the carbohydrate binding assay. Partial proteolytic digestion of photolabeled discoidin I revealed specific fragments whose labeling was completely blocked by GalNAc. This indicated that the location of carbohydrate-sensitive labeling within the structure of discoidin I was restricted. One particular tryptic fragment, Tr1, was examined in detail. These data suggest that Tr1 is derived from the carbohydrate binding site of discoidin I.

  6. Mutations of fumarase that distinguish between the active site and a nearby dicarboxylic acid binding site.

    PubMed Central

    Weaver, T.; Lees, M.; Banaszak, L.

    1997-01-01

    Two mutant forms of fumarase C from E. coli have been made using PCR and recombinant DNA. The recombinant form of the protein included a histidine arm on the C-terminal facilitating purification. Based on earlier studies, two different carboxylic acid binding sites, labeled A- and B-, were observed in crystal structures of the wild type and inhibited forms of the enzyme. A histidine at each of the sites was mutated to an asparagine. H188N at the A-site resulted in a large decrease in specific activity, while the H129N mutation at the B-site had essentially no effect. From the results, we conclude that the A-site is indeed the active site, and a dual role for H188 as a potential catalytic base is proposed. Crystal structures of the two mutant proteins produced some unexpected results. Both mutations reduced the affinity for the carboxylic acids at their respective sites. The H129N mutant should be particularly useful in future kinetic studies because it sterically blocks the B-site with the carboxyamide of asparagine assuming the position of the ligand's carboxylate. In the H188N mutation at the active site, the new asparagine side chain still interacts with an active site water that appears to have moved slightly as a result of the mutation. PMID:9098893

  7. Site-specific labeling of RNA at internal ribose hydroxyl groups: terbium-assisted deoxyribozymes at work.

    PubMed

    Büttner, Lea; Javadi-Zarnaghi, Fatemeh; Höbartner, Claudia

    2014-06-01

    A general and efficient single-step method was established for site-specific post-transcriptional labeling of RNA. Using Tb(3+) as accelerating cofactor for deoxyribozymes, various labeled guanosines were site-specifically attached to 2'-OH groups of internal adenosines in in vitro transcribed RNA. The DNA-catalyzed 2',5'-phosphodiester bond formation proceeded efficiently with fluorescent, spin-labeled, biotinylated, or cross-linker-modified guanosine triphosphates. The sequence context of the labeling site was systematically analyzed by mutating the nucleotides flanking the targeted adenosine. Labeling of adenosines in a purine-rich environment showed the fastest reactions and highest yields. Overall, practically useful yields >70% were obtained for 13 out of 16 possible nucleotide (nt) combinations. Using this approach, we demonstrate preparative labeling under mild conditions for up to ~160-nt-long RNAs, including spliceosomal U6 small nuclear RNA and a cyclic-di-AMP binding riboswitch RNA.

  8. Monitoring Protein Misfolding by Site-Specific Labeling of Proteins In Vivo

    PubMed Central

    Hsieh, Tzung-yang; Nillegoda, Nadinath B.; Tyedmers, Jens; Bukau, Bernd; Mogk, Axel; Kramer, Günter

    2014-01-01

    Incorporating fluorescent amino acids by suppression of the TAG amber codon is a useful tool for site-specific labeling of proteins and visualizing their localization in living cells. Here we use a plasmid encoded orthogonal tRNA/aminoacyl-tRNA synthetase pair to site-specifically label firefly luciferase with the environmentally sensitive fluorescent amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2- aminopropanoic acid (ANAP) and explore the detectability of conformational changes in labeled luciferase in the yeast cytoplasm. We find that ANAP labeling efficiency is greatly increased in [PSI+] cells and show that analysis of the ANAP fluorescence emission by confocal imaging allows for tracking the thermal unfolding and aggregation of luciferase in vivo. Furthermore we demonstrate that flow cytometry can be used to study conformational changes in luciferase and chaperone-mediated refolding in quantitative terms and at the level of single cells. This experimental setup for the first time allows for the direct analysis of the folding state of a protein in living cells and may serve as valuable new tool for examining mechanisms of protein folding, misfolding and aggregation. PMID:24915041

  9. Site directed spin labeling studies of Escherichia coli dihydroorotate dehydrogenase N-terminal extension

    SciTech Connect

    Couto, Sheila G.; Cristina Nonato, M.

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer EcDHODH is a membrane-associated enzyme and a promising target for drug design. Black-Right-Pointing-Pointer Enzyme's N-terminal extension is responsible for membrane association. Black-Right-Pointing-Pointer N-terminal works as a molecular lid regulating access to the protein interior. -- Abstract: Dihydroorotate dehydrogenases (DHODHs) are enzymes that catalyze the fourth step of the de novo synthesis of pyrimidine nucleotides. In this reaction, DHODH converts dihydroorotate to orotate, using a flavine mononucleotide as a cofactor. Since the synthesis of nucleotides has different pathways in mammals as compared to parasites, DHODH has gained much attention as a promising target for drug design. Escherichia coli DHODH (EcDHODH) is a family 2 DHODH that interacts with cell membranes in order to promote catalysis. The membrane association is supposedly made via an extension found in the enzyme's N-terminal. In the present work, we used site directed spin labeling (SDSL) to specifically place a magnetic probe at positions 2, 5, 19, and 21 within the N-terminal and thus monitor, by using Electron Spin Resonance (ESR), dynamics and structural changes in this region in the presence of a membrane model system. Overall, our ESR spectra show that the N-terminal indeed binds to membranes and that it experiences a somewhat high flexibility that could be related to the role of this region as a molecular lid controlling the entrance of the enzyme's active site and thus allowing the enzyme to give access to quinones that are dispersed in the membrane and that are necessary for the catalysis.

  10. Genetic code expansion enables live-cell and super-resolution imaging of site-specifically labeled cellular proteins.

    PubMed

    Uttamapinant, Chayasith; Howe, Jonathan D; Lang, Kathrin; Beránek, Václav; Davis, Lloyd; Mahesh, Mohan; Barry, Nicholas P; Chin, Jason W

    2015-04-15

    Methods to site-specifically and densely label proteins in cellular ultrastructures with small, bright, and photostable fluorophores would substantially advance super-resolution imaging. Recent advances in genetic code expansion and bioorthogonal chemistry have enabled the site-specific labeling of proteins. However, the efficient incorporation of unnatural amino acids into proteins and the specific, fluorescent labeling of the intracellular ultrastructures they form for subdiffraction imaging has not been accomplished. Two challenges have limited progress in this area: (i) the low efficiency of unnatural amino acid incorporation that limits labeling density and therefore spatial resolution and (ii) the uncharacterized specificity of intracellular labeling that will define signal-to-noise, and ultimately resolution, in imaging. Here we demonstrate the efficient production of cystoskeletal proteins (β-actin and vimentin) containing bicyclo[6.1.0]nonyne-lysine at genetically defined sites. We demonstrate their selective fluorescent labeling with respect to the proteome of living cells using tetrazine-fluorophore conjugates, creating densely labeled cytoskeletal ultrastructures. STORM imaging of these densely labeled ultrastructures reveals subdiffraction features, including nuclear actin filaments. This work enables the site-specific, live-cell, fluorescent labeling of intracellular proteins at high density for super-resolution imaging of ultrastructural features within cells.

  11. Genetic Code Expansion Enables Live-Cell and Super-Resolution Imaging of Site-Specifically Labeled Cellular Proteins

    PubMed Central

    2015-01-01

    Methods to site-specifically and densely label proteins in cellular ultrastructures with small, bright, and photostable fluorophores would substantially advance super-resolution imaging. Recent advances in genetic code expansion and bioorthogonal chemistry have enabled the site-specific labeling of proteins. However, the efficient incorporation of unnatural amino acids into proteins and the specific, fluorescent labeling of the intracellular ultrastructures they form for subdiffraction imaging has not been accomplished. Two challenges have limited progress in this area: (i) the low efficiency of unnatural amino acid incorporation that limits labeling density and therefore spatial resolution and (ii) the uncharacterized specificity of intracellular labeling that will define signal-to-noise, and ultimately resolution, in imaging. Here we demonstrate the efficient production of cystoskeletal proteins (β-actin and vimentin) containing bicyclo[6.1.0]nonyne-lysine at genetically defined sites. We demonstrate their selective fluorescent labeling with respect to the proteome of living cells using tetrazine-fluorophore conjugates, creating densely labeled cytoskeletal ultrastructures. STORM imaging of these densely labeled ultrastructures reveals subdiffraction features, including nuclear actin filaments. This work enables the site-specific, live-cell, fluorescent labeling of intracellular proteins at high density for super-resolution imaging of ultrastructural features within cells. PMID:25831022

  12. On-site preparation of technetium-99m labeled human serum albumin for clinical application.

    PubMed

    Wang, Yuh-Feng; Chuang, Mei-Hua; Chiu, Jainn-Shiun; Cham, Thau-Ming; Chung, Mei-Ing

    2007-04-01

    Technetium-99m labeled human serum albumin (Tc-99m HSA) is an important radiopharmaceutical for clinical applications, such as cardiac function tests or protein-losing gastroenteropathy assessment. However, because of transfusion-induced infectious diseases, the safety of serum products is a serious concern. In this context, serum products acquired from patients themselves are the most ideal tracer. However, the development of rapid separation and easy clinical labeling methods is not yet well established. Under such situation, products from the same ethnic group or country are now recommended by the World Health Organization as an alternative preparation. This article describes the on-site preparation of Tc-99m HSA from locally supplied serum products. Different formulations were prepared and the labeling efficiency and stability were examined. Radio-labeling efficiencies were more than 90% in all preparation protocols, except for one that omitted the stannous solution. The most cost-effective protocol contained HSA 0.1 mg, treated with stannous fluoride 0.2 mg, and mixed with Tc-99m pertechnetate 30 mCi. A biodistribution study was performed in rats using a gamma camera immediately after intravenous administration of radiolabeled HSA. Tissue/organ uptake was obtained by measuring the radioactivity in organs after sacrificing the rats at timed intervals. The biologic half-life was about 32 min, determined from sequential venous blood collections. These data indicate that our preparation of Tc-99m HSA is useful and potentially applicable clinically. In addition, this on-site preparation provides the possibility of labeling a patient's own serum for subsequent clinical application.

  13. Electrophilic, Activation-Free Fluorogenic Reagent for Labeling Bioactive Amines.

    PubMed

    Sintes, Miquel; De Moliner, Fabio; Caballero-Lima, David; Denning, David W; Read, Nick D; Kielland, Nicola; Vendrell, Marc; Lavilla, Rodolfo

    2016-06-15

    Herein we report the preparation of BODIPY mesoionic acid fluorides through a short sequence involving an isocyanide multicomponent reaction as the key synthetic step. These novel BODIPY acid fluorides are water-stable electrophilic reagents that can be used for the fluorescent derivatization of amine-containing biomolecules using mild and activation-free reaction conditions. As a proof of principle, we have labeled the antifungal natamycin and generated a novel fluorogenic probe for imaging a variety of human and plant fungal pathogens, with excellent selectivity over bacterial cells. PMID:27248580

  14. Simplifying healthful choices: a qualitative study of a physical activity based nutrition label format

    PubMed Central

    2013-01-01

    Background This study used focus groups to pilot and evaluate a new nutrition label format and refine the label design. Physical activity equivalent labels present calorie information in terms of the amount of physical activity that would be required to expend the calories in a specified food item. Methods Three focus groups with a total of twenty participants discussed food choices and nutrition labeling. They provided information on comprehension, usability and acceptability of the label. A systematic coding process was used to apply descriptive codes to the data and to identify emerging themes and attitudes. Results Participants in all three groups were able to comprehend the label format. Discussion about label format focused on issues including gender of the depicted figure, physical fitness of the figure, preference for walking or running labels, and preference for information in miles or minutes. Feedback from earlier focus groups was used to refine the labels in an iterative process. Conclusions In contrast to calorie labels, participants shown physical activity labels asked and answered, “How does this label apply to me?” This shift toward personalized understanding may indicate that physical activity labels offer an advantage over currently available nutrition labels. PMID:23742678

  15. Label-free molecular beacon system based on DNAs containing abasic sites and fluorescent ligands that bind abasic sites.

    PubMed

    Sato, Yusuke; Nishizawa, Seiichi; Teramae, Norio

    2011-10-01

    A new class of label-free molecular beacon (MB) system based on DNA strands that contain abasic (AP) sites (AP-DNA) and adopt stem-loop structures, in combination with fluorescent ligands that bind these AP sites, has been developed. Unlike a conventional MB, which requires covalent labeling of the MB with a fluorophore and a quencher, the developed system (APMB) does not require covalent attachment of signal transduction units. Detailed sensing functions of a series of APMB systems were examined with the aid of the fluorescent ligand named ATMND to provide insight into the design strategy for APMB systems. The effects of the stem length and the position of the AP site in the stem moiety on the fluorescence response of the APMB system were examined. Genotyping of a G/C SNP of PCR amplification products was successfully demonstrated with the APMB system and blue-fluorescent ATMND as a ligand. The APMB system was further extended to a system that utilized green-fluorescent lumiflavin.

  16. Kinetics and sites of destruction of /sup 111/In-oxine-labeled platelets in idiopathic thrombocytopenic purpura: a quantitative study

    SciTech Connect

    Heyns, A.D.; Loetter, M.G.; Badenhorst, P.N.; de Kock, F.; Pieters, H.; Herbst, C.; van Reenen, O.R.; Kotze, H.; Minnaar, P.C.

    1982-04-01

    Kinetics and quantification of the sites of destruction of /sup 111/In-oxine-labeled autologous platelets were investigated in eight patients with idiopathic thrombocytopenic purpura. The mean platelet count was 17 +/- 9 X 10(9)/liter; platelets were separated by differential centrifugation and labeled with 5.6 +/- 2.5 MBq /sup 111/In. Whole body and organ /sup 111/In-platelet distribution was quantitated with a scintillation camera and a computer-assisted imaging system acquisition matrix. Areas of interest were selected with the computer and organ /sup 111/In-radioactivity expressed as a percentage of whole body activity. Mean platelet survival was 49.5 +/- 29.6 hr and the survival curves were exponential. Equilibrium percentage organ /sup 111/In-radioactivity was (normal values in parentheses): spleen 33.7 +/- 8.8 (31.1 +/- 10.2); liver 16.1 +/- 9.5 (13.1 +/- 1.3); thorax 22.8 +/- 3.7 (28.2 +/- 5.6). Percentage organ /sup 111/In-activity at the time when labeled platelets had disappeared from the circulation was: spleen 44.5 +/- 16.4 (40 +/- 16); liver 16.0 +/- 11.5 (32.4 +/- 7.2); thorax 19.7 +/- 6.0 (17.7 +/- 10.3). Thorax activity corresponds to bone marrow radioactivity. Three patterns of platelet sequestration were evident. Three patients had mainly splenic sequestration, two mainly hepatic sequestration, and three diffuse reticuloendothelial system sequestration with a major component of platelets destroyed in the bone marrow. Splenectomy was performed in two patients. The pattern of /sup 111/In-platelet sequestration was not predictive of response of glucocorticoid therapy or indicative of the necessity for splenectomy. Quantitative /sup 111/In-labeled autologous platelet kinetic studies provide a new tool for the investigation of platelet disorders.U

  17. Mapping Calcium-Sensitive Regions in the Neuronal Calcium Sensor GCAP2 by Site-Specific Fluorescence Labeling.

    PubMed

    Sulmann, Stefan; Wallisch, Melanie; Scholten, Alexander; Christoffers, Jens; Koch, Karl-Wilhelm

    2016-05-10

    Myristoylation of most neuronal calcium sensor proteins, a group of EF-hand calcium-binding proteins mainly expressed in neuronal tissue, can have a strong impact on protein dynamics and functional properties. Intracellular oscillations of the free Ca(2+) concentration can trigger conformational changes in Ca(2+) sensors. The position and possible movements of the myristoyl group in the photoreceptor cell-specific Ca(2+) sensor GCAP2 are not well-defined but appear to be different from those of the highly homologous cognate GCAP1. We designed and applied a new group of diaminoterephthalate-derived fluorescent probes to label GCAP2 at a covalently attached 12-azido-dodecanoic acid (a myristoyl substitute) and at cysteine residues in critical positions. Fluorescence emission of dye-labeled GCAP2 decreased when going from low (10(-9) M) to high [Ca(2+)] (10(-3) M), reaching a half-maximal effect of fluorescence emission at 0.44 ± 0.07 μM. The modified acyl group can therefore monitor changes in the protein conformation during binding and dissociation of Ca(2+) in the physiological range of free [Ca(2+)]. However, fluorescence quenching studies showed that the dye-acyl chain was shielded from the quencher by an adjacent polypeptide region. Further probing three cysteine positions (C35, C111, and C131) by dye labeling revealed that all positions were also sensitive to a change in [Ca(2+)], but only one (C131) was sensitive to a change in [Mg(2+)]. We suggest a scenario during illumination of the photoreceptor cell in which Ca(2+) dissociates first from low and medium affinity binding sites. These steps are sensed by dyes in cysteines at positions 35 and 111. Release of Ca(2+) from high affinity sites is sensed by regions adjacent to the dye-labeled fatty acid and involves the critical conformational change leading to activating guanylate cyclase.

  18. Elucidation of the Molecular Basis of Cholecystokinin Peptide Docking to Its Receptor Using Site-Specific Intrinsic Photoaffinity Labeling and Molecular Modeling†

    PubMed Central

    Dong, Maoqing; Lam, Polo C.-H.; Pino, Delia I.; Abagyan, Ruben; Miller, Laurence J.

    2012-01-01

    G protein-coupled receptors represent the largest family of receptors and the major target of current drug development efforts. Understanding of the mechanisms of ligand binding and activation of these receptors remains limited, despite recent advances in structural determination of family members. This work focuses on the use of photoaffinity labeling and molecular modeling to elucidate the structural basis of binding a natural peptide ligand to a family A G protein-coupled receptor, the type 1 cholecystokinin receptor. Two photolabile cholecystokinin analogues were developed and characterized as representing high-affinity, fully biologically active probes with sites of covalent attachment at positions 28 and 31. The sites of receptor labeling were identified by purification, proteolytic peptide mapping, and radiochemical sequencing of labeled wild-type and mutant cholecystokinin receptors. The position 28 probe labeled second extracellular loop residue Leu199, while the position 31 probe labeled first extracellular loop residue Phe107. Along with five additional spatial approximation constraints coming from previous photoaffinity labeling studies and 12 distance restraints from fluorescence resonance energy transfer studies, these were built into two homology models of the cholecystokinin receptor, based on the recent crystal structures of the β2-adrenergic receptor and A2a-adenosine receptor. The resultant agonist ligand-occupied receptor models fully accommodate all existing experimental data and represent the best refined models of a peptide hormone receptor in this important family. PMID:19441839

  19. 21 CFR 346.52 - Labeling of permitted combinations of anorectal active ingredients.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 5 2012-04-01 2012-04-01 false Labeling of permitted combinations of anorectal active ingredients. 346.52 Section 346.52 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... HUMAN USE Labeling § 346.52 Labeling of permitted combinations of anorectal active...

  20. 21 CFR 346.52 - Labeling of permitted combinations of anorectal active ingredients.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 5 2014-04-01 2014-04-01 false Labeling of permitted combinations of anorectal active ingredients. 346.52 Section 346.52 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... HUMAN USE Labeling § 346.52 Labeling of permitted combinations of anorectal active...

  1. A fully enzymatic method for site-directed spin labeling of long RNA.

    PubMed

    Lebars, Isabelle; Vileno, Bertrand; Bourbigot, Sarah; Turek, Philippe; Wolff, Philippe; Kieffer, Bruno

    2014-09-01

    Site-directed spin labeling is emerging as an essential tool to investigate the structural and dynamical features of RNA. We propose here an enzymatic method, which allows the insertion of a paramagnetic center at a specific position in an RNA molecule. The technique is based on a segmental approach using a ligation protocol with T4 RNA ligase 2. One transcribed acceptor RNA is ligated to a donor RNA in which a thio-modified nucleotide is introduced at its 5'-end by in vitro transcription with T7 RNA polymerase. The paramagnetic thiol-specific reagent is subsequently attached to the RNA ligation product. This novel strategy is demonstrated by introducing a paramagnetic probe into the 55 nucleotides long RNA corresponding to K-turn and Specifier Loop domains from the Bacillus subtilis tyrS T-Box leader RNA. The efficiency of the coupling reaction and the quality of the resulting spin-labeled RNA were assessed by Mass Spectrometry, Electron Paramagnetic Resonance (EPR) and Nuclear Magnetic Resonance (NMR). This method enables various combinations of isotopic segmental labeling and spin labeling schemes, a strategy that will be of particular interest to investigate the structural and dynamical properties of large RNA complexes by NMR and EPR spectroscopies.

  2. Site-specific fluorescence labelling of RNA using bio-orthogonal reaction of trans-cyclooctene and tetrazine.

    PubMed

    Asare-Okai, P N; Agustin, E; Fabris, D; Royzen, M

    2014-07-25

    This communication describes a general approach for site-specific fluorescence labelling of RNA using a cytidine triphosphate (CTP) analogue derivatized with a trans-cyclooctene group. The analogue was efficiently incorporated into a model RNA strand using in vitro transcription. Bio-orthogonal reaction with fluorescein-labelled tetrazine was utilized to fluorescently tag the synthetic RNA strand.

  3. Biomolecular DNP-Supported NMR Spectroscopy using Site-Directed Spin Labeling.

    PubMed

    van der Cruijsen, Elwin A W; Koers, Eline J; Sauvée, Claire; Hulse, Raymond E; Weingarth, Markus; Ouari, Olivier; Perozo, Eduardo; Tordo, Paul; Baldus, Marc

    2015-09-01

    Dynamic nuclear polarization (DNP) has been shown to greatly enhance spectroscopic sensitivity, creating novel opportunities for NMR studies on complex and large molecular assemblies in life and material sciences. In such applications, however, site-specificity and spectroscopic resolution become critical factors that are usually difficult to control by current DNP-based approaches. We have examined in detail the effect of directly attaching mono- or biradicals to induce local paramagnetic relaxation effects and, at the same time, to produce sizable DNP enhancements. Using a membrane-embedded ion channel as an example, we varied the degree of paramagnetic labeling and the location of the DNP probes. Our results show that the creation of local spin clusters can generate sizable DNP enhancements while preserving the intrinsic benefits of paramagnetic relaxation enhancement (PRE)-based NMR approaches. DNP using chemical labeling may hence provide an attractive route to introduce molecular specificity into DNP studies in life science applications and beyond.

  4. Injection site radioactivity of (99m)Tc-labeled mannosylated dextran for sentinel lymph node mapping.

    PubMed

    Yamaguchi, Aiko; Hanaoka, Hirofumi; Pirmettis, Ioannis; Uehara, Tomoya; Tsushima, Yoshito; Papadopoulos, Minas; Arano, Yasushi

    2015-02-01

    The high and persistent radioactivity at the injection site hinders the accuracy and expansion of sentinel lymph node (SLN) mapping. We investigated the mechanism underlying the undesirable radioactivity after subcutaneous injection of (99m)Tc-labeled mannosylated dextran ((99m)Tc(CO)3-DCM20), a SLN mapping agent targeting mannose receptors on macrophages and dendritic cells, in a mouse model. Biodistribution studies were performed 1 h after subcutaneous injection of (99m)Tc(CO)3-DCM20 from the rear footpad of mice in the presence of varying molar amounts of DCM20 or DC15, a modified dextran without mannose. Biodistribution studies were also conducted after subcutaneous injection of [(125)I]radioiodinated mannosyl-neoglycoalbumin ((125)I-NMA) from the rear footpad. The distribution of fluorescence-labeled DCM20 and DC15 at the injection site was also compared 1 h after subcutaneous injection by immunofluorescent histochemistry. The radioactivity levels of (99m)Tc(CO)3-DCM20 at the injection site and popliteal lymph node, a SLN in this model, decreased with an increase in the molar amounts of DCM20, whereas no significant changes in biodistribution were observed after injection of (99m)Tc(CO)3-DCM20 with varying molar amounts of DC15. (125)I-NMA exhibited rapid elimination of radioactivity from both the popliteal lymph node and the injection site. The fluorescence-labeled DCM20 colocalized well with CD68-positive cells such as macrophages and dendritic cells at the injection site. While partial colocalization was observed between DC15 and CD68-positive cells, the signal intensity was very weak. These findings suggest that specific binding of (99m)Tc(CO)3-DCM20 to the mannose receptor on macrophages and dendritic cells would be responsible for the sustained radioactivity levels at the injection site. These results also imply that discriminated blockage of (99m)Tc(CO)3-DCM20 binding to mannose receptors at the injection sites would reduce the radioactivity at the

  5. A small ribozyme with dual-site kinase activity

    PubMed Central

    Biondi, Elisa; Maxwell, Adam W.R.; Burke, Donald H.

    2012-01-01

    Phosphoryl transfer onto backbone hydroxyls is a recognized catalytic activity of nucleic acids. We find that kinase ribozyme K28 possesses an unusually complex active site that promotes (thio)phosphorylation of two residues widely separated in primary sequence. After allowing the ribozyme to radiolabel itself by phosphoryl transfer from [γ-32P]GTP, DNAzyme-mediated cleavage yielded two radiolabeled cleavage fragments, indicating phosphorylation sites within each of the two cleavage fragments. These sites were mapped by alkaline digestion and primer extension pausing. Enzymatic digestion and mutational analysis identified nucleotides important for activity and established the active structure as being a constrained pseudoknot with unusual connectivity that may juxtapose the two reactive sites. Nuclease sensitivities for nucleotides near the pseudoknot core were altered in the presence of GTPγS, indicating donor-induced folding. The 5′ target site was more strongly favored in full-length ribozyme K28 (128 nt) than in truncated RNAs (58 nt). Electrophoretic mobilities of self-thiophosphorylated products on organomercurial gels are distinct from the 5′ mono-thiophosphorylated product produced by reaction with polynucleotide kinase, potentially indicating simultaneous labeling of both sites within individual RNA strands. Our evidence supports a single, compact structure with local dynamics, rather than global rearrangement, as being responsible for dual-site phosphorylation. PMID:22618879

  6. Pulsed EPR Distance Measurements in Soluble Proteins by Site-directed Spin-labeling (SDSL)

    PubMed Central

    de Vera, Ian Mitchelle S.; Blackburn, Mandy E.; Galiano, Luis; Fanucci, Gail E.

    2015-01-01

    The resurgence of pulsed electron paramagnetic resonance (EPR) in structural biology centers on recent improvements in distance measurements using the double electron-electron resonance (DEER) technique. This unit focuses on EPR-based distance measurements by site-directed spin-labeling (SDSL) of engineered cysteine residues in soluble proteins, with HIV-1 protease used as a model. To elucidate conformational changes in proteins, experimental protocols were optimized and existing data analysis programs were employed to derive distance distribution profiles. Experimental considerations, sample preparation and error analysis for artifact suppression are also outlined here. PMID:24510645

  7. Absolute Quantitation of Glycosylation Site Occupancy Using Isotopically Labeled Standards and LC-MS

    NASA Astrophysics Data System (ADS)

    Zhu, Zhikai; Go, Eden P.; Desaire, Heather

    2014-06-01

    N-linked glycans are required to maintain appropriate biological functions on proteins. Underglycosylation leads to many diseases in plants and animals; therefore, characterizing the extent of glycosylation on proteins is an important step in understanding, diagnosing, and treating diseases. To determine the glycosylation site occupancy, protein N-glycosidase F (PNGase F) is typically used to detach the glycan from the protein, during which the formerly glycosylated asparagine undergoes deamidation to become an aspartic acid. By comparing the abundance of the resulting peptide containing aspartic acid against the one containing non-glycosylated asparagine, the glycosylation site occupancy can be evaluated. However, this approach can give inaccurate results when spontaneous chemical deamidation of the non-glycosylated asparagine occurs. To overcome this limitation, we developed a new method to measure the glycosylation site occupancy that does not rely on converting glycosylated peptides to their deglycosylated forms. Specifically, the overall protein concentration and the non-glycosylated portion of the protein are quantified simultaneously by using heavy isotope-labeled internal standards coupled with LC-MS analysis, and the extent of site occupancy is accurately determined. The efficacy of the method was demonstrated by quantifying the occupancy of a glycosylation site on bovine fetuin. The developed method is the first work that measures the glycosylation site occupancy without using PNGase F, and it can be done in parallel with glycopeptide analysis because the glycan remains intact throughout the workflow.

  8. Site-specific one-pot dual labeling of DNA by orthogonal cycloaddition chemistry.

    PubMed

    Schoch, Juliane; Staudt, Markus; Samanta, Ayan; Wiessler, Manfred; Jäschke, Andres

    2012-07-18

    Bioorthogonal reactions are of high interest in biosciences as they allow the introduction of fluorescent dyes, affinity tags, or other unnatural moieties into biomolecules. The site-specific attachment of two or more different labels is particularly demanding and typically requires laborious multistep syntheses. Here, we report that the most popular cycloaddition in bioconjugation, the copper-catalyzed azide-alkyne click reaction (CuAAC), is fully orthogonal to the inverse electron-demand Diels-Alder reaction (DAinv). We demonstrate that both bioorthogonal reactions can be conducted concurrently in a one-pot reaction by just mixing all components. Orthogonality has been established even for highly reactive trans-cyclooctene-based dienophiles (with rate constants up to 380 000 M(-1) s(-1)). These properties allow for the convenient site-specific one-step preparation of oligonucleotide FRET probes and related reporters needed in cellular biology and biophysical chemistry.

  9. Novel Benzodiazepine Photoaffinity Probe Stereoselectively Labels a Site Deep Within the Membrane-spanning Domain of the Cholecystokinin Receptor

    PubMed Central

    Hadac, Elizabeth M.; Dawson, Eric S.; Darrow, James W.; Sugg, Elizabeth E.; Lybrand, Terry P.; Miller, Laurence J.

    2008-01-01

    An understanding of the molecular basis of drug action provides opportunities for refinement of drug properties and for development of more potent and selective molecules that act at the same biological target. In this work, we have identified the active enantiomers in racemic mixtures of structurally related benzophenone derivatives of 1,5-benzodiazepines, representing both antagonist and agonist ligands of the type A cholecystokinin receptor. The parent compounds of the 1,5-benzodiazepine CCK receptor photoaffinity ligands were originally prepared in an effort to develop orally active drugs. The enantiomeric compounds reported in this study selectively photoaffinity-labeled the CCK receptor, resulting in the identification of a site of attachment for the photolabile moiety of the antagonist probe deep within the receptor’s membrane-spanning region at Leu88, a residue within transmembrane segment two. In contrast, the agonist probe labeled a region including extracellular loop one and a portion of transmembrane segment three. The antagonist covalent attachment site to the receptor served as a guide in the construction of theoretical three-dimensional molecular models for the antagonist-receptor complex. These models provided a means for visualization of physically plausible ligand-receptor interactions in the context of all currently available biological data that address small molecule interactions with the CCK receptor. Our approach, featuring the use of novel photolabile compounds targeting the membrane-spanning receptor domain to probe the binding site region, introduces powerful tools and a strategy for direct and selective investigation of non-peptidyl ligand binding to peptide receptors. PMID:16451051

  10. Platelet labeling and positron emission tomography in the detection of sites of vascular injury

    SciTech Connect

    Yano, Y.; Brennan, K.M.; Ebbe, S.N.; Singh, M.; Moyer, B.R.; Carpenter, D.A.; Mazoyer, E.M.; Mathis, C.A.; Budinger, T.F.

    1985-05-01

    Reports show that diverse types of injury to the arterial wall elicit varying response reactions often including platelet adhesion. The authors have utilized PET and Ga-68 platelets to study mechanically induced endothelial damage in the rabbit aorta. Ga-68 (t1/2=68min) was eluted as 68-Ga-gallium chloride from a Ge-68/Ga-68 stannic oxide/lN HCl generator and purified by ether extraction from 6N HCl. Ga-68 complex with 2-mercaptopyridine-N-oxide (MPO) was formed in the presence of acetate buffer. Ga-68-MPO labeling efficiency (90-98%) was determined by a newly developed HPLC method using C-18 R/P column and methanol:citrate solvent. Separated platelets were then labelled with Ga-68-MPO and imaged with PET. The embolectomy catheter was introduced intrafemorally and pushed cranially 20 cm to the aortic arch. The bulb was inflated and the aorta scraped 3x before removing the catheter and ligating the artery. Evan's blue dye injected 30 minutes before sacrifice distinguished the damaged from undamaged aorta. As seen in gamma counter results and aortic enface autoradiographs there was a 4 fold increase in activity on the damaged aorta. Whole body PET scans showed an increase in activity along the distal aorta. Damage to the endothelial cells in vivo can be caused by several metabolic factors. The achievement of reliable platelet labeling with a positron emitter allows the authors to image the accumulation and turnover of platelets. Validation of the biological integrity after labeling is being done with Ga-67.

  11. Autoradiographic localization of /sup 3/H-paroxetine-labeled serotonin uptake sites in rat brain

    SciTech Connect

    De Souza, E.B.; Kuyatt, B.L.

    1987-01-01

    Paroxetine is a potent and selective inhibitor of serotonin uptake into neurons. Serotonin uptake sites have been identified, localized, and quantified in rat brain by autoradiography with 3H-paroxetine; 3H-paroxetine binding in slide-mounted sections of rat forebrain was of high affinity (KD = 10 pM) and the inhibition affinity constant (Ki) values of various drugs in competing 3H-paroxetine binding significantly correlated with their reported potencies in inhibiting synaptosomal serotonin uptake. Serotonin uptake sites labeled by 3H-paroxetine were highly concentrated in the dorsal and median raphe nuclei, central gray, superficial layer of the superior colliculus, lateral septal nucleus, paraventricular nucleus of the thalamus, and the islands of Calleja. High concentrations of 3H-paroxetine binding sites were found in brainstem areas containing dopamine (substantia nigra and ventral tegmental area) and norepinephrine (locus coeruleus) cell bodies. Moderate concentrations of 3H-paroxetine binding sites were present in laminae I and IV of the frontal parietal cortex, primary olfactory cortex, olfactory tubercle, regions of the basal ganglia, septum, amygdala, thalamus, hypothalamus, hippocampus, and some brainstem areas including the interpeduncular, trigeminal, and parabrachial nuclei. Lower densities of 3H-paroxetine binding sites were found in other regions of the neocortex and very low to nonsignificant levels of binding were present in white matter tracts and in the cerebellum. Lesioning of serotonin neurons with 3,4-methylenedioxyamphetamine caused large decreases in 3H-paroxetine binding. The autoradiographic distribution of 3H-paroxetine binding sites in rat brain corresponds extremely well to the distribution of serotonin terminals and cell bodies as well as with the pharmacological sites of action of serotonin.

  12. [[superscript 3]H]-Flunitrazepam-Labeled Benzodiazepine Binding Sites in the Hippocampal Formation in Autism: A Multiple Concentration Autoradiographic Study

    ERIC Educational Resources Information Center

    Guptill, Jeffrey T.; Booker, Anne B.; Gibbs, Terrell T.; Kemper, Thomas L.; Bauman, Margaret L.; Blatt, Gene J.

    2007-01-01

    Increasing evidence indicates that the GABAergic system in cerebellar and limbic structures is affected in autism. We extended our previous study that found reduced [[superscript 3]H] flunitrazepam-labeled benzodiazepine sites in the autistic hippocampus to determine whether this reduction was due to a decrease in binding site number (B [subscript…

  13. Dopamine transport sites selectively labeled by a novel photoaffinity probe: 125I-DEEP

    SciTech Connect

    Grigoriadis, D.E.; Wilson, A.A.; Lew, R.; Sharkey, J.S.; Kuhar, M.J. )

    1989-08-01

    The dopamine transporter was labeled using a photosensitive compound related to GBR-12909, {sup 125}I-1-(2-(diphenylmethoxy)ethyl)-4-(2- (4-azido-3-iodophenyl)ethyl)piperazine ({sup 125}I-DEEP). {sup 125}I-DEEP bound reversibly and with high affinity to the dopamine transport protein in the absence of light and could be covalently attached to the protein following exposure to UV light. In rat striatal homogenates, {sup 125}I-DEEP was found to incorporate covalently into a protein with apparent molecular weight of 58,000 Da. The properties of this binding protein were characteristic of the dopamine transporter since covalent attachment could be inhibited by dopamine-uptake blockers with the proper pharmacological rank order of potencies. Covalent binding was also inhibited in a stereospecific manner by (+) and (-) cocaine, as well as other cocaine analogs. The protein was not found in the cerebellum. The dopamine transporter appears to exist in a glycosylated form since photoaffinity-labeled transport sites could adsorb to wheat germ-agglutinin and could be specifically eluted from the column by beta-N-acetylglucosamine.

  14. Potential Effect of Physical Activity Calorie Equivalent (PACE) Labeling on Adult Fast Food Ordering and Exercise

    PubMed Central

    Antonelli, Ray; Viera, Anthony J.

    2015-01-01

    Introduction Numeric calorie content labels show limited efficacy in reducing the number of calories ordered from fast food meals. Physical activity calorie equivalent (PACE) labels are an alternative that may reduce the number of calories ordered in fast food meals while encouraging patrons to exercise. Methods A total of 1000 adults from 47 US states were randomly assigned via internet survey to one of four generic fast food menus: no label, calories only, calories + minutes, or calories + miles necessary to walk to burn off the calories. After completing hypothetical orders participants were asked to rate the likelihood of calorie-only and PACE labels to influence (1) food choice and (2) physical activity. Results Respondents (n = 823) ordered a median of 1580 calories from the no-label menu, 1200 from the calories-only menu, 1140 from the calories + minutes menu, and 1210 from the calories + miles menu (p = 0.0001). 40% of respondents reported that PACE labels were “very likely” to influence food item choice vs. 28% for calorie-only labels (p<0.0001). 64% of participants reported that PACE labels were “somewhat likely” or “very likely” to influence their level of physical activity vs. 49% for calorie-only labels (p<0.0001). Conclusions PACE labels may be helpful in reducing the number of calories ordered in fast food meals and may have the added benefit of encouraging exercise. PMID:26222056

  15. 21 CFR 349.79 - Labeling of permitted combinations of active ingredients.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 5 2014-04-01 2014-04-01 false Labeling of permitted combinations of active ingredients. 349.79 Section 349.79 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... § 349.79 Labeling of permitted combinations of active ingredients. Statements of identity,...

  16. 21 CFR 349.79 - Labeling of permitted combinations of active ingredients.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 5 2012-04-01 2012-04-01 false Labeling of permitted combinations of active ingredients. 349.79 Section 349.79 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... § 349.79 Labeling of permitted combinations of active ingredients. Statements of identity,...

  17. 21 CFR 349.79 - Labeling of permitted combinations of active ingredients.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 5 2013-04-01 2013-04-01 false Labeling of permitted combinations of active ingredients. 349.79 Section 349.79 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... § 349.79 Labeling of permitted combinations of active ingredients. Statements of identity,...

  18. Site-Specific N-Terminal Labeling of Peptides and Proteins using Butelase 1 and Thiodepsipeptide.

    PubMed

    Nguyen, Giang K T; Cao, Yuan; Wang, Wei; Liu, Chuan Fa; Tam, James P

    2015-12-21

    An efficient ligase with exquisite site-specificity is highly desirable for protein modification. Recently, we discovered the fastest known ligase called butelase 1 from Clitoria ternatea for intramolecular cyclization. For intermolecular ligation, butelase 1 requires an excess amount of a substrate to suppress the reverse reaction, a feature similar to other ligases. Herein, we describe the use of thiodepsipeptide substrates with a thiol as a leaving group and an unacceptable nucleophile to render the butelase-mediated ligation reactions irreversible and in high yields. Butelase 1 also accepted depsipeptides as substrates, but unlike a thiodesipeptide, the desipeptide ligation was partially reversible as butelase 1 can tolerate an alcohol group as a poor nucleophile. The thiodesipeptide method was successfully applied in N-terminal labeling of ubiquitin and green fluorescent protein using substrates with or without a biotin group in high yields. PMID:26563575

  19. Structural Characterization of Membrane-Curving Proteins: Site-Directed Spin Labeling, EPR, and Computational Refinement.

    PubMed

    Ambroso, Mark R; Haworth, Ian S; Langen, Ralf

    2015-01-01

    Endocytosis and other membrane remodeling processes require the coordinated generation of different membrane shapes. Proteins capable of manipulating lipid bilayers mediate these events using mechanisms that are not fully understood. Progress is limited by the small number of structures solved for proteins bound to different membrane shapes and tools capable of resolving such information. However, recent studies have shown site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) to be capable of obtaining high-resolution structural information for proteins bound to different membrane shapes. This technique can be applied to proteins with no known structure or proteins with structures known in solution. By refining the data obtained by EPR with computational modeling, 3D structures or structural models of membrane-bound proteins can be generated. In this chapter, we highlight the basic considerations and steps required to investigate the structures of membrane-bound proteins using SDSL, EPR, and computational refinement. PMID:26477254

  20. Analyst variability in labeling of unsupervised classifications. [test sites for Landsat 5 Thematic Mapper

    NASA Technical Reports Server (NTRS)

    Mcgwire, Kenneth C.

    1992-01-01

    Analyst variability in the labeling of unsupervised classifications is tested for Landsat 5 Thematic Mapper image products covering two test sites in southern California. The accuracy of results are tested using samples from a photo interpreted base map of the area. The significance of differences between analysts is indicated by comparing Kappa statistics derived from error matrices. Analyst variability is found to be statistically significant in most cases. Certain analysts provided consistently better results for a given study area or degree of training. This work demonstrates the potential influence of analyst bias on what would otherwise seem to be a fairly objective method and suggests that controls for this subjectivity should be factored into experimental designs.

  1. Site-Specifically Labeled Immunoconjugates for Molecular Imaging--Part 1: Cysteine Residues and Glycans.

    PubMed

    Adumeau, Pierre; Sharma, Sai Kiran; Brent, Colleen; Zeglis, Brian M

    2016-02-01

    Due to their remarkable selectivity and specificity for cancer biomarkers, immunoconjugates have emerged as extremely promising vectors for the delivery of diagnostic radioisotopes and fluorophores to malignant tissues. Paradoxically, however, these tools for precision medicine are synthesized in a remarkably imprecise way. Indeed, the vast majority of immunoconjugates are created via the random conjugation of bifunctional probes (e.g., DOTA-NCS) to amino acids within the antibody (e.g., lysines). Yet antibodies have multiple copies of these residues throughout their macromolecular structure, making control over the location of the conjugation reaction impossible. This lack of site specificity can lead to the formation of poorly defined, heterogeneous immunoconjugates with suboptimal in vivo behavior. Over the past decade, interest in the synthesis and development of site-specifically labeled immunoconjugates--both antibody-drug conjugates as well as constructs for in vivo imaging--has increased dramatically, and a number of reports have suggested that these better defined, more homogeneous constructs exhibit improved performance in vivo compared to their randomly modified cousins. In this two-part review, we seek to provide an overview of the various methods that have been developed to create site-specifically modified immunoconjugates for positron emission tomography, single photon emission computed tomography, and fluorescence imaging. We will begin with an introduction to the structure of antibodies and antibody fragments. This is followed by the core of the work: sections detailing the four different approaches to site-specific modification strategies based on cysteine residues, glycans, peptide tags, and unnatural amino acids. These discussions will be divided into two installments: cysteine residues and glycans will be detailed in Part 1 of the review, while peptide tags and unnatural amino acids will be addressed in Part 2. Ultimately, we sincerely hope

  2. Catalysis: Elusive active site in focus

    NASA Astrophysics Data System (ADS)

    Labinger, Jay A.

    2016-08-01

    The identification of the active site of an iron-containing catalyst raises hopes of designing practically useful catalysts for the room-temperature conversion of methane to methanol, a potential fuel for vehicles. See Letter p.317

  3. Genetic Incorporation of the Unnatural Amino Acid p-Acetyl Phenylalanine into Proteins for Site-Directed Spin Labeling

    PubMed Central

    Evans, Eric G.B.; Millhauser, Glenn L.

    2016-01-01

    Site-directed spin labeling (SDSL) is a powerful tool for the characterization of protein structure and dynamics; however, its application in many systems is hampered by the reliance on unique and benign cysteine substitutions for the site-specific attachment of the spin label. An elegant solution to this problem involves the use of genetically encoded unnatural amino acids (UAAs) containing reactive functional groups that are chemically orthogonal to those of the 20 amino acids found naturally in proteins. These unique functional groups can then be selectively reacted with an appropriately functionalized spin probe. In this chapter, we detail the genetic incorporation of the ketone-bearing amino acid p-acetyl phenylalanine (pAcPhe) into recombinant proteins expressed in E. coli. Incorporation of pAcPhe is followed by chemoselective reaction of the ketone side chain with a hydroxylamine-functionalized nitroxide to afford the spin-labeled side chain “K1,” and we present two protocols for successful K1 labeling of proteins bearing site-specific pAcPhe. We outline the basic requirements for pAcPhe incorporation and labeling, with an emphasis on practical aspects that must be considered by the researcher if high yields of UAA incorporation and efficient labeling reactions are to be achieved. To this end, we highlight recent advances that have led to increased yields of pAcPhe incorporation, and discuss the use of aniline-based catalysts allowing for facile conjugation of the hydroxylamine spin label under mild reaction conditions. To illustrate the utility of K1 labeling in proteins where traditional cysteine-based SDSL methods are problematic, we site-specifically K1 label the cellular prion protein at two positions in the C-terminal domain and determine the interspin distance using double electron–electron resonance EPR. Recent advances in UAA incorporation and ketone-based bioconjugation, in combination with the commercial availability of all requisite

  4. Genetic Incorporation of the Unnatural Amino Acid p-Acetyl Phenylalanine into Proteins for Site-Directed Spin Labeling.

    PubMed

    Evans, Eric G B; Millhauser, Glenn L

    2015-01-01

    Site-directed spin labeling (SDSL) is a powerful tool for the characterization of protein structure and dynamics; however, its application in many systems is hampered by the reliance on unique and benign cysteine substitutions for the site-specific attachment of the spin label. An elegant solution to this problem involves the use of genetically encoded unnatural amino acids (UAAs) containing reactive functional groups that are chemically orthogonal to those of the 20 amino acids found naturally in proteins. These unique functional groups can then be selectively reacted with an appropriately functionalized spin probe. In this chapter, we detail the genetic incorporation of the ketone-bearing amino acid p-acetyl phenylalanine (pAcPhe) into recombinant proteins expressed in E. coli. Incorporation of pAcPhe is followed by chemoselective reaction of the ketone side chain with a hydroxylamine-functionalized nitroxide to afford the spin-labeled side chain "K1," and we present two protocols for successful K1 labeling of proteins bearing site-specific pAcPhe. We outline the basic requirements for pAcPhe incorporation and labeling, with an emphasis on practical aspects that must be considered by the researcher if high yields of UAA incorporation and efficient labeling reactions are to be achieved. To this end, we highlight recent advances that have led to increased yields of pAcPhe incorporation, and discuss the use of aniline-based catalysts allowing for facile conjugation of the hydroxylamine spin label under mild reaction conditions. To illustrate the utility of K1 labeling in proteins where traditional cysteine-based SDSL methods are problematic, we site-specifically K1 label the cellular prion protein at two positions in the C-terminal domain and determine the interspin distance using double electron-electron resonance EPR. Recent advances in UAA incorporation and ketone-based bioconjugation, in combination with the commercial availability of all requisite reagents

  5. Genetic Incorporation of the Unnatural Amino Acid p-Acetyl Phenylalanine into Proteins for Site-Directed Spin Labeling.

    PubMed

    Evans, Eric G B; Millhauser, Glenn L

    2015-01-01

    Site-directed spin labeling (SDSL) is a powerful tool for the characterization of protein structure and dynamics; however, its application in many systems is hampered by the reliance on unique and benign cysteine substitutions for the site-specific attachment of the spin label. An elegant solution to this problem involves the use of genetically encoded unnatural amino acids (UAAs) containing reactive functional groups that are chemically orthogonal to those of the 20 amino acids found naturally in proteins. These unique functional groups can then be selectively reacted with an appropriately functionalized spin probe. In this chapter, we detail the genetic incorporation of the ketone-bearing amino acid p-acetyl phenylalanine (pAcPhe) into recombinant proteins expressed in E. coli. Incorporation of pAcPhe is followed by chemoselective reaction of the ketone side chain with a hydroxylamine-functionalized nitroxide to afford the spin-labeled side chain "K1," and we present two protocols for successful K1 labeling of proteins bearing site-specific pAcPhe. We outline the basic requirements for pAcPhe incorporation and labeling, with an emphasis on practical aspects that must be considered by the researcher if high yields of UAA incorporation and efficient labeling reactions are to be achieved. To this end, we highlight recent advances that have led to increased yields of pAcPhe incorporation, and discuss the use of aniline-based catalysts allowing for facile conjugation of the hydroxylamine spin label under mild reaction conditions. To illustrate the utility of K1 labeling in proteins where traditional cysteine-based SDSL methods are problematic, we site-specifically K1 label the cellular prion protein at two positions in the C-terminal domain and determine the interspin distance using double electron-electron resonance EPR. Recent advances in UAA incorporation and ketone-based bioconjugation, in combination with the commercial availability of all requisite reagents

  6. Inulin-125I-tyramine, an improved residualizing label for studies on sites of catabolism of circulating proteins

    SciTech Connect

    Maxwell, J.L.; Baynes, J.W.; Thorpe, S.R.

    1988-10-05

    Residualizing labels for protein, such as dilactitol-125I-tyramine (125I-DLT) and cellobiitol-125I-tyramine, have been used to identify the tissue and cellular sites of catabolism of long-lived plasma proteins, such as albumin, immunoglobulins, and lipoproteins. The radioactive degradation products formed from labeled proteins are relatively large, hydrophilic, resistant to lysosomal hydrolases, and accumulate in lysosomes in the cells involved in degradation of the carrier protein. However, the gradual loss of the catabolites from cells (t1/2 approximately 2 days) has limited the usefulness of residualizing labels in studies on longer lived proteins. We describe here a higher molecular weight (Mr approximately 5000), more efficient residualizing glycoconjugate label, inulin-125I-tyramine (125I-InTn). Attachment of 125I-InTn had no effect on the plasma half-life or tissue sites of catabolism of asialofetuin, fetuin, or rat serum albumin in the rat. The half-life for hepatic retention of degradation products from 125I-InTn-labeled asialofetuin was 5 days, compared to 2.3 days for 125I-DLT-labeled asialofetuin. The whole body half-lives for radioactivity from 125I-InTn-, 125I-DLT-, and 125I-labeled rat serum albumin were 7.5, 4.3, and 2.2 days, respectively. The tissue distribution of degradation products from 125I-InTn-labeled proteins agreed with results of previous studies using 125I-DLT, except that a greater fraction of total degradation products was recovered in tissues. Kinetic analyses indicated that the average half-life for retention of 125I-InTn degradation products in tissues is approximately 5 days and suggested that in vivo there are both slow and rapid routes for release of degradation products from cells.

  7. 21 CFR 333.160 - Labeling of permitted combinations of active ingredients.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 5 2014-04-01 2014-04-01 false Labeling of permitted combinations of active ingredients. 333.160 Section 333.160 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND... HUMAN USE First Aid Antibiotic Drug Products § 333.160 Labeling of permitted combinations of...

  8. Capture and Recycling of Sortase A through Site-Specific Labeling with Lithocholic Acid.

    PubMed

    Rosen, Christian B; Kwant, Richard L; MacDonald, James I; Rao, Meera; Francis, Matthew B

    2016-07-18

    Enzyme-mediated protein modification often requires large amounts of biocatalyst, adding significant costs to the process and limiting industrial applications. Herein, we demonstrate a scalable and straightforward strategy for the efficient capture and recycling of enzymes using a small-molecule affinity tag. A proline variant of an evolved sortase A (SrtA 7M) was N-terminally labeled with lithocholic acid (LA)-an inexpensive bile acid that exhibits strong binding to β-cyclodextrin (βCD). Capture and recycling of the LA-Pro-SrtA 7M conjugate was achieved using βCD-modified sepharose resin. The LA-Pro-SrtA 7M conjugate retained full enzymatic activity, even after multiple rounds of recycling. PMID:27239057

  9. Site-specific (18)F-labeling of the protein hormone leptin using a general two-step ligation procedure.

    PubMed

    Flavell, Robert R; Kothari, Paresh; Bar-Dagan, Maya; Synan, Michael; Vallabhajosula, Shankar; Friedman, Jeffrey M; Muir, Tom W; Ceccarini, Giovanni

    2008-07-16

    The protein hormone leptin acts to regulate body fat and energy expenditure. Resistance to this hormone is implicated in human obesity and its pathophysiological consequences. In order to gain insight into the mechanism of leptin resistance, an (18)F-labeled derivative was developed to study the biodistribution of the hormone using positron emission tomography (PET). A two-step, site specific ligation approach was developed for this purpose, in which an aminooxy-reactive group was incorporated at the C-terminus of leptin using expressed protein ligation (EPL), which was subsequently derivatized with [ (18)F]fluorobenzaldehyde using an aniline-accelerated radiochemical oximation reaction. The modified hormone was shown to be biologically active in vitro and in vivo, and it was applied to PET imaging in ob/ ob mice. These protocols will allow for the routine production of site-specifically (18)F radiolabeled leptin, as well as other proteins, for use in PET imaging in systems from mouse to man.

  10. Mass-tag labeling reveals site-specific and endogenous levels of protein S-fatty acylation.

    PubMed

    Percher, Avital; Ramakrishnan, Srinivasan; Thinon, Emmanuelle; Yuan, Xiaoqiu; Yount, Jacob S; Hang, Howard C

    2016-04-19

    Fatty acylation of cysteine residues provides spatial and temporal control of protein function in cells and regulates important biological pathways in eukaryotes. Although recent methods have improved the detection and proteomic analysis of cysteine fatty (S-fatty) acylated proteins, understanding how specific sites and quantitative levels of this posttranslational modification modulate cellular pathways are still challenging. To analyze the endogenous levels of protein S-fatty acylation in cells, we developed a mass-tag labeling method based on hydroxylamine-sensitivity of thioesters and selective maleimide-modification of cysteines, termed acyl-PEG exchange (APE). We demonstrate that APE enables sensitive detection of protein S-acylation levels and is broadly applicable to different classes of S-palmitoylated membrane proteins. Using APE, we show that endogenous interferon-induced transmembrane protein 3 is S-fatty acylated on three cysteine residues and site-specific modification of highly conserved cysteines are crucial for the antiviral activity of this IFN-stimulated immune effector. APE therefore provides a general and sensitive method for analyzing the endogenous levels of protein S-fatty acylation and should facilitate quantitative studies of this regulated and dynamic lipid modification in biological systems.

  11. Mass-tag labeling reveals site-specific and endogenous levels of protein S-fatty acylation.

    PubMed

    Percher, Avital; Ramakrishnan, Srinivasan; Thinon, Emmanuelle; Yuan, Xiaoqiu; Yount, Jacob S; Hang, Howard C

    2016-04-19

    Fatty acylation of cysteine residues provides spatial and temporal control of protein function in cells and regulates important biological pathways in eukaryotes. Although recent methods have improved the detection and proteomic analysis of cysteine fatty (S-fatty) acylated proteins, understanding how specific sites and quantitative levels of this posttranslational modification modulate cellular pathways are still challenging. To analyze the endogenous levels of protein S-fatty acylation in cells, we developed a mass-tag labeling method based on hydroxylamine-sensitivity of thioesters and selective maleimide-modification of cysteines, termed acyl-PEG exchange (APE). We demonstrate that APE enables sensitive detection of protein S-acylation levels and is broadly applicable to different classes of S-palmitoylated membrane proteins. Using APE, we show that endogenous interferon-induced transmembrane protein 3 is S-fatty acylated on three cysteine residues and site-specific modification of highly conserved cysteines are crucial for the antiviral activity of this IFN-stimulated immune effector. APE therefore provides a general and sensitive method for analyzing the endogenous levels of protein S-fatty acylation and should facilitate quantitative studies of this regulated and dynamic lipid modification in biological systems. PMID:27044110

  12. Low dielectric response in enzyme active site

    PubMed Central

    Mertz, Edward L.; Krishtalik, Lev I.

    2000-01-01

    The kinetics of charge transfer depend crucially on the dielectric reorganization of the medium. In enzymatic reactions that involve charge transfer, atomic dielectric response of the active site and of its surroundings determines the efficiency of the protein as a catalyst. We report direct spectroscopic measurements of the reorganization energy associated with the dielectric response in the active site of α-chymotrypsin. A chromophoric inhibitor of the enzyme is used as a spectroscopic probe. We find that water strongly affects the dielectric reorganization in the active site of the enzyme in solution. The reorganization energy of the protein matrix in the vicinity of the active site is similar to that of low-polarity solvents. Surprisingly, water exhibits an anomalously high dielectric response that cannot be described in terms of the dielectric continuum theory. As a result, sequestering the active site from the aqueous environment inside low-dielectric enzyme body dramatically reduces the dielectric reorganization. This reduction is particularly important for controlling the rate of enzymatic reactions. PMID:10681440

  13. Effects of organizational scheme and labeling on task performance in product-centered and user-centered retail Web sites.

    PubMed

    Resnick, Marc L; Sanchez, Julian

    2004-01-01

    As companies increase the quantity of information they provide through their Web sites, it is critical that content is structured with an appropriate architecture. However, resource constraints often limit the ability of companies to apply all Web design principles completely. This study quantifies the effects of two major information architecture principles in a controlled study that isolates the incremental effects of organizational scheme and labeling on user performance and satisfaction. Sixty participants with a wide range of Internet and on-line shopping experience were recruited to complete a series of shopping tasks on a prototype retail shopping Web site. User-centered labels provided a significant benefit in performance and satisfaction over labels obtained through company-centered methods. User-centered organization did not result in improved performance except when the label quality was poor. Significant interactions suggest specific guidelines for allocating resources in Web site design. Applications of this research include the design of Web sites for any commercial application, particularly E-commerce. PMID:15151158

  14. Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels.

    PubMed

    Basak, Sandip; Chatterjee, Soumili; Chakrapani, Sudha

    2016-01-01

    Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. Fundamental to these transitions is the intrinsic flexibility of the protein, which is critically modulated by membrane lipid-composition. To better understand the structural basis of channel function, it is necessary to study protein dynamics in a physiological membrane environment. Electron Paramagnetic Resonance (EPR) spectroscopy is an important tool to characterize conformational transitions between functional states. In comparison to NMR and X-ray crystallography, the information obtained from EPR is intrinsically of lower resolution. However, unlike in other techniques, in EPR there is no upper-limit to the molecular weight of the protein, the sample requirements are significantly lower, and more importantly the protein is not constrained by the crystal lattice forces. Therefore, EPR is uniquely suited for studying large protein complexes and proteins in reconstituted systems. In this article, we will discuss general protocols for site-directed spin labeling and membrane reconstitution using a prokaryotic proton-gated pentameric Ligand-Gated Ion Channel (pLGIC) from Gloeobacter violaceus (GLIC) as an example. A combination of steady-state Continuous Wave (CW) and Pulsed (Double Electron Electron Resonance-DEER) EPR approaches will be described that will enable a complete quantitative characterization of channel dynamics. PMID:27403967

  15. Synthesis of isotopically labeled P-site substrates for the ribosomal peptidyl transferase reaction

    PubMed Central

    Zhong, Minghong

    2010-01-01

    Isotopomers of the ribosomal P-site substrate, the trinucleotide peptide conjugate CCA-pcb,1 have been designed and synthesized in 26–350020steps. These include individual isotopic substitution at the α-proton, carbonyl carbon, and carbonyl oxygen of the amino acid, the O2' and O3' of the adenosine, and a remote label in the N3 and N4 of both cytidines. These isotopomers were synthesized by coupling cytidylyl-(3'5')-cytidine phosphoramidite isotopomers, as the common synthetic intermediates, with isotopically substituted A-Phe-cap-biotin (A-pcb). The isotopic enrichment is higher than 99% for 1-13C (Phe), 2-2H (Phe), and 3,4-15N2 (cytidine), 93% for 2'/3'- 18O (adenosine), and 64% for 1-18O (Phe). A new synthesis of highly enriched [1-18O2] phenylalanine has been developed. The synthesis of [3'-18O] adenosine was improved by Lewis acid aided regioselective ring opening of the epoxide and by an economical SN2-SN2 method with high isotopic enrichment (93%). Such substrates are valuable for studies of the ribosomal peptidyl transferase reaction by complete kinetic isotope effect analysis and of other biological processes catalyzed by nucleic acid related enzymes, including polymerases, reverse transcriptases, ligases, nucleases, and ribozymes. PMID:18081346

  16. High-Pressure EPR and Site-Directed Spin Labeling for Mapping Molecular Flexibility in Proteins.

    PubMed

    Lerch, Michael T; Yang, Zhongyu; Altenbach, Christian; Hubbell, Wayne L

    2015-01-01

    High hydrostatic pressure is a powerful probe of protein conformational flexibility. Pressurization reveals regions of elevated compressibility, and thus flexibility, within individual conformational states, but also shifts conformational equilibria such that "invisible" excited states become accessible for spectroscopic characterization. The central aim of this chapter is to describe recently developed instrumentation and methodologies that enable high-pressure site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) experiments on proteins and to demonstrate the information content of these experiments by highlighting specific recent applications. A brief introduction to the thermodynamics of proteins under pressure is presented first, followed by a discussion of the principles underlying SDSL-EPR detection of pressure effects in proteins, and the suitability of SDSL-EPR for this purpose in terms of timescale and ability to characterize conformational heterogeneity. Instrumentation and practical considerations for variable-pressure continuous wave EPR and pressure-resolved double electron-electron resonance (PR DEER) experiments are reviewed, and finally illustrations of data analysis using recent applications are presented. Although high-pressure SDSL-EPR is in its infancy, the recent applications presented highlight the considerable potential of the method to (1) identify compressible (flexible) regions in a folded protein; (2) determine thermodynamic parameters that relate conformational states in equilibrium; (3) populate and characterize excited states of proteins undetected at atmospheric pressure; (4) reveal the structural heterogeneity of conformational ensembles and provide distance constraints on the global structure of pressure-populated states with PR DEER.

  17. 21 CFR 346.52 - Labeling of permitted combinations of anorectal active ingredients.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... OTC drug monograph. ... 21 Food and Drugs 5 2010-04-01 2010-04-01 false Labeling of permitted combinations of anorectal active ingredients. 346.52 Section 346.52 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT...

  18. 21 CFR 346.52 - Labeling of permitted combinations of anorectal active ingredients.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... OTC drug monograph. ... 21 Food and Drugs 5 2011-04-01 2011-04-01 false Labeling of permitted combinations of anorectal active ingredients. 346.52 Section 346.52 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT...

  19. Rapid quantitative assessment of phagocytic activity of Indium-111 labeled leukocytes by chemiluminescence

    SciTech Connect

    Juni, J.E.; Petry, N.; Wahl, R.L.; Geatti, O.

    1985-05-01

    Indium-111 labeled leukocyte imaging is gaining widespread acceptance. A rapid method for assaying changes in leukocyte viability and phagocytic function during the labeling process would facilitate the evaluation of new labeling techniques and testing of labeled cells before pt injection. The authors have conducted preliminary investigations of chemiluminescence in the clinical evaluation of leukocyte labeling. The chemiluminescence assay may be performed in 30 minutes with only 0.1 ml of whole blood. Zymossan is rapidly introduced to the blood or cell suspension resulting in the emission of light which is then counted by photometer. The amount of light given off by the reaction reflects both the phagocytic function of the cells and the ability of activated phagocytes to generate activated oxygen species. They have evaluated the chemiluminescent activity of normal human leukocyte suspensions both before and after labeling with Indium-111 oxine. The chemiluminescence assay provides a rapid means of evaluating granulocyte function. Correlations of this activity with image quality may provides clues for optimization of labeling techniques.

  20. (/sup 3/H)Spiroxatrine labels a serotonin/sub 1A/-like site in the rat hippocampus

    SciTech Connect

    Nelson, D.L.; Monroe, P.J.; Lambert, G.; Yamamura, H.I.

    1987-09-28

    (/sup 3/H)Spiroxatrine was examined as a potential ligand for the labeling of 5-HT/sub 1A/ sites in the rat hippocampus. Analysis o the binding of (/sup 3/H)spiroxatrine in the absence and presence of varying concentrations of three monoamine neurotransmitters revealed that serotonin (5-HT) had high affinity for the (/sup 3/H)spiroxatrine binding sites, consistent with the labeling of 5-HT/sub 1/ sites, while dopamine and norepinephrine had very low affinity. Saturation studies of the binding of (/sup 3/H)spiroxatrine revealed a single population of sites with a K/sub d/ = 2.21 nM. Further pharmacologic characterization with the 5-HT/sub 1A/ ligands 8-hydroxy-2-(di-ni-propylamino)tetralin, ipsapirone, and WB4101 and the butyrophenone compounds spiperone and haloperidol gave results that were consistent with (/sup 3/H)spiroxatrine labeling 5-HT/sub 1A/ sites. This ligand produced stable, reproducible binding with a good ratio of specific to nonspecific binding. The binding of (/sup 3/H)spiroxatrine was sensitive to GTP, suggesting that this ligand may act as an agonist. 21 references, 5 figures, 2 tables.

  1. Myosin binding surface on actin probed by hydroxyl radical footprinting and site-directed labels.

    PubMed

    Oztug Durer, Zeynep A; Kamal, J K Amisha; Benchaar, Sabrina; Chance, Mark R; Reisler, Emil

    2011-11-25

    Actin and myosin are the two main proteins required for cell motility and muscle contraction. The structure of their strongly bound complex-rigor state-is a key for delineating the functional mechanism of actomyosin motor. Current knowledge of that complex is based on models obtained from the docking of known atomic structures of actin and myosin subfragment 1 (S1; the head and neck region of myosin) into low-resolution electron microscopy electron density maps, which precludes atomic- or side-chain-level information. Here, we use radiolytic protein footprinting for global mapping of sites across the actin molecules that are impacted directly or allosterically by myosin binding to actin filaments. Fluorescence and electron paramagnetic resonance spectroscopies and cysteine actin mutants are used for independent, residue-specific probing of S1 effects on two structural elements of actin. We identify actin residue candidates involved in S1 binding and provide experimental evidence to discriminate between the regions of hydrophobic and electrostatic interactions. Focusing on the role of the DNase I binding loop (D-loop) and the W-loop residues of actin in their interactions with S1, we found that the emission properties of acrylodan and the mobility of electron paramagnetic resonance spin labels attached to cysteine mutants of these residues change strongly and in a residue-specific manner upon S1 binding, consistent with the recently proposed direct contacts of these loops with S1. As documented in this study, the direct and indirect changes on actin induced by myosin are more extensive than known until now and attest to the importance of actin dynamics to actomyosin function. PMID:21986200

  2. Pulsed EPR Spectroscopy of 33S-Labeled Molybdenum Cofactor in Catalytically Active Bioengineered Sulfite Oxidase

    PubMed Central

    Klein, Eric L.; Belaidi, Abdel Ali; Raitsimring, Arnold M.; Davis, Amanda C.; Krämer, Tobias; Astashkin, Andrei V.; Neese, Frank; Schwarz, Günter; Enemark, John H.

    2014-01-01

    Molybdenum enzymes contain at least one pyranopterin dithiolate (molybdopterin, MPT) moiety that coordinates Mo through two dithiolate (dithiolene) sulfur atoms. For sulfite oxidase (SO), hyperfine interactions (hfi) and nuclear quadrupole interactions (nqi) of magnetic nuclei (I ≠ 0) near the Mo(V) (d1) center have been measured using high-resolution pulsed electron paramagnetic resonance (EPR) methods and interpreted with the help of the density functional theory (DFT) calculations. These have provided important insights about the active site structure and the reaction mechanism of the enzyme. However, it has not been possible to use EPR to probe the dithiolene sulfurs directly since naturally abundant 32S has no nuclear spin (I = 0). Here we describe direct incorporation of 33S (I = 3/2), the only stable magnetic sulfur isotope, into MPT using controlled in vitro synthesis with purified proteins. The electron spin echo envelope modulation (ESEEM) spectra from 33S-labeled MPT in this catalytically active SO variant are dominated by the ‘inter-doublet’ transition arising from the strong nuclear quadrupole interaction, as also occurs for the 33S-labeled exchangeable equatorial sulfite ligand [Klein, E. L., et al., Inorg. Chem. 2012, 51, 1408 – 1418]. The estimated experimental hfi and nqi parameters for 33S (aiso = 3 MHz and e2Qq/h = 25 MHz) are in good agreement with those predicted by DFT. In addition, the DFT calculations show that the two 33S atoms are indistinguishable by EPR and reveal a strong intermixing between their out-of-plane pz orbitals and the dxy orbital of Mo(V). PMID:24387640

  3. Dense small molecule labeling enables activator-dependent STORM by proximity mapping.

    PubMed

    Chen, Ye; Gu, Min; Gunning, Peter W; Russell, Sarah M

    2016-09-01

    Stochastic optical reconstruction microscopy (STORM) enables high-resolution imaging, but multi-channel 3D imaging is problematic because of chromatic aberrations and alignment errors. The use of activator-dependent STORM in which spectrally distinct activators can be coupled with a single reporter can circumvent such issues. However, the standard approach of linking activators and reporters to a single antibody molecule is hampered by low labeling density and the large size of the antibody. We proposed that small molecule labels might enable activator-dependent STORM if the reporter or activator were linked to separate small molecules that bound within 3.5 nm of each other. This would greatly increase the labeling density and therefore improve resolution. We tested various mixtures of phalloidin- or mCling-conjugated fluorophore to demonstrate this feasibility. The specific activation was dependent on the choice of activator, its density, a matching activating laser and its power. In addition to providing an effective means of multi-channel 3D STORM imaging, this method also provides information about the local proximity between labels, potentially enabling super-resolved mapping of the conformation of the labeled structures. PMID:27246003

  4. Dense small molecule labeling enables activator-dependent STORM by proximity mapping.

    PubMed

    Chen, Ye; Gu, Min; Gunning, Peter W; Russell, Sarah M

    2016-09-01

    Stochastic optical reconstruction microscopy (STORM) enables high-resolution imaging, but multi-channel 3D imaging is problematic because of chromatic aberrations and alignment errors. The use of activator-dependent STORM in which spectrally distinct activators can be coupled with a single reporter can circumvent such issues. However, the standard approach of linking activators and reporters to a single antibody molecule is hampered by low labeling density and the large size of the antibody. We proposed that small molecule labels might enable activator-dependent STORM if the reporter or activator were linked to separate small molecules that bound within 3.5 nm of each other. This would greatly increase the labeling density and therefore improve resolution. We tested various mixtures of phalloidin- or mCling-conjugated fluorophore to demonstrate this feasibility. The specific activation was dependent on the choice of activator, its density, a matching activating laser and its power. In addition to providing an effective means of multi-channel 3D STORM imaging, this method also provides information about the local proximity between labels, potentially enabling super-resolved mapping of the conformation of the labeled structures.

  5. Active site of tripeptidyl peptidase II from human erythrocytes is of the subtilisin type.

    PubMed Central

    Tomkinson, B; Wernstedt, C; Hellman, U; Zetterqvist, O

    1987-01-01

    The present report presents evidence that the amino acid sequence around the serine of the active site of human tripeptidyl peptidase II is of the subtilisin type. The enzyme from human erythrocytes was covalently labeled at its active site with [3H]diisopropyl fluorophosphate, and the protein was subsequently reduced, alkylated, and digested with trypsin. The labeled tryptic peptides were purified by gel filtration and repeated reversed-phase HPLC, and their amino-terminal sequences were determined. Residue 9 contained the radioactive label and was, therefore, considered to be the active serine residue. The primary structure of the part of the active site (residues 1-10) containing this residue was concluded to be Xaa-Thr-Gln-Leu-Met-Asx-Gly-Thr-Ser-Met. This amino acid sequence is homologous to the sequence surrounding the active serine of the microbial peptidases subtilisin and thermitase. These data demonstrate that human tripeptidyl peptidase II represents a potentially distinct class of human peptidases and raise the question of an evolutionary relationship between the active site of a mammalian peptidase and that of the subtilisin family of serine peptidases. PMID:3313395

  6. Active-Q: Validation of the Web-Based Physical Activity Questionnaire Using Doubly Labeled Water

    PubMed Central

    Trolle Lagerros, Ylva; Christensen, Sara Elisabeth; Möller, Elisabeth; Wright, Antony; Sjölander, Arvid; Bälter, Katarina

    2012-01-01

    Background Increased use of the Internet provides new opportunities for collecting data in large studies. The aim of our new Web-based questionnaire, Active-Q, is to assess total physical activity and inactivity in adults. Active-Q assesses habitual activity during the past year via questions in four different domains: (1) daily occupation, (2) transportation to and from daily occupation, (3) leisure time activities, and (4) sporting activities. Objective The objective of our study is to validate Active-Q’s energy expenditure estimates using the doubly labeled water (DLW) method, and to assess the reproducibility of Active-Q by comparing the results of the questionnaire completed by the same group on two occasions. Methods The validity and reproducibility of Active-Q were assessed in a group of 37 individuals, aged 20 to 65 years. Active-Q was distributed via email to the participants. The total energy expenditure of the participants was assessed using DLW for 11 consecutive days. Results The median time to complete Active-Q was 6.1 minutes. The majority of participants (27/37, 73%) reported that the questionnaire was “easy” or “very easy” to answer. On average, Active-Q overestimated the total daily energy expenditure by 440 kJ compared with the DLW. The Spearman correlation between the two methods was r = 0.52 (P < .001). The intraclass correlation coefficient for total energy expenditure between the results of Active-Q completed on two occasions was 0.83 (95% CI 0.73-0.93). Conclusions Active-Q is a valid and reproducible method of assessing total energy expenditure. It is also a user-friendly method and suitable for Web-based data collection in large epidemiological studies. PMID:22356755

  7. Selective labeling of serotonin uptake sites in rat brain by (/sup 3/H)citalopram contrasted to labeling of multiple sites by (/sup 3/H)imipramine

    SciTech Connect

    D'Amato, R.J.; Largent, B.L.; Snowman, A.M.; Snyder, S.H.

    1987-07-01

    Citalopram is a potent and selective inhibitor of neuronal serotonin uptake. In rat brain membranes (/sup 3/H)citalopram demonstrates saturable and reversible binding with a KD of 0.8 nM and a maximal number of binding sites (Bmax) of 570 fmol/mg of protein. The drug specificity for (/sup 3/H)citalopram binding and synaptosomal serotonin uptake are closely correlated. Inhibition of (/sup 3/H)citalopram binding by both serotonin and imipramine is consistent with a competitive interaction in both equilibrium and kinetic analyses. The autoradiographic pattern of (/sup 3/H)citalopram binding sites closely resembles the distribution of serotonin. By contrast, detailed equilibrium-saturation analysis of (/sup 3/H)imipramine binding reveals two binding components, i.e., high affinity (KD = 9 nM, Bmax = 420 fmol/mg of protein) and low affinity (KD = 553 nM, Bmax = 8560 fmol/mg of protein) sites. Specific (/sup 3/H)imipramine binding, defined as the binding inhibited by 100 microM desipramine, is displaced only partially by serotonin. Various studies reveal that the serotonin-sensitive portion of binding corresponds to the high affinity sites of (/sup 3/H)imipramine binding whereas the serotonin-insensitive binding corresponds to the low affinity sites. Lesioning of serotonin neurons with p-chloroamphetamine causes a large decrease in (/sup 3/H)citalopram and serotonin-sensitive (/sup 3/H)imipramine binding with only a small effect on serotonin-insensitive (/sup 3/H)imipramine binding. The dissociation rate of (/sup 3/H)imipramine or (/sup 3/H)citalopram is not altered by citalopram, imipramine or serotonin up to concentrations of 10 microM. The regional distribution of serotonin sensitive (/sup 3/H)imipramine high affinity binding sites closely resembles that of (/sup 3/H)citalopram binding.

  8. Disclosure Pattern of Self-Labeled People Living with HIV/AIDS on Chinese Social Networking Site: An Exploratory Study.

    PubMed

    Han, Jin; Tian, Xianyun; Yu, Guang; He, Fang

    2016-08-01

    HIV/AIDS is an important public health issue in China. The number of people living with HIV/AIDS (PLWHA) has been increasing since the introduction of highly active antiretroviral therapy. PLWHA's life quality is becoming an important issue, with lack of research in China. In this study, a group of PLWHA (n = 663) was identified using HIV/AIDS relevant usernames on a Chinese social networking site (Weibo) to study their daily living situations. We found that more than 99.10% of PLWHA were male, among whom 90.80% turned out to be homosexual. They had significantly more fans and followees, but fewer postings compared to the general population. The mean age of the PLWHA was 28.96 (SD = 5.05) years old, and southwest and northwest China had a high prevalence of HIV/AIDS. In addition, PLWHA's postings were coded and we found that more than half of the postings (n = 769, 51.03%) contained strong emotions. Less than one-fifth of the postings were directly related to HIV/AIDS topics (n = 269, 17.85%), while seeking emotional support, such as looking for stable partnership, was ranked as the first priority of support seeking. In summary, we found that the majority of the self-labeled PLWHA were likely to be men who have sex with men. They used Weibo to share their daily life events and seek emotional support. Implications for promoting HIV/AIDS education and prevention through Chinese social networking sites were also discussed. PMID:27494331

  9. Potential Effect of Physical Activity Calorie Equivalent Labeling on Parent Fast Food Decisions

    PubMed Central

    Antonelli, Ray

    2015-01-01

    OBJECTIVES: Menu labels displaying food energy in physical activity calorie equivalents (PACE) is a possible strategy to encourage ordering meals with fewer calories and promoting physical activity. Potential effects of such labeling for children have never been examined. METHODS: We conducted a national survey of 1000 parents randomized to 1 of 4 fast food menus: no labels, calories only, calories plus minutes, or calories plus miles needed to walk to burn the calories. Respondents were asked to imagine they were in a fast food restaurant and place an order for their child. At the survey’s conclusion, all respondents were shown a calorie-only label and both PACE labels and asked to rate the likelihood each label would influence them to encourage their child to exercise. RESULTS: We excluded respondents whose meals totaled 0 calories or >4000 calories, leaving 823 parents in the analysis. The mean age of the child for whom the meal was “ordered” was 9.5 years. Parents whose menus displayed no label ordered an average of 1294 calories, whereas those shown calories only, calories plus minutes, or calories plus miles ordered 1066, 1060, and 1099 calories, respectively (P = .0001). Only 20% of parents reported that calories-only labeling would be “very likely” to prompt them to encourage their children to exercise versus 38% for calories plus minutes (P < .0001) and 37% for calories plus miles (P < .0001). CONCLUSIONS: PACE labeling may influence parents’ decisions on what fast food items to order for their children and encourage them to get their children to exercise. PMID:25624379

  10. Site-specific labeling of RNA by combining genetic alphabet expansion transcription and copper-free click chemistry.

    PubMed

    Someya, Tatsuhiko; Ando, Ami; Kimoto, Michiko; Hirao, Ichiro

    2015-08-18

    Site-specific labeling of long-chain RNAs with desired molecular probes is an imperative technique to facilitate studies of functional RNA molecules. By genetic alphabet expansion using an artificial third base pair, called an unnatural base pair, we present a post-transcriptional modification method for RNA transcripts containing an incorporated azide-linked unnatural base at specific positions, using a copper-free click reaction. The unnatural base pair between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa) functions in transcription. Thus, we chemically synthesized a triphosphate substrate of 4-(4-azidopentyl)-pyrrole-2-carbaldehyde (N3-PaTP), which can be site-specifically introduced into RNA, opposite Ds in templates by T7 transcription. The N3-Pa incorporated in the transcripts was modified with dibenzocyclooctyne (DIBO) derivatives. We demonstrated the transcription of 17-, 76- and 260-mer RNA molecules and their site-specific labeling with Alexa 488, Alexa 594 and biotin. This method will be useful for preparing RNA molecules labeled with any functional groups of interest, toward in vivo experiments.

  11. Validation of binding of SE-75 labeled sucralfate to sites of gastrointestinal ulceration

    SciTech Connect

    Maurer, A.H.; Knight, L.C.; Kollman, M.; Krevsky, B.; Pleet, D.; D'Ercole, F.; Siegel, J.A.; Fisher, R.S.; Malmud, L.S.

    1985-05-01

    This study was performed to determine if and for how long sucralfate (SU) binds selectively to sites of gastro-intestinal (GI) ulceration. Se-Su was prepared by sulfating sucrose with tracer Se-75 and precipitating it as the basic Al salt. All patients (pts) had endoscopy to confirm the presence of either: esophagitis (n=5), gastritis (GA) (n=5), gastric ulcers (GU) (n=5), duodenal ulcers (DU) (n=5), or no ulceration (NU) (n=5). Following an overnight fast the pts swallowed 1 gm with 100 ..mu..Ci of Se-SU and were imaged continuously over 24 hours or until no activity remained in the upper GI tract. Pts with GU visually demonstrated focal SU binding at the ulcers for an average of 3.9 +- 1.1 hrs. with a mean GET of 68 +- 25 min. Mean GET for pts with DU was prolonged, 171 +- 63 min, however focal binding at duodenal ulcers was not seen. All pts with GA had diffuse retention of SU in the stomach with a mean GET of 118 +- 34 min. Focal binding of SU at all sites of esophagitis was seen with a T-1/2 of 65 +- 32 min at the ulcerations. In conclusion these data support the theory that the mechanism of ulcer healing with SU is related to its ability to adhere to the ulcer site forming a protective barrier. In addition Se-SU is a potential ulcer imaging agent which can be used to noninvasively assess healing.

  12. Active Sites Environmental Monitoring Program: Action levels

    SciTech Connect

    Ashwood, J.S.; Ashwood, T.L.

    1991-10-01

    The Active Sites Environmental Monitoring Program (ASEMP) was established at Oak Ridge National Laboratory to provide for early leak detection and to monitor performance of the active low-level waste disposal facilities in Solid Waste Storage Area (SWSA) 6 and the transuranic waste storage areas in SWSA 5 North. Early leak detection is accomplished by sampling runoff, groundwater, and perched water in burial trenches. Sample results are compared to action levels that represent background contamination by naturally occurring and fallout-derived radionuclides. 15 refs., 3 figs., 12 tabs.

  13. Mapping membrane protein backbone dynamics: a comparison of site-directed spin labeling with NMR 15N-relaxation measurements.

    PubMed

    Lo, Ryan H; Kroncke, Brett M; Solomon, Tsega L; Columbus, Linda

    2014-10-01

    The ability to detect nanosecond backbone dynamics with site-directed spin labeling (SDSL) in soluble proteins has been well established. However, for membrane proteins, the nitroxide appears to have more interactions with the protein surface, potentially hindering the sensitivity to backbone motions. To determine whether membrane protein backbone dynamics could be mapped with SDSL, a nitroxide was introduced at 55 independent sites in a model polytopic membrane protein, TM0026. Electron paramagnetic resonance spectral parameters were compared with NMR (15)N-relaxation data. Sequential scans revealed backbone dynamics with the same trends observed for the R1 relaxation rate, suggesting that nitroxide dynamics remain coupled to the backbone on membrane proteins.

  14. Dual Mode Fluorophore-Doped Nickel Nitrilotriacetic Acid-Modified Silica Nanoparticles Combine Histidine-Tagged Protein Purification with Site-Specific Fluorophore Labeling

    PubMed Central

    Kim, Sung Hoon; Jeyakumar, M.; Katzenellenbogen, John A.

    2008-01-01

    We present the first example of a fluorophore-doped nickel chelate surface- modified silica nanoparticle that functions in a dual mode, combining histidine-tagged protein purification with site-specific fluorophore labeling. Tetramethylrhodamine (TMR)-doped silica nanoparticles, estimated to contain 700–900 TMRs per ca. 23-nm particle, were surface modified with nitrilotriacetic acid (NTA), producing TMR-SiO2-NTA-Ni+2. Silica-embedded TMR retains very high quantum yield, is resistant to quenching by buffer components and is modestly quenched and only to a certain depth (ca. 2 nm) by surface-attached Ni+2. When exposed to a bacterial lysate containing estrogen receptor α ligand binding domain (ERα) as a minor component, these beads showed very high specificity binding, enabling protein purification in one step. The capacity and specificity of these beads for binding a his-tagged protein were characterized by electrophoresis, radiometric counting, and MALDI-TOF MS. ERα, bound to TMR-SiO2-NTA-Ni++ beads in a site-specific manner, exhibited good activity for ligand binding and for ligand-induced binding to coactivators in solution FRET experiments and protein microarray fluorometric and FRET assays. This dual-mode type TMR-SiO2-NTA-Ni++ system represents a powerful combination of one-step histidine-tagged protein purification and site-specific labeling with multiple fluorophore species. BRIEFS Tetramethylrhodamine-doped silica nanoparticles surface modified with nitrilotriacetic acid are dual-mode agents that can be used to purify and site-specifically fluorophore label his-tagged proteins in one step for fluorometric and FRET experiments. PMID:17910454

  15. Characterization of an active, fluorescein-labelled kinesin.

    PubMed

    Marya, P K; Fraylich, P E; Eagles, P A

    1990-10-01

    Kinesin was isolated from bovine intradural nerve roots and conjugated with 5-(iodoacetamido)fluorescein. The modified kinesin (AF-kinesin) supports the movement of organelles along microtubules at rates comparable with those obtained using unmodified kinesin. AF-kinesin was purified by high-performance liquid chromatography. SDS/PAGE analysis of the purified fraction showed the presence of a fluorescent band at the position of the 125-kDa kinesin heavy chain. This protein promoted microtubule gliding with MgATP and with MgGTP at rates comparable to those of unlabelled kinesin. AF-kinesin had a fluorescein/protein ratio of one. Video microscopy at low light levels was used to monitor the interactions between the analogue and microtubules. AF-kinesin binds to microtubules in the presence of adenosine 5'-[beta, gamma-imino]triphosphate or ADP. Brief incubation of the microtubule. AF-kinesin complex with 10 mM ATP or GTP completely removes the labelled molecule. AF-kinesin can be inactivated in its ability to cause microtubule gliding by irradiating it with light that bleaches the bound fluorophore. When the protein is damaged in this way it still binds to microtubules and does so in the presence of ATP. PMID:2146115

  16. Comparison of doubly labeled water with respirometry at low- and high-activity levels

    SciTech Connect

    Westerterp, K.R.; Brouns, F.; Saris, W.H.; ten Hoor, F.

    1988-07-01

    In previous studies the doubly labeled water method for measuring energy expenditure in free-living humans has been validated against respirometry under sedentary conditions. In the present investigation, energy expenditure is measured simultaneously with doubly labeled water and respirometry at low- and high-activity levels. Over 6 days, five subjects were measured doing mainly sedentary activities like desk work; their average daily metabolic rate was 1.40 +/- 0.09 (SD) times sleeping metabolic rate. Four subjects were measured twice over 3.5 days, including 2 days with heavy bicycle ergometer work, resulting in an average daily metabolic rate of 2.61 +/- 0.25 (SD) times sleeping metabolic rate. At the low-activity level, energy expenditures from the doubly labeled water method were on the average 1.4 +/- 3.9% (SD) larger than those from respirometry. At the high-activity level, the doubly labeled water method yielded values that were 1.0 +/- 7.0% (SD) lower than those from respirometry. Results demonstrate the utility of the doubly labeled water method for the determination of energy expenditure in the range of activity levels in daily life.

  17. An Efficient Site-Specific Method for Irreversible Covalent Labeling of Proteins with a Fluorophore

    PubMed Central

    Liu, Jiaquan; Hanne, Jeungphill; Britton, Brooke M.; Shoffner, Matthew; Albers, Aaron E.; Bennett, Jared; Zatezalo, Rachel; Barfield, Robyn; Rabuka, David; Lee, Jong-Bong; Fishel, Richard

    2015-01-01

    Fluorophore labeling of proteins while preserving native functions is essential for bulk Förster resonance energy transfer (FRET) interaction and single molecule imaging analysis. Here we describe a versatile, efficient, specific, irreversible, gentle and low-cost method for labeling proteins with fluorophores that appears substantially more robust than a similar but chemically distinct procedure. The method employs the controlled enzymatic conversion of a central Cys to a reactive formylglycine (fGly) aldehyde within a six amino acid Formylglycine Generating Enzyme (FGE) recognition sequence in vitro. The fluorophore is then irreversibly linked to the fGly residue using a Hydrazinyl-Iso-Pictet-Spengler (HIPS) ligation reaction. We demonstrate the robust large-scale fluorophore labeling and purification of E.coli (Ec) mismatch repair (MMR) components. Fluorophore labeling did not alter the native functions of these MMR proteins in vitro or in singulo. Because the FGE recognition sequence is easily portable, FGE-HIPS fluorophore-labeling may be easily extended to other proteins. PMID:26582263

  18. An Efficient Site-Specific Method for Irreversible Covalent Labeling of Proteins with a Fluorophore.

    PubMed

    Liu, Jiaquan; Hanne, Jeungphill; Britton, Brooke M; Shoffner, Matthew; Albers, Aaron E; Bennett, Jared; Zatezalo, Rachel; Barfield, Robyn; Rabuka, David; Lee, Jong-Bong; Fishel, Richard

    2015-01-01

    Fluorophore labeling of proteins while preserving native functions is essential for bulk Förster resonance energy transfer (FRET) interaction and single molecule imaging analysis. Here we describe a versatile, efficient, specific, irreversible, gentle and low-cost method for labeling proteins with fluorophores that appears substantially more robust than a similar but chemically distinct procedure. The method employs the controlled enzymatic conversion of a central Cys to a reactive formylglycine (fGly) aldehyde within a six amino acid Formylglycine Generating Enzyme (FGE) recognition sequence in vitro. The fluorophore is then irreversibly linked to the fGly residue using a Hydrazinyl-Iso-Pictet-Spengler (HIPS) ligation reaction. We demonstrate the robust large-scale fluorophore labeling and purification of E.coli (Ec) mismatch repair (MMR) components. Fluorophore labeling did not alter the native functions of these MMR proteins in vitro or in singulo. Because the FGE recognition sequence is easily portable, FGE-HIPS fluorophore-labeling may be easily extended to other proteins. PMID:26582263

  19. Characterization of active sites in zeolite catalysts

    SciTech Connect

    Eckert, J.; Bug, A.; Nicol, J.M.

    1997-11-01

    This is the final report of a three-year, Laboratory Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). Atomic-level details of the interaction of adsorbed molecules with active sites in catalysts are urgently needed to facilitate development of more effective and/or environmentally benign catalysts. To this end the authors have carried out neutron scattering studies combined with theoretical calculations of the dynamics of small molecules inside the cavities of zeolite catalysts. The authors have developed the use of H{sub 2} as a probe of adsorption sites by observing the hindered rotations of the adsorbed H{sub 2} molecule, and they were able to show that an area near the four-rings is the most likely adsorption site for H{sub 2} in zeolite A while adsorption of H{sub 2} near cations located on six-ring sites decreases in strength as Ni {approximately} Co > Ca > Zn {approximately} Na. Vibrational and rotational motions of ethylene and cyclopropane adsorption complexes were used as a measure for zeolite-adsorbate interactions. Preliminary studies of the binding of water, ammonia, and methylamines were carried out in a number of related guest-host materials.

  20. Identification of the active-site serine in human lecithin: cholesterol acyltransferase

    SciTech Connect

    Farooqui, J.; Wohl, R.C.; Kezdy, F.J.; Scanu, A.M.

    1987-05-01

    Lecithin:cholesterol acyltransferase (LCAT) from human plasma reacts stoichiometrically with diisopropylphosphorofluoridate (DFP) resulting in the complete loss of transacylase activity. Purified LCAT was covalently labeled with (TH) DFP and the labeled protein was reduced and carboxymethylated. Cyanogen bromide cleavage followed by gel permeation chromatography yielded a peptide of 4-5 KDa (LCAT CNBr-III) containing most of the radioactive label. Preliminary studies comparing the amino acid composition of the LCAT-CNBr-III with the sequence of LCAT indicate that this peptide corresponds to fragment 168-220. Automated Edman degradation of the radioactive peptide recovered a radioactive PTC-amino acid at cycle 14. Of all predicted CNBr fragments only peptide 168-220 contained a serine at residue 14 from the amino terminus of the peptide. The authors conclude that serine 181 is the active site serine of LCAT.

  1. Ligand-free palladium-mediated site-specific protein labeling inside gram-negative bacterial pathogens.

    PubMed

    Li, Jie; Lin, Shixian; Wang, Jie; Jia, Shang; Yang, Maiyun; Hao, Ziyang; Zhang, Xiaoyu; Chen, Peng R

    2013-05-15

    Palladium, a key transition metal in advancing modern organic synthesis, mediates diverse chemical conversions including many carbon-carbon bond formation reactions between organic compounds. However, expanding palladium chemistry for conjugation of biomolecules such as proteins, particularly within their native cellular context, is still in its infancy. Here we report the site-specific protein labeling inside pathogenic Gram-negative bacterial cells via a ligand-free palladium-mediated cross-coupling reaction. Two rationally designed pyrrolysine analogues bearing an aliphatic alkyne or an iodophenyl handle were first encoded in different enteric bacteria, which offered two facial handles for palladium-mediated Sonogashira coupling reaction on proteins within these pathogens. A GFP-based bioorthogonal reaction screening system was then developed, allowing evaluation of both the efficiency and the biocompatibilty of various palladium reagents in promoting protein-small molecule conjugation. The identified simple compound-Pd(NO3)2 exhibited high efficiency and biocompatibility for site-specific labeling of proteins in vitro and inside living E. coli cells. This Pd-mediated protein coupling method was further utilized to label and visualize a Type-III Secretion (T3S) toxin-OspF in Shigella cells. Our strategy may be generally applicable for imaging and tracking various virulence proteins within Gram-negative bacterial pathogens.

  2. Activity Recognition Using Community Data to Complement Small Amounts of Labeled Instances †

    PubMed Central

    Garcia-Ceja, Enrique; Brena, Ramon F.

    2016-01-01

    Human Activity Recognition (HAR) is an important part of ambient intelligence systems since it can provide user-context information, thus allowing a greater personalization of services. One of the problems with HAR systems is that the labeling process for the training data is costly, which has hindered its practical application. A common approach is to train a general model with the aggregated data from all users. The problem is that for a new target user, this model can perform poorly because it is biased towards the majority type of users and does not take into account the particular characteristics of the target user. To overcome this limitation, a user-dependent model can be trained with data only from the target user that will be optimal for this particular user; however, this requires a considerable amount of labeled data, which is cumbersome to obtain. In this work, we propose a method to build a personalized model for a given target user that does not require large amounts of labeled data. Our method uses data already labeled by a community of users to complement the scarce labeled data of the target user. Our results showed that the personalized model outperformed the general and the user-dependent models when labeled data is scarce. PMID:27314355

  3. FDA cigarette warning labels lower craving and elicit frontoinsular activation in adolescent smokers

    PubMed Central

    Do, Kathy T.

    2015-01-01

    Cigarette smoking is an economically and epidemiologically expensive public health concern. Most adult smokers become addicted during adolescence, rendering it a crucial period for prevention and intervention. Although litigation claims have delayed implementation, graphic warning labels proposed by the U.S. Food and Drug Administration (FDA) may be a promising way to achieve this goal. We aimed to determine the efficacy of the labels in reducing in-scanner craving and to characterize the neurobiological responses in adolescent and adult smokers and non-smokers. While undergoing functional magnetic resonance imaging, thirty-nine 13- to 18-year-old adolescent and forty-one 25- to 30-year-old adult smokers and non-smokers rated their desire to smoke when presented with emotionally graphic warning labels and comparison non-graphic labels. Compared with adult smokers, adolescent smokers exhibited greater craving reduction in response to the warning labels. Although smokers evinced overall blunted recruitment of insula and dorsolateral prefrontal cortex (DLPFC) relative to non-smokers, an effect that was stronger in adolescent smokers, parametrically increasing activation of these regions was associated with greater craving reduction. Functional connectivity analyses suggest that greater DLPFC regulation of limbic regions predicted cigarette craving. These data underscore a prominent role of frontoinsular circuitry in predicting the efficacy of FDA graphic warning labels in craving reduction in adult and adolescent smokers. PMID:25887154

  4. FDA cigarette warning labels lower craving and elicit frontoinsular activation in adolescent smokers.

    PubMed

    Do, Kathy T; Galván, Adriana

    2015-11-01

    Cigarette smoking is an economically and epidemiologically expensive public health concern. Most adult smokers become addicted during adolescence, rendering it a crucial period for prevention and intervention. Although litigation claims have delayed implementation, graphic warning labels proposed by the U.S. Food and Drug Administration (FDA) may be a promising way to achieve this goal. We aimed to determine the efficacy of the labels in reducing in-scanner craving and to characterize the neurobiological responses in adolescent and adult smokers and non-smokers. While undergoing functional magnetic resonance imaging, thirty-nine 13- to 18-year-old adolescent and forty-one 25- to 30-year-old adult smokers and non-smokers rated their desire to smoke when presented with emotionally graphic warning labels and comparison non-graphic labels. Compared with adult smokers, adolescent smokers exhibited greater craving reduction in response to the warning labels. Although smokers evinced overall blunted recruitment of insula and dorsolateral prefrontal cortex (DLPFC) relative to non-smokers, an effect that was stronger in adolescent smokers, parametrically increasing activation of these regions was associated with greater craving reduction. Functional connectivity analyses suggest that greater DLPFC regulation of limbic regions predicted cigarette craving. These data underscore a prominent role of frontoinsular circuitry in predicting the efficacy of FDA graphic warning labels in craving reduction in adult and adolescent smokers.

  5. FDA cigarette warning labels lower craving and elicit frontoinsular activation in adolescent smokers.

    PubMed

    Do, Kathy T; Galván, Adriana

    2015-11-01

    Cigarette smoking is an economically and epidemiologically expensive public health concern. Most adult smokers become addicted during adolescence, rendering it a crucial period for prevention and intervention. Although litigation claims have delayed implementation, graphic warning labels proposed by the U.S. Food and Drug Administration (FDA) may be a promising way to achieve this goal. We aimed to determine the efficacy of the labels in reducing in-scanner craving and to characterize the neurobiological responses in adolescent and adult smokers and non-smokers. While undergoing functional magnetic resonance imaging, thirty-nine 13- to 18-year-old adolescent and forty-one 25- to 30-year-old adult smokers and non-smokers rated their desire to smoke when presented with emotionally graphic warning labels and comparison non-graphic labels. Compared with adult smokers, adolescent smokers exhibited greater craving reduction in response to the warning labels. Although smokers evinced overall blunted recruitment of insula and dorsolateral prefrontal cortex (DLPFC) relative to non-smokers, an effect that was stronger in adolescent smokers, parametrically increasing activation of these regions was associated with greater craving reduction. Functional connectivity analyses suggest that greater DLPFC regulation of limbic regions predicted cigarette craving. These data underscore a prominent role of frontoinsular circuitry in predicting the efficacy of FDA graphic warning labels in craving reduction in adult and adolescent smokers. PMID:25887154

  6. Motions of the Substrate Recognition Duplex in a Group I Intron Assessed by Site-Directed Spin Labeling

    SciTech Connect

    Grant, Gian Paola G; Boyd, Nathan; Herschlag, Daniel; Qin, Peter Z

    2009-03-11

    The Tetrahymena group I intron recognizes its oligonucleotide substrate in a two-step process. First, a substrate recognition duplex, called the P1 duplex, is formed. The P1 duplex then docks into the prefolded ribozyme core by forming tertiary contacts. P1 docking controls both the rate and the fidelity of substrate cleavage and has been extensively studied as a model for the formation of RNA tertiary structure. However, previous work has been limited to studying millisecond or slower motions. Here we investigated nanosecond P1 motions in the context of the ribozyme using site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy. A nitroxide spin label (R5a) was covalently attached to a specific site of the substrate oligonucleotide, the labeled substrate was bound to a prefolded ribozyme to form the P1 duplex, and X-band EPR spectroscopy was used to monitor nitroxide motions in the 0.1-50 ns regime. Using substrates that favor the docked or the undocked states, it was established that R5a was capable of reporting P1 duplex motions. Using R5a-labeled substrates it was found that the J1/2 junction connecting P1 to the ribozyme core controls nanosecond P1 mobility in the undocked state. This may account for previous observations that J1/2 mutations weaken substrate binding and give rise to cryptic cleavage. This study establishes the use of SDSL to probe nanosecond dynamic behaviors of individual helices within large RNA and RNA/protein complexes. This approach may help in understanding the relationship between RNA structure, dynamics, and function.

  7. Site-Specifically Labeled Immunoconjugates for Molecular Imaging--Part 2: Peptide Tags and Unnatural Amino Acids.

    PubMed

    Adumeau, Pierre; Sharma, Sai Kiran; Brent, Colleen; Zeglis, Brian M

    2016-04-01

    Molecular imaging using radioisotope- or fluorophore-labeled antibodies is increasingly becoming a critical component of modern precision medicine. Yet despite this promise, the vast majority of these immunoconjugates are synthesized via the random coupling of amine-reactive bifunctional probes to lysines within the antibody, a process that can result in heterogeneous and poorly defined constructs with suboptimal pharmacological properties. In an effort to circumvent these issues, the last 5 years have played witness to a great deal of research focused on the creation of effective strategies for the site-specific attachment of payloads to antibodies. These chemoselective modification methods yield immunoconjugates that are more homogenous and better defined than constructs created using traditional synthetic approaches. Moreover, site-specifically labeled immunoconjugates have also been shown to exhibit superior in vivo behavior compared to their randomly modified cousins. The over-arching goal of this two-part review is to provide a broad yet detailed account of the various site-specific bioconjugation approaches that have been used to create immunoconjugates for positron emission tomography (PET), single photon emission computed tomography (SPECT), and fluorescence imaging. In Part 1, we covered site-specific bioconjugation techniques based on the modification of cysteine residues and the chemoenzymatic manipulation of glycans. In Part 2, we will detail two families of bioconjugation approaches that leverage biochemical tools to achieve site-specificity. First, we will discuss modification methods that employ peptide tags either as sites for enzyme-catalyzed ligations or as radiometal coordination architectures. And second, we will examine bioconjugation strategies predicated on the incorporation of unnatural or non-canonical amino acids into antibodies via genetic engineering. Finally, we will compare the advantages and disadvantages of the modification

  8. Site-Specifically Labeled Immunoconjugates for Molecular Imaging—Part 2: Peptide Tags and Unnatural Amino Acids

    PubMed Central

    Adumeau, Pierre; Sharma, Sai Kiran; Brent, Colleen; Zeglis, Brian M.

    2016-01-01

    Molecular imaging using radioisotope- or fluorophore-labeled antibodies is increasingly becoming a critical component of modern precision medicine. Yet despite this promise, the vast majority of these immunoconjugates are synthesized via the random coupling of amine-reactive bifunctional probes to lysines within the antibody, a process that can result in heterogeneous and poorly defined constructs with suboptimal pharmacological properties. In an effort to circumvent these issues, the last 5 years have played witness to a great deal of research focused on the creation of effective strategies for the site-specific attachment of payloads to antibodies. These chemoselective modification methods yield immunoconjugates that are more homogenous and better defined than constructs created using traditional synthetic approaches. Moreover, site-specifically labeled immunoconjugates have also been shown to exhibit superior in vivo behavior compared to their randomly modified cousins. The over-arching goal of this two-part review is to provide a broad yet detailed account of the various site-specific bioconjugation approaches that have been used to create immunoconjugates for positron emission tomography (PET), single photon emission computed tomography (SPECT), and fluorescence imaging. In Part 1, we covered site-specific bioconjugation techniques based on the modification of cysteine residues and the chemoenzymatic manipulation of glycans. In Part 2, we will detail two families of bioconjugation approaches that leverage biochemical tools to achieve site-specificity. First, we will discuss modification methods that employ peptide tags either as sites for enzyme-catalyzed ligations or as radiometal coordination architectures. And second, we will examine bioconjugation strategies predicated on the incorporation of unnatural or non-canonical amino acids into antibodies via genetic engineering. Finally, we will compare the advantages and disadvantages of the modification

  9. Active Sites Environmental Monitoring Program: Program plan

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1992-02-01

    The Active Sites Environmental Monitoring Program (ASEMP), initiated in 1989, provides early detection and performance monitoring of transuranic (TRU) waste and active low-level waste (LLW) facilities at Oak Ridge National Laboratory (ORNL) in accordance with US Department of Energy (DOE) Order 5820.2A. Active LLW facilities in Solid Waste Storage Area (SWSA) 6 include Tumulus I and Tumulus II, the Interim Waste Management Facility (IWMF), LLW silos, high-range wells, asbestos silos, and fissile wells. The tumulus pads and IWMF are aboveground, high-strength concrete pads on which concrete vaults containing metal boxes of LLW are placed; the void space between the boxes and vaults is filled with grout. Eventually, these pads and vaults will be covered by an engineered multilayered cap. All other LLW facilities in SWSA 6 are below ground. In addition, this plan includes monitoring of the Hillcut Disposal Test Facility (HDTF) in SWSA 6, even though this facility was completed prior to the data of the DOE order. In SWSA 5 North, the TRU facilities include below-grade engineered caves, high-range wells, and unlined trenches. All samples from SWSA 6 are screened for alpha and beta activity, counted for gamma-emitting isotopes, and analyzed for tritium. In addition to these analytes, samples from SWSA 5 North are analyzed for specific transuranic elements.

  10. In vivo biotinylation and incorporation of a photo-inducible unnatural amino acid to an antibody-binding domain improve site-specific labeling of antibodies.

    PubMed

    Kanje, Sara; Hober, Sophia

    2015-04-01

    Antibodies are important molecules in many research fields, where they play a key role in various assays. Antibody labeling is therefore of great importance. Currently, most labeling techniques take advantage of certain amino acid side chains that commonly appear throughout proteins. This makes it hard to control the position and exact degree of labeling of each antibody. Hence, labeling of the antibody may affect the antibody-binding site. This paper presents a novel protein domain based on the IgG-binding domain C2 of streptococcal protein G, containing the unnatural amino acid BPA, that can cross-link other molecules. This novel domain can, with improved efficiency compared to previously reported similar domains, site-specifically cross-link to IgG at the Fc region. An efficient method for simultaneous in vivo incorporation of BPA and specific biotinylation in a flask cultivation of Escherichia coli is described. In comparison to a traditionally labeled antibody sample, the C2-labeled counterpart proved to have a higher proportion of functional antibodies when immobilized on a solid surface and the same limit of detection in an ELISA. This method of labeling is, due to its efficiency and simplicity, of high interest for all antibody-based assays where it is important that labeling does not interfere with the antibody-binding site.

  11. Mapping of the acetylcholine binding site of the nicotinic acetylcholine receptor: ( sup 3 H)nicotine as an agonist photoaffinity label

    SciTech Connect

    Middleton, R.E.; Cohen, J.B. )

    1991-07-16

    The agonist ({sup 3}H)nicotine was used as a photoaffinity label for the acetylcholine binding sties on the Torpedo nicotinic acetylcholine receptor (AChR). ({sup 3}H)Nicotine binds at equilibrium with K{sub eq} = 0.6 {mu}M to the agonist binding sites. Irradiation with 254-nm light of AChR-rich membranes equilibrated with ({sup 3}H)nicotine resulted in covalent incorporation into the {alpha}- and {gamma}-subunits, which was inhibited by agonists and competitive antagonists but not by noncompetitive antagonists. Inhibition of labeling by d-tubocurarine demonstrated that the {alpha}-subunit was labeled via both agonist sites but the {gamma}-subunit was labeled only via the site that binds d-tubocurarine with high affinity. Chymotryptic digestion of the {alpha}-subunit confirmed that Try-198 was the principal amino acid labeled by ({sup 3}H)nicotine. This confirmation required a novel radiosequencing strategy employing o-phthalaldehyde ({sup 3}H)Nicotine, which is the first photoaffinity agonist used, labels primarily Tyr-198 in contrast to competitive antagonist affinity labels, which label primarily Tyr-190 and Cys-192/Cys-193.

  12. Agonist interactions with 5-HT3 receptor recognition sites in the rat entorhinal cortex labelled by structurally diverse radioligands.

    PubMed Central

    Barnes, J. M.; Barnes, N. M.; Costall, B.; Jagger, S. M.; Naylor, R. J.; Robertson, D. W.; Roe, S. Y.

    1992-01-01

    1. The pharmacological properties of 5-HT3 receptor recognition sites labelled with [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330 in membranes prepared from the rat entorhinal cortex were investigated to assess the presence of cooperativity within the 5-HT3 receptor complex. 2. In rat entorhinal cortex homogenates, [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330 labelled homogeneous densities of recognition sites (defined by granisetron, 10 microM) with high affinity (Bmax = 75 +/- 5, 53 +/- 5, 92 +/- 6 and 79 +/- 6 fmol mg-1 protein, respectively; pKd = 9.41 +/- 0.04, 8.69 +/- 0.14, 8.81 +/- 0.06 and 10.14 +/- 0.04 for [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330, respectively, n = 3-8). 3. Quipazine and granisetron competed for the binding of each of the radioligands in the rat entorhinal cortex preparation at low nanomolar concentrations (pIC50; quipazine 9.38-8.51, granisetron 8.62-8.03), whilst the agonists, 5-hydroxytryptamine (5-HT), phenylbiguanide (PBG) and 2-methyl-5-HT competed at sub-micromolar concentrations (pIC50; 5-HT 7.16-6.42, PBG 7.52-6.40, 2-methyl-5-HT 7.38-6.09). 4. Competition curves generated with increasing concentrations of quipazine, PBG, 5-HT and 2-methyl-5-HT displayed Hill coefficients greater than unity when the 5-HT3 receptor recognition sites in the entorhinal cortex preparation were labelled with [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330. These competing compounds displayed Hill coefficients of around unity when the sites were labelled with [3H]-(S)-zacopride. Competition for the binding of [3H]-(S)-zacopride, [3H]-LY278,584, [3H]-granisetron and [3H]-GR67330 by granisetron generated Hill coefficients around unity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1559139

  13. 21 CFR 349.79 - Labeling of permitted combinations of active ingredients.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... established for the individual ingredients in the applicable OTC drug monograph. ... 21 Food and Drugs 5 2011-04-01 2011-04-01 false Labeling of permitted combinations of active ingredients. 349.79 Section 349.79 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND...

  14. 21 CFR 349.79 - Labeling of permitted combinations of active ingredients.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... established for the individual ingredients in the applicable OTC drug monograph. ... 21 Food and Drugs 5 2010-04-01 2010-04-01 false Labeling of permitted combinations of active ingredients. 349.79 Section 349.79 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND...

  15. 21 CFR 346.52 - Labeling of permitted combinations of anorectal active ingredients.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 5 2013-04-01 2013-04-01 false Labeling of permitted combinations of anorectal active ingredients. 346.52 Section 346.52 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS FOR HUMAN USE ANORECTAL DRUG PRODUCTS FOR...

  16. Activity-dependent fluorescent labeling of bacterial cells expressing the TOL pathway

    SciTech Connect

    William K. Keener; Mary E. Watwood

    2005-01-01

    3-Ethynylbenzoate functions as an activity-dependent, fluorogenic and chromogenic probe for Pseudomonas putida mt-2, which is known to degrade toluene via conversion to benzoate, followed by meta ring fission of the intermediate, catechol. This direct physiological analysis allows the fluorescent labeling of cells whose toluene-degrading enzymes have been induced by an aromatic substrate.

  17. Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation

    PubMed Central

    Pleiner, Tino; Bates, Mark; Trakhanov, Sergei; Lee, Chung-Tien; Schliep, Jan Erik; Chug, Hema; Böhning, Marc; Stark, Holger; Urlaub, Henning; Görlich, Dirk

    2015-01-01

    Nanobodies are single-domain antibodies of camelid origin. We generated nanobodies against the vertebrate nuclear pore complex (NPC) and used them in STORM imaging to locate individual NPC proteins with <2 nm epitope-label displacement. For this, we introduced cysteines at specific positions in the nanobody sequence and labeled the resulting proteins with fluorophore-maleimides. As nanobodies are normally stabilized by disulfide-bonded cysteines, this appears counterintuitive. Yet, our analysis showed that this caused no folding problems. Compared to traditional NHS ester-labeling of lysines, the cysteine-maleimide strategy resulted in far less background in fluorescence imaging, it better preserved epitope recognition and it is site-specific. We also devised a rapid epitope-mapping strategy, which relies on crosslinking mass spectrometry and the introduced ectopic cysteines. Finally, we used different anti-nucleoporin nanobodies to purify the major NPC building blocks – each in a single step, with native elution and, as demonstrated, in excellent quality for structural analysis by electron microscopy. The presented strategies are applicable to any nanobody and nanobody-target. DOI: http://dx.doi.org/10.7554/eLife.11349.001 PMID:26633879

  18. Topology of OxlT, the oxalate transporter of Oxalobacter formigenes, determined by site-directed fluorescence labeling.

    PubMed

    Ye, L; Jia, Z; Jung, T; Maloney, P C

    2001-04-01

    The topology of OxlT, the oxalate:formate exchange protein of Oxalobacter formigenes, was established by site-directed fluorescence labeling, a simple strategy that generates topological information in the context of the intact protein. Accessibility of cysteine to the fluorescent thiol-directed probe Oregon green maleimide (OGM) was examined for a panel of 34 single-cysteine variants, each generated in a His(9)-tagged cysteine-less host. The reaction with OGM was readily scored by examining the fluorescence profile after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of material purified by Ni2+ linked affinity chromatography. A position was assigned an external location if its single-cysteine derivative reacted with OGM added to intact cells; a position was designated internal if OGM labeling required cell lysis. We also showed that labeling of external, but not internal, positions was blocked by prior exposure of cells to the impermeable and nonfluorescent thiol-specific agent ethyltrimethylammonium methanethiosulfonate. Of the 34 positions examined in this way, 29 were assigned unambiguously to either an internal or external location; 5 positions could not be assigned, since the target cysteine failed to react with OGM. There was no evidence of false-positive assignment. Our findings document a simple and rapid method for establishing the topology of a membrane protein and show that OxlT has 12 transmembrane segments, confirming inferences from hydropathy analysis.

  19. (19)F-labeling of the adenine H2-site to study large RNAs by NMR spectroscopy.

    PubMed

    Sochor, F; Silvers, R; Müller, D; Richter, C; Fürtig, B; Schwalbe, H

    2016-01-01

    In comparison to proteins and protein complexes, the size of RNA amenable to NMR studies is limited despite the development of new isotopic labeling strategies including deuteration and ligation of differentially labeled RNAs. Due to the restricted chemical shift dispersion in only four different nucleotides spectral resolution remains limited in larger RNAs. Labeling RNAs with the NMR-active nucleus (19)F has previously been introduced for small RNAs up to 40 nucleotides (nt). In the presented work, we study the natural occurring RNA aptamer domain of the guanine-sensing riboswitch comprising 73 nucleotides from Bacillus subtilis. The work includes protocols for improved in vitro transcription of 2-fluoroadenosine-5'-triphosphat (2F-ATP) using the mutant P266L of the T7 RNA polymerase. Our NMR analysis shows that the secondary and tertiary structure of the riboswitch is fully maintained and that the specific binding of the cognate ligand hypoxanthine is not impaired by the introduction of the (19)F isotope. The thermal stability of the (19)F-labeled riboswitch is not altered compared to the unmodified sequence, but local base pair stabilities, as measured by hydrogen exchange experiments, are modulated. The characteristic change in the chemical shift of the imino resonances detected in a (1)H,(15)N-HSQC allow the identification of Watson-Crick base paired uridine signals and the (19)F resonances can be used as reporters for tertiary and secondary structure transitions, confirming the potential of (19)F-labeling even for sizeable RNAs in the range of 70 nucleotides.

  20. Correlation between phosphatidylinositol labeling and contraction in rabbit aorta: effect of alpha-1 adrenergic activation

    SciTech Connect

    Villalobos-Molina, R.; Uc, M.; Hong, E.; Garcia-Sainz, J.A.

    1982-07-01

    Activation of rabbit aortic strips with alpha adrenergic agonists increased the labeling (with (/sup 32/P)Pi) of phosphatidylinositol (PI) and phosphatidic acid and contracted the vascular preparations in dose-related fashion. Epinephrine, norepinephrine and methoxamine produced maximal effects, whereas clonidine behaved as partial agonist and B-HT 933 (2-amino-6-ethyl-4,5,7,8-tetrahydro-6H-oxazole-(5,4-d) azepin dihydrochloride) was almost without activity in the two experimental models used. Phenylephrine was a full agonist in producing contraction, but failed to elicit the maximal increase in PI labeling. The EC50 values to produce contraction of aortic strips were lower for all agonists than those required to increase the incorporation of radioactive phosphate into PI, but there was a good correlation between the two sets of data. The increased PI labeling and contraction of aortic strips induced by epinephrine were antagonized by prazosin and yohimbine in dose-related fashion, but the first alpha blocker was about three orders of magnitude more potent than the second in antagonizing the two effects. The present results indicate that both stimulation of PI labeling and contraction are mediated through activation of alpha-1 adrenoceptors in rabbit aorta.

  1. A cyclopropene-modified nucleotide for site-specific RNA labeling using genetic alphabet expansion transcription.

    PubMed

    Eggert, F; Kath-Schorr, S

    2016-06-01

    Site-specific RNA modification with methyl cyclopropene moieties is performed by T7 in vitro transcription. An existing unnatural base is functionalized with a cyclopropene moiety and used in transcription reactions to produce site-specifically cyclopropene-modified RNA molecules. The posttranscriptional inverse electron demand Diels-Alder cycloaddition reaction with a selected tetrazine-fluorophore conjugate is demonstrated.

  2. A cyclopropene-modified nucleotide for site-specific RNA labeling using genetic alphabet expansion transcription.

    PubMed

    Eggert, F; Kath-Schorr, S

    2016-06-01

    Site-specific RNA modification with methyl cyclopropene moieties is performed by T7 in vitro transcription. An existing unnatural base is functionalized with a cyclopropene moiety and used in transcription reactions to produce site-specifically cyclopropene-modified RNA molecules. The posttranscriptional inverse electron demand Diels-Alder cycloaddition reaction with a selected tetrazine-fluorophore conjugate is demonstrated. PMID:27181840

  3. Label-free fluorescent molecular beacon based on a small fluorescent molecule non-covalently bound to the intentional gap site in the stem moiety.

    PubMed

    Gao, Qiang; Lin, Kai; Zhang, Hongge; Qi, Honglan; Zhang, Chengxiao

    2010-12-15

    A label-free fluorescent molecular beacon (MB) based on a fluorescent molecule, 5,6,7-trimethyl-1,8-naphthyridin-2-ylamine (ATMND) which is non-covalently bound to the intentional gap site in the stem moiety of the label-free MB, was developed. In the absence of a cDNA, ATMND fluorescence is significantly quenched because it binds to the unpaired cytosine at the gap site by hydrogen bonding. As a result, the label-free MB shows almost no fluorescence. Upon hybridization with cDNA, the label-free MB undergoes a conformational change to destroy the gap site. This results in an effective fluorescent enhancement because of the release of the ATMND from the gap site to the solution. Fluorescence titration shows that ATMND strongly binds to the cytosine at the gap site (K(11)>10(6)). Circular-dichroism spectroscopy indicates that the binding of ATMND at the gap site of the stem moiety does not induce a significant conformational change to the hairpin DNA. Under optimal conditions, the fluorescent intensity of the label-free MB increases with an increase in cDNA concentration from 50 nM to 1.5 μM. A detection limit of 20 nM cDNA was achieved. A single mismatched target ss-DNA can be effectively discriminated from cDNA. The advantage of the label-free MB is that both its ends can be left free to introduce other useful functionalities. In addition, the label-free MB synthesis introduced in this paper is relatively simple and inexpensive because no label is required.

  4. Characterization and sequencing of the active site of 1-aminocyclopropane-1-carboxylate synthase

    SciTech Connect

    Yip, Wing-Kin; Dong, Jian-Guo; Yang, S.F. ); Kenny, J.W.; Thompson, G.A. )

    1990-10-01

    The pyridoxal phosphate (PLP)-dependent 1-aminocyclopropane-1-carboxylic acid (ACC) synthase the key enzyme in ethylene biosynthesis, is inactivated by its substrate S-adenosylmethionine (AdoMet). Apple ACC synthase was purified with an immunoaffinity gel, and its active site was probed with NaB{sup 3}H{sub 4} or Ado({sup 14}C)Met. Peptide sequencing of both {sup 3}H- and {sup 14}C-labeled peptides revealed a common dodecapeptide of Ser-Leu-Ser-Xaa-Asp-Leu-Gly-Leu-Pro-Gly-Phe-Arg, where Xaa was the modified, radioactive residue in each case. Acid hydrolysis of the {sup 3}H-labeled enzyme released radioactive N-pyridoxyllysine, indicating that the active-site peptide contained lysine at position 4. Mass spectrometry of the {sup 14}C-labeled peptide indicated a protonated molecular ion at m/z 1390.6, from which the mass of Xaa was calculated to be 229, a number that is equivalent to the mass of a lysine residue alkylated by the 2-aminobutyrate portion of AdoMet, as we previously proposed. These results indicate that the same active-site lysine binds the PLP and convalently links to the 2-aminobutyrate portion of AdoMet during inactivation. The active site of tomato ACC synthase was probed in the same manner with Ado ({sup 14}C)Met. Sequencing of the tomato active-site peptide revealed two highly conserved dodecapeptides; the minor peptide possessed a sequence identical to that of the apple enzyme, whereas the major peptide differed from the minor peptide in that methionine replaced leucine at position 6.

  5. Visualization of Active Glucocerebrosidase in Rodent Brain with High Spatial Resolution following In Situ Labeling with Fluorescent Activity Based Probes

    PubMed Central

    Herrera Moro Chao, Daniela; Kallemeijn, Wouter W.; Marques, Andre R. A.; Orre, Marie; Ottenhoff, Roelof; van Roomen, Cindy; Foppen, Ewout; Renner, Maria C.; Moeton, Martina; van Eijk, Marco; Boot, Rolf G.; Kamphuis, Willem; Hol, Elly M.; Aten, Jan; Overkleeft, Hermen S.; Kalsbeek, Andries; Aerts, Johannes M. F. G.

    2015-01-01

    Gaucher disease is characterized by lysosomal accumulation of glucosylceramide due to deficient activity of lysosomal glucocerebrosidase (GBA). In cells, glucosylceramide is also degraded outside lysosomes by the enzyme glucosylceramidase 2 (GBA2) of which inherited deficiency is associated with ataxias. The interest in GBA and glucosylceramide metabolism in the brain has grown following the notion that mutations in the GBA gene impose a risk factor for motor disorders such as α-synucleinopathies. We earlier developed a β-glucopyranosyl-configured cyclophellitol-epoxide type activity based probe (ABP) allowing in vivo and in vitro visualization of active molecules of GBA with high spatial resolution. Labeling occurs through covalent linkage of the ABP to the catalytic nucleophile residue in the enzyme pocket. Here, we describe a method to visualize active GBA molecules in rat brain slices using in vivo labeling. Brain areas related to motor control, like the basal ganglia and motor related structures in the brainstem, show a high content of active GBA. We also developed a β-glucopyranosyl cyclophellitol-aziridine ABP allowing in situ labeling of GBA2. Labeled GBA2 in brain areas can be identified and quantified upon gel electrophoresis. The distribution of active GBA2 markedly differs from that of GBA, being highest in the cerebellar cortex. The histological findings with ABP labeling were confirmed by biochemical analysis of isolated brain areas. In conclusion, ABPs offer sensitive tools to visualize active GBA and to study the distribution of GBA2 in the brain and thus may find application to establish the role of these enzymes in neurodegenerative disease conditions such as α-synucleinopathies and cerebellar ataxia. PMID:26418157

  6. Metabolism of radio-labelled C3: effects of in vivo activation in rabbits

    PubMed Central

    Charlesworth, J. A.; Williams, D. G.; Naish, P.; Lachmann, P. J.; Peters, D. K.

    1974-01-01

    Turnover studies were performed in rabbits using biologically screened, highly purified, radio-labelled human C3 and C3c. Experiments were also carried out using agents known to activate the complement system in vivo—cobra venom factor, human nephritic serum and nephrotoxic antibody to rabbit glomerular basement membrane. Activation of labelled C3 by cobra factor provided information regarding the metabolic behaviour of C3d. The fractional catabolic ratio (FCR) of human C3 in normal rabbits was 2·4–2·8% of the plasma pool per hr. FCR for C3c was 4.5% per hr. Activation of C3 in vivo consistently resulted in accelerated disappearance of plasma radioactivity. Analysis of the plasma and total body radioactivity curves indicated that both hypercatabolism and extravascular sequestration of radioactivity were responsible for this phenomenon. PMID:4468849

  7. High-activity radio-iodine labeling of conventional and stealth liposomes.

    PubMed

    Mougin-Degraef, Marie; Jestin, Emmanuelle; Bruel, Damien; Remaud-Le Saëc, Patricia; Morandeau, Laurence; Faivre-Chauvet, Alain; Barbet, Jacques

    2006-01-01

    A new method to label preformed liposomes with high activities of radiohalogenated compounds has been developed. It uses activated esters of simple synthetic molecules that may be readily halogenated, such as Bolton-Hunter reagent (BH), and arginine-containing liposomes. BH, in the form of an activated ester, crosses the liposome membrane to react with arginine inside the liposomes, as demonstrated by thin-layer chromatography and by the fact that saline-containing liposomes, or hydrolyzed BH or the water soluble sulfo-BH afforded only marginal encapsulation yields. Under optimized conditions, between 37 and 55 degrees C, 62 +/- 4% (mean +/- SD) of radiolabeled BH were consistently encapsulated in the liposomes within 30 min. In molar amounts, this corresponds to a mean of 56 nmol of BH per micromol of lipids. Based on achievable specific activity, up to 2.8 GBq of iodine-131 could be entrapped per micromol of lipids. Leakage of radioactivity was very low, with less than 5% of the encapsulated activity released within 6 days at 4 degrees C in phosphate-buffered saline and less than 50% within 24 h in human serum at 37 degrees C. The labeling stability, and the fact that both conventional and PEGylated liposomes can be readily labeled with high doses of radioactivity, will make this technique useful for in vivo targeting applications, such as tumor detection (using iodine-123 or iodine-124) or therapy (with iodine-131 or astatine-211). PMID:16556552

  8. A coupled protein and probe engineering approach for selective inhibition and activity based probe labeling of the caspases

    PubMed Central

    Xiao, Junpeng; Broz, Petr; Puri, Aaron W.; Deu, Edgar; Morell, Montse; Monack, Denise M.; Bogyo, Matthew

    2013-01-01

    Caspases are cysteine proteases that play essential roles in apoptosis and inflammation. Unfortunately, their highly conserved active sites and overlapping substrate specificities make it difficult to use inhibitors or activity-based probes to study the function, activation, localization and regulation of individual members of this family. Here we describe a strategy to engineer a caspase to contain a latent nucleophile that can be targeted by a probe containing a suitably placed electrophile, thereby allowing specific, irreversible inhibition and labeling of only the engineered protease. To accomplish this, we have identified a non-conserved residue on the small subunit of all caspases that is near the substrate-binding pocket and that can be mutated to a non-catalytic cysteine residue. We demonstrate that an active site probe containing an irreversible binding acrylamide electrophile can specifically target this cysteine residue. Here we validate the approach using the apoptotic mediator, caspase-8 and the inflammasome effector, caspase-1. We show that the engineered enzymes are functionally identical to the wild type enzymes and that the approach allows specific inhibition and direct imaging of the engineered targets in cells. Therefore, this method can be used to image localization and activation as well as the functional contributions of individual caspase proteases to the process of cell death or inflammation. PMID:23701470

  9. Allosteric nature of P2X receptor activation probed by photoaffinity labelling

    PubMed Central

    Bhargava, Y; Rettinger, J; Mourot, A

    2012-01-01

    BACKGROUND AND PURPOSE In P2X receptors, agonist binding at the interface between neighbouring subunits is efficiently transduced to ion channel gating. However, the relationship between binding and gating is difficult to study because agonists continuously bind and unbind. Here, we covalently incorporated agonists in the binding pocket of P2X receptors and examined how binding site occupancy affects the ability of the channel to gate. EXPERIMENTAL APPROACH We used a strategy for tethering agonists to their ATP-binding pocket, while simultaneously probing ion channel gating using electrophysiology. The agonist 2′,3′-O-(4-benzoylbenzoyl)-ATP (BzATP), a photoaffinity analogue of ATP, enabled us to trap rat homomeric P2X2 receptor and a P2X2/1 receptor chimera in different agonist-bound states. UV light was used to control the degree of covalent occupancy of the receptors. KEY RESULTS Irradiation of the P2X2/1 receptor chimera – BzATP complex resulted in a persistent current that lasted even after extensive washout, consistent with photochemical tethering of the agonist BzATP and trapping of the receptors in an open state. Partial labelling with BzATP primed subsequent agonist binding and modulated gating efficiency for both full and partial agonists. CONCLUSIONS AND IMPLICATIONS Our photolabelling strategy provides new molecular insights into the activation mechanism of the P2X receptor. We show here that priming with full agonist molecules leads to an increase in gating efficiency after subsequent agonist binding. PMID:22725669

  10. Elucidating the design principles of photosynthetic electron-transfer proteins by site-directed spin labeling EPR spectroscopy.

    PubMed

    Ishara Silva, K; Jagannathan, Bharat; Golbeck, John H; Lakshmi, K V

    2016-05-01

    Site-directed spin labeling electron paramagnetic resonance (SDSL EPR) spectroscopy is a powerful tool to determine solvent accessibility, side-chain dynamics, and inter-spin distances at specific sites in biological macromolecules. This information provides important insights into the structure and dynamics of both natural and designed proteins and protein complexes. Here, we discuss the application of SDSL EPR spectroscopy in probing the charge-transfer cofactors in photosynthetic reaction centers (RC) such as photosystem I (PSI) and the bacterial reaction center (bRC). Photosynthetic RCs are large multi-subunit proteins (molecular weight≥300 kDa) that perform light-driven charge transfer reactions in photosynthesis. These reactions are carried out by cofactors that are paramagnetic in one of their oxidation states. This renders the RCs unsuitable for conventional nuclear magnetic resonance spectroscopy investigations. However, the presence of native paramagnetic centers and the ability to covalently attach site-directed spin labels in RCs makes them ideally suited for the application of SDSL EPR spectroscopy. The paramagnetic centers serve as probes of conformational changes, dynamics of subunit assembly, and the relative motion of cofactors and peptide subunits. In this review, we describe novel applications of SDSL EPR spectroscopy for elucidating the effects of local structure and dynamics on the electron-transfer cofactors of photosynthetic RCs. Because SDSL EPR Spectroscopy is uniquely suited to provide dynamic information on protein motion, it is a particularly useful method in the engineering and analysis of designed electron transfer proteins and protein networks. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.

  11. Elucidating the design principles of photosynthetic electron-transfer proteins by site-directed spin labeling EPR spectroscopy.

    PubMed

    Ishara Silva, K; Jagannathan, Bharat; Golbeck, John H; Lakshmi, K V

    2016-05-01

    Site-directed spin labeling electron paramagnetic resonance (SDSL EPR) spectroscopy is a powerful tool to determine solvent accessibility, side-chain dynamics, and inter-spin distances at specific sites in biological macromolecules. This information provides important insights into the structure and dynamics of both natural and designed proteins and protein complexes. Here, we discuss the application of SDSL EPR spectroscopy in probing the charge-transfer cofactors in photosynthetic reaction centers (RC) such as photosystem I (PSI) and the bacterial reaction center (bRC). Photosynthetic RCs are large multi-subunit proteins (molecular weight≥300 kDa) that perform light-driven charge transfer reactions in photosynthesis. These reactions are carried out by cofactors that are paramagnetic in one of their oxidation states. This renders the RCs unsuitable for conventional nuclear magnetic resonance spectroscopy investigations. However, the presence of native paramagnetic centers and the ability to covalently attach site-directed spin labels in RCs makes them ideally suited for the application of SDSL EPR spectroscopy. The paramagnetic centers serve as probes of conformational changes, dynamics of subunit assembly, and the relative motion of cofactors and peptide subunits. In this review, we describe novel applications of SDSL EPR spectroscopy for elucidating the effects of local structure and dynamics on the electron-transfer cofactors of photosynthetic RCs. Because SDSL EPR Spectroscopy is uniquely suited to provide dynamic information on protein motion, it is a particularly useful method in the engineering and analysis of designed electron transfer proteins and protein networks. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson. PMID:26334844

  12. Affinity labeling of Escherichia coli phenylalanyl-tRNA synthetase at the binding site for tRNA

    SciTech Connect

    Hountondji, C.; Schmitter, J.M.; Beauvallet, C.; Blanquet, S.

    1987-08-25

    Periodate-oxidized tRNA/sup Phe/ (tRNA/sub ox//sup Phe/) behaves as a specific affinity label of tetrameric Escherichia coli phenylalanyl-tRNA synthetase (PheRS). Reaction of the ..cap alpha../sub 2/..beta../sub 2/ enzyme with tRNA/sub ox//sup Phe/ results in the loss of tRNA/sup Phe/ aminoacylation activity with covalent attachment of 2 mol of tRNA dialdehyde/mol of enzyme, in agreement with the stoichiometry of tRNA binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the PheRS-(/sup 14/C)tRNA/sub ox//sup Phe/ covalent complex indicates that the large (..cap alpha.., M/sub r/ 87K) subunit of the enzyme interacts with the 3'-adenosine of tRNA/sub ox//sup Phe/. The (/sup 14/C)tRNA-labeled chymotryptic peptides of PheRS were purified by both gel filtration and reverse-phase high-performance liquid chromatography. The radioactivity was almost equally distributed among three peptides: Met-Lys(Ado)-Phe, Ala-Asp-Lys(Ado)-Leu, and Lys-Ile-Lys(Ado)-Ala. These sequences correspond to residues 1-3, 59-62, and 104-107, respectively, in the N-terminal region of the 795 amino acid sequence of the ..cap alpha.. subunit. It is noticeable that the labeled peptide Ala-Asp-Lys-Leu is adjacent to residues 63-66 (Arg-Val-Thr-Lys). The latter sequence was just predicted to resemble the proposed consensus tRNA CCA binding region Lys-Met-Ser-Lys-Ser, as deduced from previous affinity labeling studies on E. coli methionyl- and tyrosyl-tRNA synthetases.

  13. Control of active sites in flocculation: Concept of equivalent active sites''

    SciTech Connect

    Behl, S.; Moudgil, B.M. . Dept. of Materials Science and Engineering)

    1993-12-01

    Flocculation and dispersion of solids are strong functions of the amount and conformation of the adsorbed polymer. Regions of dispersion and flocculation of solids with particular polymer molecules may be deduced from saturation adsorption data. The concept of equivalent active sites'' is proposed to explain flocculation and dispersion behavior irrespective of the amount or conformation of the adsorbed polymer. The concept has been further extended to study the selective flocculation process.

  14. EPR Studies of Functionally Active, Nitroxide Spin-Labeled Peptide Analogs of the C-terminus of a G-Protein Alpha Subunit

    PubMed Central

    Van Eps, Ned; Anderson, Lori L.; Kisselev, Oleg G.; Baranski, Thomas J.; Hubbell, Wayne L.; Marshall, Garland R.

    2010-01-01

    The C-terminal tail of the transducin alpha subunit, Gtα(340–350), is known to bind and stabilize the active conformation of rhodopsin upon photoactivation (R*). Five spin-labeled analogs of Gtα(340–350) demonstrated native-like activity in their ability to bind and stabilize R*. The spin label 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) was employed at interior sites within the peptide, whereas a Proxyl (3-carboxyl-2,2,5,5-tetramethyl-pyrrolidinyloxy) spin label was employed at the amino terminus of the peptide. Upon binding to R*, the electron paramagnetic resonance spectrum of TOAC343-Gtα(340–350) revealed greater immobilization of the nitroxide when compared to that of the N-terminal modified Proxyl-Gtα(340–350) analog. A double-labeled Proxyl/TOAC348-Gtα(340–350) was examined by DEER spectroscopy to determine the distribution of distances between the two nitroxides in the peptides when in solution and when bound to R*. TOAC and Proxyl spin labels in this GPCR-G-protein α-peptide system provide unique biophysical probes that can be used to explore the structure and conformational changes at the rhodopsin-G-protein interface. PMID:20695526

  15. Mapping protein conformational heterogeneity under pressure with site-directed spin labeling and double electron-electron resonance.

    PubMed

    Lerch, Michael T; Yang, Zhongyu; Brooks, Evan K; Hubbell, Wayne L

    2014-04-01

    The dominance of a single native state for most proteins under ambient conditions belies the functional importance of higher-energy conformational states (excited states), which often are too sparsely populated to allow spectroscopic investigation. Application of high hydrostatic pressure increases the population of excited states for study, but structural characterization is not trivial because of the multiplicity of states in the ensemble and rapid (microsecond to millisecond) exchange between them. Site-directed spin labeling in combination with double electron-electron resonance (DEER) provides long-range (20-80 Å) distance distributions with angstrom-level resolution and thus is ideally suited to resolve conformational heterogeneity in an excited state populated under high pressure. DEER currently is performed at cryogenic temperatures. Therefore, a method was developed for rapidly freezing spin-labeled proteins under pressure to kinetically trap the high-pressure conformational ensemble for subsequent DEER data collection at atmospheric pressure. The methodology was evaluated using seven doubly-labeled mutants of myoglobin designed to monitor selected interhelical distances. For holomyoglobin, the distance distributions are narrow and relatively insensitive to pressure. In apomyoglobin, on the other hand, the distributions reveal a striking conformational heterogeneity involving specific helices in the pressure range of 0-3 kbar, where a molten globule state is formed. The data directly reveal the amplitude of helical fluctuations, information unique to the DEER method that complements previous rate determinations. Comparison of the distance distributions for pressure- and pH-populated molten globules shows them to be remarkably similar despite a lower helical content in the latter. PMID:24707053

  16. A neurosteroid analogue photolabeling reagent labels the colchicine binding site on tubulin: A mass spectrometric analysis

    PubMed Central

    Chen, Zi-Wei; Chen, Li-Hai; Akentieva, Natalia; Lichti, Cheryl F.; Darbandi, Ramin; Hastings, Randy; Covey, Douglas F.; Reichert, David E.; Townsend, R. Reid; Evers, Alex S.

    2013-01-01

    Previous studies have shown that the neurosteroid analogue, 6-Azi-pregnanolone (6-AziP), photolabels voltage-dependent anion channels and proteins of approximately 55 kDa in rat brain membranes. The present study used two dimensional electrophoresis and nano-electrospray ionization ion trap mass spectrometry (nano-ESI-MS) to identify the 55 kDa proteins (pI 4.8) as isoforms of β-tubulin. This identification was confirmed by immuno-blot and immunoprecipitation of photolabeled protein with anti-β-tubulin antibody and by the demonstration that 6-AziP photolabels purified bovine brain tubulin in a concentration-dependent pattern. To identify the photolabeling sites, purified bovine brain tubulin was photolabeled with 6-AziP, digested with trypsin, and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI). A 6-AziP adduct of TAVCDIPPR (m/z=1287.77), a β-tubulin specific peptide, was detected by MALDI. High resolution LC-MS/MS analysis identified that 6-AziP was covalently bound to cysteine 354 (Cys-354), previously identified as a colchicine binding site. 6-AziP photolabeling was inhibited by 2-methoxyestradiol, an endogenous derivative of estradiol thought to bind to the colchicine site. Structural modeling predicted that neurosteroids could dock in this colchicine site at the interface between α- and β-tubulin with the photolabeling group of 6-AziP positioned proximate to Cys-354. PMID:22451060

  17. Site-specific labeling of proteins and peptides with trans-cyclooctene containing handles capable of tetrazine ligation.

    PubMed

    Wollack, James W; Monson, Benjamin J; Dozier, Jonathan K; Dalluge, Joseph J; Poss, Kristina; Hilderbrand, Scott A; Distefano, Mark D

    2014-08-01

    There is a growing library of functionalized non-natural substrates for the enzyme protein farnesyltransferase (PFTase). PFTase covalently attaches these functionalized non-natural substrates to proteins ending in the sequence CAAX, where C is a cysteine that becomes alkylated, A represents an aliphatic amino acid, and X is Ser, Met, Ala, or Gln. Reported substrates include a variety of functionalities that allow modified proteins to undergo subsequent bioconjugation reactions. To date the most common strategy used in this approach has been copper catalyzed azide-alkyne cycloaddition (CuAAC). While being fast and bioorthogonal CuAAC has limited use in live cell experiments due to copper's toxicity.(1) Here, we report the synthesis of trans-cyclooctene geranyl diphosphate. This substrate can be synthesized from geraniol in six steps and be enzymatically transferred to peptides and proteins that end in a CAAX sequence. Proteins and peptides site-specially modified with trans-cyclooctene geranyl diphosphate were subsequently targeted for further modification via tetrazine ligation. As tetrazine ligation is bioorthogonal, fast, and is contingent on ring strain rather than the addition of a copper catalyst, this labeling strategy should prove useful for labeling proteins where the presence of copper may hinder solubility or biological reactivity.

  18. Labelling of endogenous target protein via N-S acyl transfer-mediated activation of N-sulfanylethylanilide.

    PubMed

    Denda, Masaya; Morisaki, Takuya; Kohiki, Taiki; Yamamoto, Jun; Sato, Kohei; Sagawa, Ikuko; Inokuma, Tsubasa; Sato, Youichi; Yamauchi, Aiko; Shigenaga, Akira; Otaka, Akira

    2016-07-14

    The ligand-dependent incorporation of a reporter molecule (e.g., fluorescence dye or biotin) onto a endogenous target protein has emerged as an important strategy for elucidating protein function using various affinity-based labelling reagents consisting of reporter, ligand and reactive units. Conventional labelling reagents generally use a weakly activated reactive unit, which can result in the non-specific labelling of proteins in a ligand-independent manner. In this context, the activation of a labelling reagent through a targeted protein-ligand interaction could potentially overcome the problems associated with conventional affinity-based labelling reagents. We hypothesized that this type of protein-ligand-interaction-mediated activation could be accomplished using N-sulfanylethylanilide (SEAlide) as the reactive unit in the labelling reagent. Electrophilically unreactive amide-type SEAlide can be activated by its conversion to the corresponding active thioester in the presence of a phosphate salt, which can act as an acid-base catalyst. It has been suggested that protein surfaces consisting of hydrophilic residues such as amino, carboxyl and imidazole groups could function as acid-base catalysts. We therefore envisioned that a SEAlide-based labelling reagent (SEAL) bearing SEAlide as a reactive unit could be activated through the binding of the SEAL with a target protein. Several SEALs were readily prepared in this study using standard 9-fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase protocols. These SEAL systems were subsequently applied to the ligand-dependent labelling of human carbonic anhydrase (hCA) and cyclooxyganese 1. Although we have not yet obtained any direct evidence for the target protein-mediated activation of the SEAlide unit, our results for the reaction of these SEALs with hCA1 or butylamine indirectly support our hypothesis. The SEALs reported in this study represent valuable new entries to the field of affinity-based labelling reagents

  19. ( sup 3 H)opipramol labels a novel binding site and sigma receptors in rat brain membranes

    SciTech Connect

    Ferris, C.D.; Hirsch, D.J.; Brooks, B.P.; Snowman, A.M.; Snyder, S.H. )

    1991-02-01

    Opipramol (OP), a clinically effective antidepressant with a tricyclic structure, is inactive as an inhibitor of biogenic amine uptake. ({sup 3}H)Opipramol binds saturably to rat brain membranes (apparent KD = 4 nM, Bmax = 3 pmol/mg of protein). ({sup 3}H)Opipramol binding can be differentiated into haloperidol-sensitive and -resistant components, with Ki values for haloperidol of 1 nM (Bmax = 1 pmol/mg of protein) and 350 nM (Bmax = 1.9 pmol/mg of protein), respectively. The drug specificity of the haloperidol-sensitive component is the same as that of sigma receptors labeled with (+)-({sup 3}H)3-(3-hydroxyphenyl)-N-(1-propyl)piperdine. The haloperidol-resistant component does not correspond to any known neurotransmitter receptor or uptake recognition site. It displays high affinity for phenothiazines and related structures such as perphenazine, clopenthixol, and flupenthixol, whose potencies are comparable to that of opipramol. Because certain of these drugs are more potent at the haloperidol-resistant opipramol site than in exerting any other action, it is possible that this opipramol-selective site may mediate their therapeutic effects.

  20. Calcium structural transition of human cardiac troponin C in reconstituted muscle fibres as studied by site-directed spin labelling.

    PubMed

    Nakamura, Motoyoshi; Ueki, Shoji; Hara, Hideyuki; Arata, Toshiaki

    2005-04-22

    The in situ structure of human cardiac troponin C (hcTnC) has been studied with site-directed, spin labelling, electron paramagnetic resonance (SDSL-EPR). Analysis of the in situ structures of hcTnC is essential for elucidating the molecular mechanism behind its Ca(2+)-sensitive regulation. We prepared two hcTnC mutants (C35S and C84S) containing one native cysteine residue (84 and 35, respectively) for spin labelling. The mutants were labelled with a methane thiosulfonate spin label (MTSSL) and the TnC was reconstituted into permeabilized muscle fibres. The mobility of Cys84-MTSSL changed markedly after addition of Ca2+, while that of the Cys35 residue did not change in the monomer state or in fibres. The rotational correlation time of Cys84-MTSSL decreased from 32ns to 13ns upon Ca(2+)-binding in the monomer state, whereas in fibres the spectrum of Cys84-MTSSL was resolved into mobile (16ns) and immobile (35ns) components and the addition of Ca2+ increased the immobile component. Moreover, the accessibility of Cys84-MTSSL to molecular oxygen increased slightly in the presence of Ca2+. These data suggest that Cys35 remains in the same location regardless of the addition of Ca2+, whereas Cys84 is located at the position that interacts with B and C helices of hcTnC and interacts with troponin I (TnI) at high concentrations of Ca2+. We determined the distances between Cys35 and Cys84 by measuring pulsed electron-electron double resonance spectra. The distances were 26.0 angstroms and 27.2 angstroms in the monomer state and in fibres, respectively, and the addition of Ca2+ decreased the distance to 23.2 angstroms in fibres but only slightly in the monomer state, showing that Ca2+ binding to the N-domain of hcTnC induced a larger structural change in muscle fibres than in the monomer state.

  1. Using Ensemble Decisions and Active Selection to Improve Low-Cost Labeling for Multi-View Data

    NASA Technical Reports Server (NTRS)

    Rebbapragada, Umaa; Wagstaff, Kiri L.

    2011-01-01

    This paper seeks to improve low-cost labeling in terms of training set reliability (the fraction of correctly labeled training items) and test set performance for multi-view learning methods. Co-training is a popular multiview learning method that combines high-confidence example selection with low-cost (self) labeling. However, co-training with certain base learning algorithms significantly reduces training set reliability, causing an associated drop in prediction accuracy. We propose the use of ensemble labeling to improve reliability in such cases. We also discuss and show promising results on combining low-cost ensemble labeling with active (low-confidence) example selection. We unify these example selection and labeling strategies under collaborative learning, a family of techniques for multi-view learning that we are developing for distributed, sensor-network environments.

  2. Dissecting the active site of a photoreceptor protein

    NASA Astrophysics Data System (ADS)

    Hoff, Wouter; Hara, Miwa; Ren, Jie; Moghadam, Farzaneh; Xie, Aihua; Kumauchi, Masato

    While enzymes are quite large molecules, functionally important chemical events are often limited to a small region of the protein: the active site. The physical and chemical properties of residues at such active sites are often strongly altered compared to the same groups dissolved in water. Understanding such effects is important for unraveling the mechanisms underlying protein function and for protein engineering, but has proven challenging. Here we report on our ongoing efforts on using photoactive yellow protein (PYP), a bacterial photoreceptor, as a model system for such effects. We will report on the following questions: How many residues affect active site properties? Are these residues in direct physical contact with the active site? Can functionally important residues be recognized in the crystal structure of a protein? What structural resolution is needed to understand active sites? What spectroscopic techniques are most informative? Which weak interactions dominate active site properties?

  3. 75 FR 12546 - Agency Information Collection Activities; Proposed Collection; Comment Request; Cosmetic Labeling...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-16

    ... Collection; Comment Request; Cosmetic Labeling Regulations AGENCY: Food and Drug Administration, HHS. ACTION... on information collection provisions in FDA's cosmetic labeling regulations. DATES: Submit written or... appropriate, and other forms of information technology. Cosmetic Labeling Regulations--21 CFR Part 701...

  4. Identification of active oxalotrophic bacteria by Bromodeoxyuridine DNA labeling in a microcosm soil experiments.

    PubMed

    Bravo, Daniel; Martin, Gaëtan; David, Maude M; Cailleau, Guillaume; Verrecchia, Eric; Junier, Pilar

    2013-11-01

    The oxalate-carbonate pathway (OCP) leads to a potential carbon sink in terrestrial environments. This process is linked to the activity of oxalotrophic bacteria. Although isolation and molecular characterizations are used to study oxalotrophic bacteria, these approaches do not give information on the active oxalotrophs present in soil undergoing the OCP. The aim of this study was to assess the diversity of active oxalotrophic bacteria in soil microcosms using the Bromodeoxyuridine (BrdU) DNA labeling technique. Soil was collected near an oxalogenic tree (Milicia excelsa). Different concentrations of calcium oxalate (0.5%, 1%, and 4% w/w) were added to the soil microcosms and compared with an untreated control. After 12 days of incubation, a maximal pH of 7.7 was measured for microcosms with oxalate (initial pH 6.4). At this time point, a DGGE profile of the frc gene was performed from BrdU-labeled soil DNA and unlabeled soil DNA. Actinobacteria (Streptomyces- and Kribbella-like sequences), Gammaproteobacteria and Betaproteobacteria were found as the main active oxalotrophic bacterial groups. This study highlights the relevance of Actinobacteria as members of the active bacterial community and the identification of novel uncultured oxalotrophic groups (i.e. Kribbella) active in soils.

  5. Absorption of enzymatically active sup 125 I-labeled bovine milk xanthine oxidase fed to rabbits

    SciTech Connect

    Rzucidlo, S.J. ); Zikakis, J.P. )

    1990-05-01

    Rabbits fed a regular laboratory diet supplemented with a high-fat milk containing xanthine oxidase (XO) were studied to determine the presence of active XO in the blood. A pilot feeding study, where rabbits consumed a high-fat diet containing xanthine oxidase, showed a correlation between dairy food consumption and XO activity in the blood. Antibody to dietary XO was also found. In a second study, rabbits were fed ad libitum the high-fat milk and blood serum samples were tested weekly for XO activity. No elevation in serum XO activity was found. A third study showed that serum XO activity was increased when rabbits were force fed the high-fat milk. The final study consisted of force feeding {sup 125}I-labeled XO to one rabbit to ascertain whether the observed increase in serum XO was due to dietary or endogenous XO. Isoelectric focusing of sera collected from the test rabbit strongly suggested that at least a portion of the serum XO contained the radioactive label. This is the first direct evidence showing the uptake of dietary active XO from the gut.

  6. Mars Surveyor Project Landing Site Activities

    NASA Technical Reports Server (NTRS)

    Gulick, Virginia C.; Briggs, Geoffrey; Saunders, R. Stephen; Gilmore, Martha; Soderblom, Larry

    1999-01-01

    The Mars Surveyor Program --now a cooperative program led by NASA and CNES along with other international partners -- is underway. It has the primary science objective of furthering our understanding of the biological potential and possible biological history of Mars and has the complementary objective of improving our understanding of martian climate evolution and planetary history The missions will develop technology and acquire data necessary for eventual human Exploration. Launches of orbiters, landers and rovers will take place in 2001 and in 2003; in 2005 a complete system will be launched capable of returning samples to Earth by 2008. A key aspect of the program is the selection of landing sites. This abstract 1) reports on the status of the landing site selection process that begins with the 2001 lander mission and 2) outlines be opportunities for the Mars community to provide input into the landing site selection process.

  7. Mars Surveyor Project Landing Site Activities

    NASA Technical Reports Server (NTRS)

    Gulick, V. C.; Briggs, Geoffrey; Saunders, R. Stephen; Gilmore, Martha; Soderblom, Larry

    1999-01-01

    The Mars Surveyor Program -- now a cooperative program led by NASA and CNES along with other international partners -- is underway. It has the primary science objective of furthering our understanding of the biological potential and possible biological history of Mars and has the complementary objective of improving our understanding of martian climate evolution and planetary history. The missions will develop technology and acquire data necessary for eventual human exploration. Launches of orbiters, landers and rovers will take place in 2001 and in 2003; in 2005 a complete system will be launched capable of returning samples to Earth by 2008. A key aspect of the program is the selection of landing sites. This abstract 1) reports on the status of the landing site selection process that begins with the 2001 lander mission and 2) outlines the opportunities for the Mars community to provide input into the landing site selection process.

  8. HPLC-purified 2-(/sup 125/I)iodomelatonin labels multiple binding sites in hamster brain

    SciTech Connect

    Niles, L.P.; Pickering, D.S.; Sayer, B.G.

    1987-09-30

    Binding of 2-(/sup 125/I)iodomelatonin in hamster brain synaptosomal membranes at 0 degrees C is rapid, saturable, reversible and sensitive to heat and trypsin treatment. Computer resolution of curvilinear Scatchard plots yielded high- and low-affinity components as follows: Kd1 = 0.32 +/- 0.14 nM, Bmax1 = 5.6 +/- 1.7 fmol/mg protein and Kd2 = 10.5 +/- 3.2 nM, Bmax2 = 123 +/- 33 fmol/mg protein (n = 3). Competition experiments indicated that 2-iodomelatonin and prazosin are the most potent inhibitors of high-affinity binding. Unlike prazosin, several alpha-adrenergic agents and various neurotransmitters were ineffective. These findings suggest that prazosin may be a potent antagonist at a unique, non-alpha-adrenergic, high-affinity binding site for melatonin.

  9. Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

    DOE PAGES

    Liu, Daniel S.; Nivon, Lucas G.; Richter, Florian; Goldman, Peter J.; Deerinck, Thomas J.; Yao, Jennifer Z.; Richardson, Douglas; Phipps, William S.; Ye, Anne Z.; Ellisman, Mark H.; et al

    2014-10-13

    In this study, chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of themore » intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.« less

  10. Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

    SciTech Connect

    Liu, Daniel S.; Nivon, Lucas G.; Richter, Florian; Goldman, Peter J.; Deerinck, Thomas J.; Yao, Jennifer Z.; Richardson, Douglas; Phipps, William S.; Ye, Anne Z.; Ellisman, Mark H.; Drennan, Catherine L.; Baker, David; Ting, Alice Y.

    2014-10-13

    In this study, chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.

  11. Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

    PubMed Central

    Liu, Daniel S.; Nivón, Lucas G.; Richter, Florian; Goldman, Peter J.; Deerinck, Thomas J.; Yao, Jennifer Z.; Richardson, Douglas; Phipps, William S.; Ye, Anne Z.; Ellisman, Mark H.; Drennan, Catherine L.; Baker, David; Ting, Alice Y.

    2014-01-01

    Chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies. PMID:25313043

  12. Catalysis-dependent selenium incorporation and migration in the nitrogenase active site iron-molybdenum cofactor

    PubMed Central

    Spatzal, Thomas; Perez, Kathryn A; Howard, James B; Rees, Douglas C

    2015-01-01

    Dinitrogen reduction in the biological nitrogen cycle is catalyzed by nitrogenase, a two-component metalloenzyme. Understanding of the transformation of the inert resting state of the active site FeMo-cofactor into an activated state capable of reducing dinitrogen remains elusive. Here we report the catalysis dependent, site-selective incorporation of selenium into the FeMo-cofactor from selenocyanate as a newly identified substrate and inhibitor. The 1.60 Å resolution structure reveals selenium occupying the S2B site of FeMo-cofactor in the Azotobacter vinelandii MoFe-protein, a position that was recently identified as the CO-binding site. The Se2B-labeled enzyme retains substrate reduction activity and marks the starting point for a crystallographic pulse-chase experiment of the active site during turnover. Through a series of crystal structures obtained at resolutions of 1.32–1.66 Å, including the CO-inhibited form of Av1-Se2B, the exchangeability of all three belt-sulfur sites is demonstrated, providing direct insights into unforeseen rearrangements of the metal center during catalysis. DOI: http://dx.doi.org/10.7554/eLife.11620.001 PMID:26673079

  13. Catalysis-dependent selenium incorporation and migration in the nitrogenase active site iron-molybdenum cofactor.

    PubMed

    Spatzal, Thomas; Perez, Kathryn A; Howard, James B; Rees, Douglas C

    2015-12-16

    Dinitrogen reduction in the biological nitrogen cycle is catalyzed by nitrogenase, a two-component metalloenzyme. Understanding of the transformation of the inert resting state of the active site FeMo-cofactor into an activated state capable of reducing dinitrogen remains elusive. Here we report the catalysis dependent, site-selective incorporation of selenium into the FeMo-cofactor from selenocyanate as a newly identified substrate and inhibitor. The 1.60 Å resolution structure reveals selenium occupying the S2B site of FeMo-cofactor in the Azotobacter vinelandii MoFe-protein, a position that was recently identified as the CO-binding site. The Se2B-labeled enzyme retains substrate reduction activity and marks the starting point for a crystallographic pulse-chase experiment of the active site during turnover. Through a series of crystal structures obtained at resolutions of 1.32-1.66 Å, including the CO-inhibited form of Av1-Se2B, the exchangeability of all three belt-sulfur sites is demonstrated, providing direct insights into unforeseen rearrangements of the metal center during catalysis.

  14. Activation of Inhibitors by Sortase Triggers Irreversible Modification of the Active Site*S

    PubMed Central

    Maresso, Anthony W.; Wu, Ruiying; Kern, Justin W.; Zhang, Rongguang; Janik, Dorota; Missiakas, Dominique M.; Duban, Mark-Eugene; Joachimiak, Andrzej; Schneewind, Olaf

    2011-01-01

    Sortases anchor surface proteins to the cell wall of Gram-positive pathogens through recognition of specific motif sequences. Loss of sortase leads to large reductions in virulence, which identifies sortase as a target for the development of antibacterials. By screening 135,625 small molecules for inhibition, we report here that aryl (β-amino)ethyl ketones inhibit sortase enzymes from staphylococci and bacilli. Inhibition of sortases occurs through an irreversible, covalent modification of their active site cysteine. Sortases specifically activate this class of molecules via β-elimination, generating a reactive olefin intermediate that covalently modifies the cysteine thiol. Analysis of the three-dimensional structure of Bacillus anthracis sortase B with and without inhibitor provides insights into the mechanism of inhibition and reveals binding pockets that can be exploited for drug discovery. PMID:17545669

  15. The bifunctional active site of s-adenosylmethionine synthetase. Roles of the active site aspartates.

    PubMed

    Taylor, J C; Markham, G D

    1999-11-12

    S-Adenosylmethionine (AdoMet) synthetase catalyzes the biosynthesis of AdoMet in a unique enzymatic reaction. Initially the sulfur of methionine displaces the intact tripolyphosphate chain (PPP(i)) from ATP, and subsequently PPP(i) is hydrolyzed to PP(i) and P(i) before product release. The crystal structure of Escherichia coli AdoMet synthetase shows that the active site contains four aspartate residues. Aspartate residues Asp-16* and Asp-271 individually provide the sole protein ligand to one of the two required Mg(2+) ions (* denotes a residue from a second subunit); aspartates Asp-118 and Asp-238* are proposed to interact with methionine. Each aspartate has been changed to an uncharged asparagine, and the metal binding residues were also changed to alanine, to assess the roles of charge and ligation ability on catalytic efficiency. The resultant enzyme variants all structurally resemble the wild type enzyme as indicated by circular dichroism spectra and are tetramers. However, all have k(cat) reductions of approximately 10(3)-fold in AdoMet synthesis, whereas the MgATP and methionine K(m) values change by less than 3- and 8-fold, respectively. In the partial reaction of PPP(i) hydrolysis, mutants of the Mg(2+) binding residues have >700-fold reduced catalytic efficiency (k(cat)/K(m)), whereas the D118N and D238*N mutants are impaired less than 35-fold. The catalytic efficiency for PPP(i) hydrolysis by Mg(2+) site mutants is improved by AdoMet, like the wild type enzyme. In contrast AdoMet reduces the catalytic efficiency for PPP(i) hydrolysis by the D118N and D238*N mutants, indicating that the events involved in AdoMet activation are hindered in these methionyl binding site mutants. Ca(2+) uniquely activates the D271A mutant enzyme to 15% of the level of Mg(2+), in contrast to the approximately 1% Ca(2+) activation of the wild type enzyme. This indicates that the Asp-271 side chain size is a discriminator between the activating ability of Ca(2+) and the

  16. Antigen Binding and Site-Directed Labeling of Biosilica-Immobilized Fusion Proteins Expressed in Diatoms.

    PubMed

    Ford, Nicole R; Hecht, Karen A; Hu, DeHong; Orr, Galya; Xiong, Yijia; Squier, Thomas C; Rorrer, Gregory L; Roesijadi, Guritno

    2016-03-18

    The diatom Thalassiosira pseudonana was genetically modified to express biosilica-targeted fusion proteins comprising either enhanced green fluorescent protein (EGFP) or single chain antibodies engineered with a tetracysteine tagging sequence. Of interest were the site-specific binding of (1) the fluorescent biarsenical probe AsCy3 and AsCy3e to the tetracysteine tagged fusion proteins and (2) high and low molecular mass antigens, the Bacillus anthracis surface layer protein EA1 or small molecule explosive trinitrotoluene (TNT), to biosilica-immobilized single chain antibodies. Analysis of biarsenical probe binding using fluorescence and structured illumination microscopy indicated differential colocalization with EGFP in nascent and mature biosilica, supporting the use of either EGFP or bound AsCy3 and AsCy3e in studying biosilica maturation. Large increases in the lifetime of a fluorescent analogue of TNT upon binding single chain antibodies provided a robust signal capable of discriminating binding to immobilized antibodies in the transformed frustule from nonspecific binding to the biosilica matrix. In conclusion, our results demonstrate an ability to engineer diatoms to create antibody-functionalized mesoporous silica able to selectively bind chemical and biological agents for the development of sensing platforms.

  17. Label-free molecular beacon for real-time monitoring of DNA polymerase activity.

    PubMed

    Ma, Changbei; Liu, Haisheng; Wang, Jun; Jin, Shunxin; Wang, Kemin

    2016-05-01

    Traditional methods for assaying DNA polymerase activity are discontinuous, time consuming, and laborious. Here, we report a new approach for label-free and real-time monitoring of DNA polymerase activity using a Thioflavin T (ThT) probe. In the presence of DNA polymerase, the DNA primer could be elongated through polymerase reaction to open MB1, leading to the release of the G-quartets. These then bind to ThT to form ThT/G-quadruplexes with an obvious fluorescence generation. It exhibits a satisfying detection result for the activity of DNA polymerase with a low detection limit of 0.05 unit/ml. In addition, no labeling with a fluorophore or a fluorophore-quencher pair is required; this method is fairly simple, fast, and low cost. Furthermore, the proposed method was also applied to assay the inhibition of DNA polymerase activity. This approach may offer potential applications in drug screening, clinical diagnostics, and some other related biomedical research. PMID:26894757

  18. Label-free molecular beacon for real-time monitoring of DNA polymerase activity.

    PubMed

    Ma, Changbei; Liu, Haisheng; Wang, Jun; Jin, Shunxin; Wang, Kemin

    2016-05-01

    Traditional methods for assaying DNA polymerase activity are discontinuous, time consuming, and laborious. Here, we report a new approach for label-free and real-time monitoring of DNA polymerase activity using a Thioflavin T (ThT) probe. In the presence of DNA polymerase, the DNA primer could be elongated through polymerase reaction to open MB1, leading to the release of the G-quartets. These then bind to ThT to form ThT/G-quadruplexes with an obvious fluorescence generation. It exhibits a satisfying detection result for the activity of DNA polymerase with a low detection limit of 0.05 unit/ml. In addition, no labeling with a fluorophore or a fluorophore-quencher pair is required; this method is fairly simple, fast, and low cost. Furthermore, the proposed method was also applied to assay the inhibition of DNA polymerase activity. This approach may offer potential applications in drug screening, clinical diagnostics, and some other related biomedical research.

  19. The active site of ribulose-bisphosphate carboxylase/oxygenase

    SciTech Connect

    Hartman, F.C.

    1991-01-01

    The active site of ribulose-bisphosphate carboxylase/oxygenase requires interacting domains of adjacent, identical subunits. Most active-site residues are located within the loop regions of an eight-stranded {beta}/{alpha}-barrel which constitutes the larger C-terminal domain; additional key residues are located within a segment of the smaller N-terminal domain which partially covers the mouth of the barrel. Site-directed mutagenesis of the gene encoding the enzyme from Rhodospirillum rubrum has been used to delineate functions of active-site residues. 6 refs., 2 figs.

  20. In vivo labeling of 5-hydroxytryptamine uptake sites in mouse brain with ( sup 3 H)-6-nitroquipazine

    SciTech Connect

    Hashimoto, K.; Goromaru, T. )

    1990-10-01

    6-Nitroquipazine (DU 24565; 6-nitro 2-piperazinylquinoline) is a very potent 5-hydroxytryptamine (5-HT; serotonin) uptake inhibitor. It has been demonstrated very recently that (3H)-6-nitroquipazine is a suitable radioligand for studying 5-HT uptake sites. The present study evaluates (3H)6-nitroquipazine as a radioligand for in vivo labeling of 5-HT uptake sites in mouse brain. Very high uptake of radioactivity in the brain after i.v. administration of (3H)-6-nitroquipazine was shown. Regional distribution of the radioactivity in mouse brain 3 hr after injection of (3H)-6-nitroquipazine was in the order (highest to lowest) hypothalamus greater than midbrain greater than striatum greater than hippocampus greater than cerebral cortex greater than medulla oblongata greater than cerebellum. The regional distribution of in vivo (3H)-6-nitroquipazine binding in mouse brain was highly correlated with that in rat brain obtained from previous in vitro binding studies. Coadministration of carrier 6-nitroquipazine (5 mg/kg) significantly decreased the radioactivity in the hypothalamus, whereas that in the cerebellum and cerebral cortex was increased. Because the cerebellum has very low density of (3H)-6-nitroquipazine binding sites, the radioactivity in the cerebellum could, therefore, reflect the amount on nonspecific binding and free ligand. Kinetic studies showed highest in vivo specific binding 1 hr after injection of (3H)-6-nitroquipazine and slow clearance of specific binding. Specific binding in the hypothalamus was inhibited in a stereoselective manner by the stereoisomers of norzimelidine. Furthermore, specific binding in the hypothalamus was reduced by several 5-HT uptake inhibitors, in a dose-dependent manner.

  1. Structure of the high-affinity binding site for noncompetitive blockers of the acetylcholine receptor: serine-262 of the delta subunit is labeled by [3H]chlorpromazine.

    PubMed Central

    Giraudat, J; Dennis, M; Heidmann, T; Chang, J Y; Changeux, J P

    1986-01-01

    The membrane-bound acetylcholine receptor from Torpedo marmorata was photolabeled by the noncompetitive channel blocker [3H]chlorpromazine under equilibrium conditions in the presence of agonist. Incorporation of radioactivity into all subunits occurred and was reduced by addition of phencyclidine, a specific ligand for the high-affinity site for noncompetitive blockers. The delta subunit was purified and digested with trypsin, and the resulting fragments were fractionated by reversed-phase HPLC. The labeled peptide could not be purified to homogeneity because of its marked hydrophobic character, but a combination of differential CNBr subcleavage and cosequencing of partially purified fragments enabled us to identify Ser-262 as being labeled by [3H]chlorpromazine. The labeling of this particular residue was prevented by phencyclidine and thus took place at the level of, or in proximity to, the high-affinity site for noncompetitive blockers. Ser-262 is located in a hydrophobic and potentially transmembrane segment termed MII. Images PMID:3085104

  2. Savannah River Site prioritization of transition activities

    SciTech Connect

    Finley, R.H.

    1993-11-01

    Effective management of SRS conversion from primarily a production facility to other missions (or Decontamination and Decommissioning (D&D)) requires a systematic and consistent method of prioritizing the transition activities. This report discusses the design of a prioritizing method developed to achieve systematic and consistent methods of prioritizing these activities.

  3. DOE site performance assessment activities. Radioactive Waste Technical Support Program

    SciTech Connect

    Not Available

    1990-07-01

    Information on performance assessment capabilities and activities was collected from eight DOE sites. All eight sites either currently dispose of low-level radioactive waste (LLW) or plan to dispose of LLW in the near future. A survey questionnaire was developed and sent to key individuals involved in DOE Order 5820.2A performance assessment activities at each site. The sites surveyed included: Hanford Site (Hanford), Idaho National Engineering Laboratory (INEL), Los Alamos National Laboratory (LANL), Nevada Test Site (NTS), Oak Ridge National Laboratory (ORNL), Paducah Gaseous Diffusion Plant (Paducah), Portsmouth Gaseous Diffusion Plant (Portsmouth), and Savannah River Site (SRS). The questionnaire addressed all aspects of the performance assessment process; from waste source term to dose conversion factors. This report presents the information developed from the site questionnaire and provides a comparison of site-specific performance assessment approaches, data needs, and ongoing and planned activities. All sites are engaged in completing the radioactive waste disposal facility performance assessment required by DOE Order 5820.2A. Each site has achieved various degrees of progress and have identified a set of critical needs. Within several areas, however, the sites identified common needs and questions.

  4. Safety Oversight of Decommissioning Activities at DOE Nuclear Sites

    SciTech Connect

    Zull, Lawrence M.; Yeniscavich, William

    2008-01-15

    The Defense Nuclear Facilities Safety Board (Board) is an independent federal agency established by Congress in 1988 to provide nuclear safety oversight of activities at U.S. Department of Energy (DOE) defense nuclear facilities. The activities under the Board's jurisdiction include the design, construction, startup, operation, and decommissioning of defense nuclear facilities at DOE sites. This paper reviews the Board's safety oversight of decommissioning activities at DOE sites, identifies the safety problems observed, and discusses Board initiatives to improve the safety of decommissioning activities at DOE sites. The decommissioning of former defense nuclear facilities has reduced the risk of radioactive material contamination and exposure to the public and site workers. In general, efforts to perform decommissioning work at DOE defense nuclear sites have been successful, and contractors performing decommissioning work have a good safety record. Decommissioning activities have recently been completed at sites identified for closure, including the Rocky Flats Environmental Technology Site, the Fernald Closure Project, and the Miamisburg Closure Project (the Mound site). The Rocky Flats and Fernald sites, which produced plutonium parts and uranium materials for defense needs (respectively), have been turned into wildlife refuges. The Mound site, which performed R and D activities on nuclear materials, has been converted into an industrial and technology park called the Mound Advanced Technology Center. The DOE Office of Legacy Management is responsible for the long term stewardship of these former EM sites. The Board has reviewed many decommissioning activities, and noted that there are valuable lessons learned that can benefit both DOE and the contractor. As part of its ongoing safety oversight responsibilities, the Board and its staff will continue to review the safety of DOE and contractor decommissioning activities at DOE defense nuclear sites.

  5. Site-directed isotope labeling and FTIR spectroscopy: assignment of tyrosine bands in the bR-->M difference spectrum of bacteriorhodopsin.

    PubMed

    Liu, X M; Sonar, S; Lee, C P; Coleman, M; RajBhandary, U L; Rothschild, K J

    1995-01-01

    Fourier transform infrared difference spectroscopy has been used extensively to probe structural changes in bacteriorthodopsin and other retinal proteins. However, the absence of a general method to assign bands to individual chemical groups in a protein has limited the application of this technique. While site-directed mutagenesis has been successful in special cases for such assignments, in general, this approach induces perturbations in the structure and function of the protein, thereby preventing unambiguous band assignments. A new approach has recently been reported (Sonar et al., Nature Struct. Biol. 1 (1994) 512-517) which involves cell-free expression of bacteriorhodopsin and site-directed isotope labeling (SDIL). We have now used this method to re-examine bands assigned in the bR-->M difference spectrum to tyrosine residues. Our results show that out of 11 tyrosines in bR, only Tyr 185 is structurally active. This work further demonstrates the power of SDIL and FTIR to probe conformational changes at the level of individual amino acid residues in proteins.

  6. Conformation of T4 lysozyme in solution. Hinge-bending motion and the substrate-induced conformational transition studied by site-directed spin labeling.

    PubMed

    Mchaourab, H S; Oh, K J; Fang, C J; Hubbell, W L

    1997-01-14

    T4 lysozyme and mutants thereof crystallize in different conformations that are related to each other by a bend about a hinge in the molecule. This observation suggests that the wild type protein may undergo a hinge-bending motion in solution to allow substrate access to an otherwise closed active site cleft [Faber, H.R., & Matthews, B.W. (1990) Nature 348, 263-266]. To test this hypothesis, either single or pairs of nitroxide side chains were introduced into the protein to monitor tertiary contact interactions and inter-residue distances, respectively, in solution. A set of constraints for these structural parameters was derived from a reference state, a covalent enzyme-substrate adduct where the enzyme is locked in the closed state. In the absence of substrate, differences in both inter-residue distances and tertiary contact interactions relative to this reference state are consistent with a hinge-bending motion that opens the active site cleft. Quantitative analysis of spin-spin interactions between nitroxide pairs reveals an 8 A relative domain movement upon substrate binding. In addition, it is demonstrated that the I3P mutation, which produces a large hinge-bending angle in the crystal, has no effect on the solution conformation. Thus, the hinge motion is not the result of the mutation but is an integral part of T4 lysozyme catalysis in solution, as suggested recently [Zhang, X.J., Wozniak, J.A., & Matthews, B.W. (1995) J. Mol. Biol. 250, 527-552]. The strategy employed here, based on site-directed spin labeling, should be generally applicable to the study of protein conformation and conformational changes in solution.

  7. Three-step preparation and purification of phosphorus-33-labeled creatine phosphate of high specific activity

    SciTech Connect

    Savabi, F.; Geiger, P.J.; Bessman, S.P.

    1984-03-01

    Rabbit heart mitochondria were used as a source of enzymes for the synthesis of phosphorus-labeled creatine phosphate. This method is based on the coupled reaction between mitochondrial oxidative phosphorylation and mitochondrial-bound creatine kinase. It is possible to convert more than 90% of the inorganic phosphate (P/sub i/) to creatine phosphate. The method used only small amounts of adenine nucleotides which led to a product with only slight nucleotide contamination. This could be removed by activated charcoal extraction. For further purification, a method for the removal of residual P/sub i/ is described. 20 references.

  8. Synthesis of nucleoside 5'-tetraphosphates containing terminal fluorescent labels via activated cyclic trimetaphosphate.

    PubMed

    Mohamady, Samy; Taylor, Scott D

    2014-03-01

    2'-Deoxynucleotide 5'-tetraphosphates in which a fluorescent label is attached to the terminal phosphate are used as key reagents in high-throughput DNA sequencing techniques and in single nucleotide polymorphism typing assays. We demonstrate that this class of compounds can be prepared by reacting fluorophores such as 7-hydroxy-4-methylcoumarin, methylfluorescein, fluorescein and resorufin with an activated form of cyclic trimetaphosphate to give intermediate 11. Reaction of 11 with 2'-deoxynucleoside 5'-monophosphates or a nucleoside 5'-monophosphate gave the target compounds in good yield. PMID:24552623

  9. Ionizable Side Chains at Catalytic Active Sites of Enzymes

    PubMed Central

    Jimenez-Morales, David; Liang, Jie

    2012-01-01

    Catalytic active sites of enzymes of known structure can be well defined by a modern program of computational geometry. The CASTp program was used to define and measure the volume of the catalytic active sites of 573 enzymes in the Catalytic Site Atlas database. The active sites are identified as catalytic because the amino acids they contain are known to participate in the chemical reaction catalyzed by the enzyme. Acid and base side chains are reliable markers of catalytic active sites. The catalytic active sites have 4 acid and 5 base side chains, in an average volume of 1072 Å3. The number density of acid side chains is 8.3 M (in chemical units); the number density of basic side chains is 10.6 M. The catalytic active site of these enzymes is an unusual electrostatic and steric environment in which side chains and reactants are crowded together in a mixture more like an ionic liquid than an ideal infinitely dilute solution. The electrostatics and crowding of reactants and side chains seems likely to be important for catalytic function. In three types of analogous ion channels, simulation of crowded charges accounts for the main properties of selectivity measured in a wide range of solutions and concentrations. It seems wise to use mathematics designed to study interacting complex fluids when making models of the catalytic active sites of enzymes. PMID:22484856

  10. Multisubstrate Isotope Labeling and Metagenomic Analysis of Active Soil Bacterial Communities

    PubMed Central

    Verastegui, Y.; Cheng, J.; Engel, K.; Kolczynski, D.; Mortimer, S.; Lavigne, J.; Montalibet, J.; Romantsov, T.; Hall, M.; McConkey, B. J.; Rose, D. R.; Tomashek, J. J.; Scott, B. R.

    2014-01-01

    ABSTRACT Soil microbial diversity represents the largest global reservoir of novel microorganisms and enzymes. In this study, we coupled functional metagenomics and DNA stable-isotope probing (DNA-SIP) using multiple plant-derived carbon substrates and diverse soils to characterize active soil bacterial communities and their glycoside hydrolase genes, which have value for industrial applications. We incubated samples from three disparate Canadian soils (tundra, temperate rainforest, and agricultural) with five native carbon (12C) or stable-isotope-labeled (13C) carbohydrates (glucose, cellobiose, xylose, arabinose, and cellulose). Indicator species analysis revealed high specificity and fidelity for many uncultured and unclassified bacterial taxa in the heavy DNA for all soils and substrates. Among characterized taxa, Actinomycetales (Salinibacterium), Rhizobiales (Devosia), Rhodospirillales (Telmatospirillum), and Caulobacterales (Phenylobacterium and Asticcacaulis) were bacterial indicator species for the heavy substrates and soils tested. Both Actinomycetales and Caulobacterales (Phenylobacterium) were associated with metabolism of cellulose, and Alphaproteobacteria were associated with the metabolism of arabinose; members of the order Rhizobiales were strongly associated with the metabolism of xylose. Annotated metagenomic data suggested diverse glycoside hydrolase gene representation within the pooled heavy DNA. By screening 2,876 cloned fragments derived from the 13C-labeled DNA isolated from soils incubated with cellulose, we demonstrate the power of combining DNA-SIP, multiple-displacement amplification (MDA), and functional metagenomics by efficiently isolating multiple clones with activity on carboxymethyl cellulose and fluorogenic proxy substrates for carbohydrate-active enzymes. PMID:25028422

  11. Structural arrangement of tRNA binding sites on Escherichia coli ribosomes, as revealed from data on affinity labelling with photoactivatable tRNA derivatives.

    PubMed

    Graifer, D M; Babkina, G T; Matasova, N B; Vladimirov, S N; Karpova, G G; Vlassov, V V

    1989-07-01

    A systematic study of protein environment of tRNA in ribosomes in model complexes representing different translation steps was carried out using the affinity labelling of the ribosomes with tRNA derivatives bearing aryl azide groups scattered statistically over tRNA guanine residues. Analysis of the proteins crosslinked to tRNA derivatives showed that the location of the derivatives in the aminoacyl (A) site led to the labelling of the proteins S5 and S7 in all complexes studied, whereas the labelling of the proteins S2, S8, S9, S11, S14, S16, S17, S18, S19, S21 as well as L9, L11, L14, L15, L21, L23, L24, L29 depended on the state of tRNA in A site. Similarly, the location of tRNA derivatives in the peptidyl (P) site resulted in the labelling of the proteins L27, S11, S13 and S19 in all states, whereas the labelling of the proteins S5, S7, S9, S12, S14, S20, S21 as well as L2, L13, L14, L17, L24, L27, L31, L32, L33 depended on the type of complex. The derivatives of tRNA(fMet) were found to crosslink to S1, S3, S5, S7, S9, S14 and L1, L2, L7/L12, L27. Based on the data obtained, a general principle of the dynamic functioning of ribosomes has been proposed: (i) the formation of each type of ribosomal complex is accompanied by changes in mutual arrangement of proteins - 'conformational adjustment' of the ribosome - and (ii) a ribosome can dynamically change its internal structure at each step of initiation and elongation; on the 70 S ribosome there are no rigidly fixed structures forming tRNA-binding sites (primarily A and P sites).

  12. Circular dichroism and site-directed spin labeling reveal structural and dynamical features of high-pressure states of myoglobin

    PubMed Central

    Lerch, Michael T.; Horwitz, Joseph; McCoy, John; Hubbell, Wayne L.

    2013-01-01

    Excited states of proteins may play important roles in function, yet are difficult to study spectroscopically because of their sparse population. High hydrostatic pressure increases the equilibrium population of excited states, enabling their characterization [Akasaka K (2003) Biochemistry 42:10875–85]. High-pressure site-directed spin-labeling EPR (SDSL-EPR) was developed recently to map the site-specific structure and dynamics of excited states populated by pressure. To monitor global secondary structure content by circular dichroism (CD) at high pressure, a modified optical cell using a custom MgF2 window with a reduced aperture is introduced. Here, a combination of SDSL-EPR and CD is used to map reversible structural transitions in holomyoglobin and apomyoglobin (apoMb) as a function of applied pressure up to 2 kbar. CD shows that the high-pressure excited state of apoMb at pH 6 has helical content identical to that of native apoMb, but reversible changes reflecting the appearance of a conformational ensemble are observed by SDSL-EPR, suggesting a helical topology that fluctuates slowly on the EPR time scale. Although the high-pressure state of apoMb at pH 6 has been referred to as a molten globule, the data presented here reveal significant differences from the well-characterized pH 4.1 molten globule of apoMb. Pressure-populated states of both holomyoglobin and apoMb at pH 4.1 have significantly less helical structure, and for the latter, that may correspond to a transient folding intermediate. PMID:24248390

  13. Circular dichroism and site-directed spin labeling reveal structural and dynamical features of high-pressure states of myoglobin.

    PubMed

    Lerch, Michael T; Horwitz, Joseph; McCoy, John; Hubbell, Wayne L

    2013-12-01

    Excited states of proteins may play important roles in function, yet are difficult to study spectroscopically because of their sparse population. High hydrostatic pressure increases the equilibrium population of excited states, enabling their characterization [Akasaka K (2003) Biochemistry 42:10875-85]. High-pressure site-directed spin-labeling EPR (SDSL-EPR) was developed recently to map the site-specific structure and dynamics of excited states populated by pressure. To monitor global secondary structure content by circular dichroism (CD) at high pressure, a modified optical cell using a custom MgF2 window with a reduced aperture is introduced. Here, a combination of SDSL-EPR and CD is used to map reversible structural transitions in holomyoglobin and apomyoglobin (apoMb) as a function of applied pressure up to 2 kbar. CD shows that the high-pressure excited state of apoMb at pH 6 has helical content identical to that of native apoMb, but reversible changes reflecting the appearance of a conformational ensemble are observed by SDSL-EPR, suggesting a helical topology that fluctuates slowly on the EPR time scale. Although the high-pressure state of apoMb at pH 6 has been referred to as a molten globule, the data presented here reveal significant differences from the well-characterized pH 4.1 molten globule of apoMb. Pressure-populated states of both holomyoglobin and apoMb at pH 4.1 have significantly less helical structure, and for the latter, that may correspond to a transient folding intermediate. PMID:24248390

  14. Characterization of biomimetic calcium phosphate labeled with fluorescent dextran for quantification of osteoclastic activity.

    PubMed

    Maria, Salwa M; Prukner, Christiane; Sheikh, Zeeshan; Müller, Frank A; Komarova, Svetlana V; Barralet, Jake E

    2015-07-01

    Bone resorbing osteoclasts represent an important therapeutic target for diseases associated with bone and joint destruction, such as rheumatoid arthritis, periodontitis, and osteoporosis. The quantification of osteoclast resorptive activity in vitro is widely used for screening new anti-resorptive medications. The aim of this paper was to develop a simplified semi-automated method for the quantification of osteoclastic resorption using fluorescently labeled biomimetic mineral layers which can replace time intensive, often subjective and clearly non-sustainable use of translucent slices of tusks from vulnerable or endangered species such as the elephant. Osteoclasts were formed from RAW 264.7 mouse monocyte cell line using the pro-resorptive cytokine receptor activator of nuclear factor kappa-B ligand (RANKL). We confirmed that fluorescent labeling did not interfere with the biomimetic features of hydroxyapatite, and developed an automated method for quantifying osteoclastic resorption. Correlation between our assay and traditional manual measurement techniques was found to be very strong (R(2)=0.99). In addition, we modified the technique to provide depth and volume data of the resorption pits by confocal imaging at defined depths. Thus, our method allows automatic quantification of total osteoclastic resorption as well as additional data not obtainable by the current tusk slice technique offering a better alternative for high throughput screening of potential antiresorptives.

  15. Active site histidine in spinach ribulosebisphosphate carboxylase/oxygenase modified by diethyl pyrocarbonate

    SciTech Connect

    Igarashi, Y.; McFadden, B.A.; el-Gul, T.

    1985-07-16

    (TH) Diethyl pyrocarbonate was synthesized from (TH) ethanol prepared by the reduction of acetaldehyde by NaB3H4. Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from spinach was inactivated with this reagent at pH 7.0 the presence of 20 mM MgS , and tryptic peptides that contained modified histidine residues were isolated by reverse-phase high-performance liquid chromatography. Labeling of the enzyme was conducted in the presence and absence of the competitive inhibitor sedoheptulose 1,7-bisphosphate. The amount of one peptide that was heavily labeled in the absence of this compound was reduced 10-fold in its presence. The labeled residue was histidine-298. This result, in combination with earlier experiments, suggests that His-298 in spinach RuBisCO is located in the active site domain and is essential to enzyme activity. This region of the primary structure is strongly conserved in seven other ribulosebisphosphate carboxylases from divergent sources.

  16. Preliminary studies of acetylcholinesterase activity in the rat brain using N-phenylferrocenecarboxamide labelled by the technetium-99m.

    PubMed

    Mejri, Najoua; Said, Nadia Malek; Guizani, Sihem; Essouissi, Imen; Saidi, Mouldi

    2013-05-01

    There is currently great interest in developing radiolabeled substrates for acetylcholinesterase that would be useful in the in vivo imaging of patients with Alzheimer's disease. The reduction of acetylcholinesterase (AChE) activity in the brain has been measured in dementia disorders such as Alzheimer's disease and dementia with Lewy bodies using (11)C and (18)F-labeled acetylcholine analogues. Our aim was to develop a new 99mTc-labeled acetylcholine analogue: N-phenylferrocenecarboxamide labelled with technetium-99m (99mTc-TPCC) to study acetylcholinesterase activity. In vivo and in vitro studies demonstrated that the labelled compound was a substrate for acetylcholinesterase. The hydrolytic rate of this substrate was measured and the specificity was evaluated using the inhibitor BW 284 C51. In rat experiments, the 99mTc-TPCC showed desirable properties for studying the acetylcholinesterase in the rat brain: high hydrolytic rate and a moderate specificity of the substrate for acetylcholinesterase.

  17. Conformational Properties of Seven Toac-Labeled Angiotensin I Analogues Correlate with Their Muscle Contraction Activity and Their Ability to Act as ACE Substrates

    PubMed Central

    Teixeira, Luis Gustavo D.; Malavolta, Luciana; Bersanetti, Patrícia A.; Schreier, Shirley; Carmona, Adriana K.; Nakaie, Clovis R.

    2015-01-01

    Conformational properties of the angiotensin II precursor, angiotensin I (AngI) and analogues containing the paramagnetic amino acid TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) at positions 0, 1, 3, 5, 8, 9, and 10, were examined by EPR, CD, and fluorescence. The conformational data were correlated to their activity in muscle contraction experiments and to their properties as substrates of the angiotensin I-converting enzyme (ACE). Biological activity studies indicated that TOAC0-AngI and TOAC1-AngI maintained partial potency in guinea pig ileum and rat uterus. Kinetic parameters revealed that only derivatives labeled closer to the N-terminus (positions 0, 1, 3, and 5) were hydrolyzed by ACE, indicating that peptides bearing the TOAC moiety far from the ACE cleavage site (Phe8-His9 peptide bond) were susceptible to hydrolysis, albeit less effectively than the parent compound. CD spectra indicated that AngI exhibited a flexible structure resulting from equilibrium between different conformers. While the conformation of N-terminally-labeled derivatives was similar to that of the native peptide, a greater propensity to acquire folded structures was observed for internally-labeled, as well as C-terminally labeled, analogues. These structures were stabilized in secondary structure-inducing agent, TFE. Different analogues gave rise to different β-turns. EPR spectra in aqueous solution also distinguished between N-terminally, internally-, and C-terminally labeled peptides, yielding narrower lines, indicative of greater mobility for the former. Interestingly, the spectra of peptides labeled at, or close, to the C-terminus, showed that the motion in this part of the peptides was intermediate between that of N-terminally and internally-labeled peptides, in agreement with the suggestion of turn formation provided by the CD spectra. Quenching of the Tyr4 fluorescence by the differently positioned TOAC residues corroborated the data obtained by the

  18. Nitroxide spin labels as EPR reporters of the relaxation and magnetic properties of the heme-copper site in cytochrome bo3, E. coli.

    PubMed

    Oganesyan, Vasily S; White, Gaye F; Field, Sarah; Marritt, Sophie; Gennis, Robert B; Yap, Lai Lai; Thomson, Andrew J

    2010-11-01

    A nitroxide spin label (SL) has been used to probe the electron spin relaxation times and the magnetic states of the oxygen-binding heme-copper dinuclear site in Escherichia coli cytochrome bo(3), a quinol oxidase (QO), in different oxidation states. The spin lattice relaxation times, T(1), of the SL are enhanced by the paramagnetic metal sites in QO and hence show a strong dependence on the oxidation state of the latter. A new, general form of equations and a computer simulation program have been developed for the calculation of relaxation enhancement by an arbitrary fast relaxing spin system of S ≥ 1/2. This has allowed us to obtain an accurate estimate of the transverse relaxation time, T (2), of the dinuclear coupled pair Fe(III)-Cu(B)(II) in the oxidized form of QO that is too short to measure directly. In the case of the F' state, the relaxation properties of the heme-copper center have been shown to be consistent with a ferryl [Fe(IV)=O] heme and Cu(B)(II) coupled by approximately 1.5-3 cm(-1) to a radical. The magnitude suggests that the coupling arises from a radical form of the covalently linked tyrosine-histidine ligand to Cu(II) with unpaired spin density primarily on the tyrosine component. This work demonstrates that nitroxide SLs are potentially valuable tools to probe both the relaxation and the magnetic properties of multinuclear high-spin paramagnetic active sites in proteins that are otherwise not accessible from direct EPR measurements.

  19. Labelling a single particle for positron emission particle tracking using direct activation and ion-exchange techniques

    NASA Astrophysics Data System (ADS)

    Fan, X.; Parker, D. J.; Smith, M. D.

    2006-06-01

    Positron emission particle tracking (PEPT) is a non-invasive technique used for obtaining dynamic information within multiphase systems. It involves tracking a single radioactively labelled tracer particle. The tracking efficiency and representative of PEPT data are crucially dependent on the amount of radioactivity labelled in a single particle, as well as the physical and chemical properties of a tracer. This paper will discuss the effect of tracer properties on PEPT data and two labelling techniques, direct activation and ion-exchange, in detail. In direct activation, particles are directly bombarded using a 33 MeV 3He beam. A few of the oxygen atoms in the particles are then converted into 18F radioisotope. Direct activation can be used to label a particle with a size range from 1 to 10 mm, but the material must be able to resist a high temperature. The ion-exchange technique can be used to label smaller resin particles with a size ranging from 60 to 1000 μm. The radioactivity labelled in a single resin bead is controlled by ion-exchange properties of the resin material, anions present in the radioactive water and processing time.

  20. 78 FR 4153 - Agency Information Collection Activities; Proposed Collection; Comment Request; Food Labeling...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-01-18

    ... Collection; Comment Request; Food Labeling; Notification Procedures for Statements on Dietary Supplements... requiring manufacturers, packers, and distributors of dietary supplements to notify us that they are marketing a dietary supplement product that bears on its label or in its labeling a statement provided...

  1. Label free detection of optogenetically stimulated cellular activity by low coherence interferometry (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Satpathy, Sarmishtha; Batabyal, Subrata; Dave, Digant P.; Mohanty, Samarendra K.

    2016-03-01

    Detecting cellular activity in sub-millisecond timescale and micrometer resolution without using invasive means has been a long standing goal in the study of cellular networks. Here, we have employed phase sensitive low coherence interferometry for detecting optogenetically stimulated activity of cells. Nanoscale changes in optical path length (due to change in refractive index and changes in cell thickness) occur when cells are activated, which we aim to detect by phase sensitive low coherence interferometry. A low coherence interferometry and patch-clamp electrophysiology systems were integrated with an inverted fluorescence microscope. Blue laser beam was coupled to the electrophysiology-interferometric detection system for optogenetic stimulation. The phase-sensitive measurements were carried out on Channelrhodopsin-2 sensitized cells (identified by YFP fluorescence) as well as control cells in reflection mode for different intensities and exposures of optogenetic stimulation beam. This method offers good temporal and spatial resolution without using exogenous labeling. Results of studies on all optical stimulation and detection of cellular activity will be presented. Interpretation of the optical activity signals will be discussed in context with changes in cell physiology during stimulation. We will also discuss the potential sources of various artifacts in optical/electrical detection of cellular activity during optical stimulation.

  2. Label-free electrochemical impedance detection of kinase and phosphatase activities using carbon nanofiber nanoelectrode arrays

    PubMed Central

    Li, Yifen; Syed, Lateef; Liu, Jianwei; Hua, Duy H.; Li, Jun

    2012-01-01

    We demonstrate the feasibility of a label-free electrochemical method to detect the kinetics of phosphorylation and dephosphorylation of surface-attached peptides catalyzed by kinase and phosphatase, respectively. The peptides with a sequence specific to c-Src tyrosine kinase and protein tyrosine phosphatase 1B (PTP1B) were first validated with ELISA-based protein tyrosine kinase assay and then functionalized on vertically aligned carbon nanofiber (VACNF) nanoelectrode arrays (NEAs). Real-time electrochemical impedance spectroscopy (REIS) measurements showed reversible impedance changes upon the addition of c-Src kinase and PTP1B phosphatase. Only a small and unreliable impedance variation was observed during the peptide phosphorylation, but a large and fast impedance decrease was observed during the peptide dephosphorylation at different PTP1B concentrations. The REIS data of dephosphorylation displayed a well-defined exponential decay following the Michaelis-Menten heterogeneous enzymatic model with a specific constant, kcat/Km, of (2.1 ± 0.1) × 107 M−1 s−1. Consistent values of the specific constant was measured at PTP1B concentration varying from 1.2 to 2.4 nM with the corresponding electrochemical signal decay constant varying from 38.5 to 19.1 s. This electrochemical method can be potentially used as a label-free method for profiling enzyme activities in fast reactions. PMID:22935373

  3. Active site - a site of binding of affinity inhibitors in baker's yeast inorganic pyrophosphatase

    SciTech Connect

    Svyato, I.E.; Sklyankina, V.A.; Avaeva, S.M.

    1986-03-20

    The interaction of the enzyme-substrate complex with methyl phosphate, O-phosphoethanolamine, O-phosphopropanolamine, N-acetylphosphoserine, and phosphoglyolic acid, as well as pyrophosphatase, modified by monoesters of phosphoric acid, with pyrophosphate and tripolyphosphate, was investigated. It was shown that the enzyme containing the substrate in the active site does not react with monophosphates, but modified pyrophosphatase entirely retains the ability to bind polyanions to the regulatory site. It is concluded that the inactivation of baker's yeast inorganic pyrophosphatase by monoesters of phosphoric acid, which are affinity inhibitors of it, is the result of modification of the active site of the enzyme.

  4. A novel approach to predict active sites of enzyme molecules.

    PubMed

    Chou, Kuo-Chen; Cai, Yu-dong

    2004-04-01

    Enzymes are critical in many cellular signaling cascades. With many enzyme structures being solved, there is an increasing need to develop an automated method for identifying their active sites. However, given the atomic coordinates of an enzyme molecule, how can we predict its active site? This is a vitally important problem because the core of an enzyme molecule is its active site from the viewpoints of both pure scientific research and industrial application. In this article, a topological entity was introduced to characterize the enzymatic active site. Based on such a concept, the covariant discriminant algorithm was formulated for identifying the active site. As a paradigm, the serine hydrolase family was demonstrated. The overall success rate by jackknife test for a data set of 88 enzyme molecules was 99.92%, and that for a data set of 50 independent enzyme molecules was 99.91%. Meanwhile, it was shown through an example that the prediction algorithm can also be used to find any typographic error of a PDB file in annotating the constituent amino acids of catalytic triad and to suggest a possible correction. The very high success rates are due to the introduction of a covariance matrix in the prediction algorithm that makes allowance for taking into account the coupling effects among the key constituent atoms of active site. It is anticipated that the novel approach is quite promising and may become a useful high throughput tool in enzymology, proteomics, and structural bioinformatics. PMID:14997541

  5. Structure of membrane-bound α-synuclein from site-directed spin labeling and computational refinement

    PubMed Central

    Jao, Christine C.; Hegde, Balachandra G.; Chen, Jeannie; Haworth, Ian S.; Langen, Ralf

    2008-01-01

    α-Synuclein is known to play a causative role in Parkinson disease. Although its physiological functions are not fully understood, α-synuclein has been shown to interact with synaptic vesicles and modulate neurotransmitter release. However, the structure of its physiologically relevant membrane-bound state remains unknown. Here we developed a site-directed spin labeling and EPR-based approach for determining the structure of α-synuclein bound to a lipid bilayer. Continuous-wave EPR was used to assign local secondary structure and to determine the membrane immersion depth of lipid-exposed residues, whereas pulsed EPR was used to map long-range distances. The structure of α-synuclein was built and refined by using simulated annealing molecular dynamics restrained by the immersion depths and distances. We found that α-synuclein forms an extended, curved α-helical structure that is over 90 aa in length. The monomeric helix has a superhelical twist similar to that of right-handed coiled-coils which, like α-synuclein, contain 11-aa repeats, but which are soluble, oligomeric proteins (rmsd = 0.82 Å). The α-synuclein helix extends parallel to the curved membrane in a manner that allows conserved Lys and Glu residues to interact with the zwitterionic headgroups, while uncharged residues penetrate into the acyl chain region. This structural arrangement is significantly different from that of α-synuclein in the presence of the commonly used membrane-mimetic detergent SDS, which induces the formation of two antiparallel helices. Our structural analysis emphasizes the importance of studying membrane protein structure in a bilayer environment. PMID:19066219

  6. Mechanistic study for immobilization of cysteine-labeled oligopeptides on UV-activated surfaces.

    PubMed

    Ong, Lian Hao; Ding, Xiaokang; Yang, Kun-Lin

    2014-10-01

    In this study, we report immobilization of cysteine-labeled oligopeptides on UV activated surfaces decorated with N,N-dimethyl-n-octadecyl-3-aminopropyltrimethoxysilyl chloride (DMOAP). Our result shows that cysteine group, regardless of its position in the oligopeptide, is essential for successful immobilization of oligopeptide on the UV-activated surface. A possible reaction mechanism is nucleophilic addition of thiolates to surface aldehyde groups generated during UV activation. By using this technique, we are able to incorporate anchoring points into oligopeptides through cysteine residues. Furthermore, immobilized oligopeptides on the UV-activated surface is very stable even under harsh washing conditions. Finally, we show that an HPQ-containing oligopeptide can be immobilized on the UV-activated surface, but the final surface density and its ability to bind streptavidin are affected by the position of cysteine and HPQ. An oligopeptide with a cysteine at the N-terminus and a HPQ motif at the C-terminus gives the highest binding signal in the streptavidin-binding assay. This result is potentially useful for the development of functional oligopeptide microarrays for detecting target protein molecules.

  7. Fluorescence labelling of phosphatase activity in digestive glands of carnivorous plants.

    PubMed

    Płachno, B J; Adamec, L; Lichtscheidl, I K; Peroutka, M; Adlassnig, W; Vrba, J

    2006-11-01

    A new ELF (enzyme labelled fluorescence) assay was applied to detect phosphatase activity in glandular structures of 47 carnivorous plant species, especially Lentibulariaceae, in order to understand their digestive activities. We address the following questions: (1) Are phosphatases produced by the plants and/or by inhabitants of the traps? (2) Which type of hairs/glands is involved in the production of phosphatases? (3) Is this phosphatase production a common feature among carnivorous plants or is it restricted to evolutionarily advanced species? Our results showed activity of the phosphatases in glandular structures of the majority of the plants tested, both from the greenhouse and from sterile culture. In addition, extracellular phosphatases can also be produced by trap inhabitants. In Utricularia, activity of phosphatase was detected in internal glands of 27 species from both primitive and advanced sections and different ecological groups. Further positive reactions were found in Genlisea, Pinguicula, Aldrovanda, Dionaea, Drosera, Drosophyllum, Nepenthes, and Cephalotus. In Utricularia and Genlisea, enzymatic secretion was independent of stimulation by prey. Byblis and Roridula are usually considered as "proto-carnivores", lacking digestive enzymes. However, we found high activity of phosphatases in both species. Thus, they should be classified as true carnivores. We suggest that the inflorescence of Byblis and some Pinguicula species might also be an additional "carnivorous organ", which can trap a prey, digest it, and finally absorb available nutrients.

  8. Synthesis of water-dispersible zinc oxide quantum dots with antibacterial activity and low cytotoxicity for cell labeling

    NASA Astrophysics Data System (ADS)

    Hsu, Shan-hui; Lin, Ying Yi; Huang, Sherry; Lem, Kwok Wai; Huong Nguyen, Dinh; Lee, Dai Soo

    2013-11-01

    Typical photoluminescent semiconductor nanoparticles, called quantum dots (QDs), have potential applications in biological labeling. When used to label stem cells, QDs may impair the differentiation capacity of the stem cells. In this study, we synthesized zinc oxide (ZnO) QDs in methanol with an average size of ∼2 nm. We then employed two different types of polyethylene glycol (PEG) molecules (SH-PEG-NH2 and NH2-PEG-NH2) to conjugate ZnO QDs and made them water-dispersible. Fourier transform infrared spectroscopy spectra indicated the attachment of PEG molecules on ZnO QDs. No obvious size alteration was observed for ZnO QDs after PEG conjugation. The water-dispersible ZnO QDs still retained the antibacterial activity and fluorescence intensity. The cytotoxicity evaluation revealed that ZnO QDs at higher concentrations decreased cell viability but were generally safe at 30 ppm or below. Cell lines of hepatocytes (HepG2), osteoblasts (MC3T3-E1) and mesenchymal stem cells (MSCs) were successfully labeled by the water-dispersible ZnO QDs at 30 ppm. The ZnO QD-labeled MSCs maintained their stemness and differentiation capacity. Therefore, we conclude that the water-dispersible ZnO QDs developed in this study have antibacterial activity, low cytotoxicity, and proper labeling efficiency, and can be used to label a variety of cells including stem cells.

  9. Synthesis of water-dispersible zinc oxide quantum dots with antibacterial activity and low cytotoxicity for cell labeling.

    PubMed

    Hsu, Shan-hui; Lin, Ying Yi; Huang, Sherry; Lem, Kwok Wai; Nguyen, Dinh Huong; Lee, Dai Soo

    2013-11-29

    Typical photoluminescent semiconductor nanoparticles, called quantum dots (QDs), have potential applications in biological labeling. When used to label stem cells, QDs may impair the differentiation capacity of the stem cells. In this study, we synthesized zinc oxide (ZnO) QDs in methanol with an average size of ∼2 nm. We then employed two different types of polyethylene glycol (PEG) molecules (SH-PEG-NH2 and NH2-PEG-NH2) to conjugate ZnO QDs and made them water-dispersible. Fourier transform infrared spectroscopy spectra indicated the attachment of PEG molecules on ZnO QDs. No obvious size alteration was observed for ZnO QDs after PEG conjugation. The water-dispersible ZnO QDs still retained the antibacterial activity and fluorescence intensity. The cytotoxicity evaluation revealed that ZnO QDs at higher concentrations decreased cell viability but were generally safe at 30 ppm or below. Cell lines of hepatocytes (HepG2), osteoblasts (MC3T3-E1) and mesenchymal stem cells (MSCs) were successfully labeled by the water-dispersible ZnO QDs at 30 ppm. The ZnO QD-labeled MSCs maintained their stemness and differentiation capacity. Therefore, we conclude that the water-dispersible ZnO QDs developed in this study have antibacterial activity, low cytotoxicity, and proper labeling efficiency, and can be used to label a variety of cells including stem cells.

  10. Alkyl isocyanates as active site-directed inactivators of guinea pig liver transglutaminase.

    PubMed

    Gross, M; Whetzel, N K; Folk, J E

    1975-10-10

    Alkyl isocyanates are effective inactivators of guinea pig liver transglutaminase. Based on the specificity of the reaction the protection against inactivation by glutamine substrate, and the essential nature of calcium for the inactivation reaction, it is concluded that these reagents act as amide substrate analogs and, thus function in an active site-specific manner. Support for the contention that inactivation results from alkyl thiocarbamate ester formation through the single active site sulfhydryl group of the enzyme is (a) the loss of one free--SH group and the incorporation of 1 mol of reagent/mol of enzyme in the reaction, (b) similarity in chemical properties of the inactive enzyme derivative formed to those previously reported for another alkyl thiocarbamoylenzyme and an alkyl thiocarbamoylcysteine derivative, and (c) the finding that labeled peptides from digests of [methyl-14C]thiocarbamoyltransglutaminase and those from digests of iodoacetamide-inactivated enzyme occupy similar positions on peptide maps. Transglutaminase was found to be inactivated neither by urethan anlogs of its active ester substrates nor by urea analogs of its amide substrates. It is concluded on the basis of these findings that inactive carbamoylenzyme derivatives are formed only by direct addition of the transglutaminase active--SH group to the isocyanate C--N double bond, and not, like several serine active site enzymes, by nucleophilic displacement with urethan analogs of substrate, or by nucleophilic displacement with urea analogs of substrate. PMID:240837

  11. Growth exponents in surface models with non-active sites

    NASA Astrophysics Data System (ADS)

    Santos, M.; Figueiredo, W.; Aarão Reis, F. D. A.

    2006-11-01

    In this work, we studied the role played by the inactive sites present on the substrate of a growing surface. In our model, one particle sticks at the surface if the site where it falls is an active site. However, we allow the deposited particle to diffuse along the surface in accordance with some mechanism previously defined. Using Monte Carlo simulations, and some analytical results, we have investigated the model in (1+1) and (2+1) dimensions considering different relaxation mechanisms. We show that the consideration of non-active sites is a crucial point in the model. In fact, we have seen that the saturation regime is not observed for any value of the density of inactive sites. Besides, the growth exponent β turns to be one, at long times, whatever the mechanism of diffusion we consider in one and two dimensions.

  12. Chemo-enzymatic synthesis of site-specific isotopically labeled nucleotides for use in NMR resonance assignment, dynamics and structural characterizations

    PubMed Central

    Longhini, Andrew P.; LeBlanc, Regan M.; Becette, Owen; Salguero, Carolina; Wunderlich, Christoph H.; Johnson, Bruce A.; D'Souza, Victoria M.; Kreutz, Christoph; Dayie, T. Kwaku

    2016-01-01

    Stable isotope labeling is central to NMR studies of nucleic acids. Development of methods that incorporate labels at specific atomic positions within each nucleotide promises to expand the size range of RNAs that can be studied by NMR. Using recombinantly expressed enzymes and chemically synthesized ribose and nucleobase, we have developed an inexpensive, rapid chemo-enzymatic method to label ATP and GTP site specifically and in high yields of up to 90%. We incorporated these nucleotides into RNAs with sizes ranging from 27 to 59 nucleotides using in vitro transcription: A-Site (27 nt), the iron responsive elements (29 nt), a fluoride riboswitch from Bacillus anthracis (48 nt), and a frame-shifting element from a human corona virus (59 nt). Finally, we showcase the improvement in spectral quality arising from reduced crowding and narrowed linewidths, and accurate analysis of NMR relaxation dispersion (CPMG) and TROSY-based CEST experiments to measure μs-ms time scale motions, and an improved NOESY strategy for resonance assignment. Applications of this selective labeling technology promises to reduce difficulties associated with chemical shift overlap and rapid signal decay that have made it challenging to study the structure and dynamics of large RNAs beyond the 50 nt median size found in the PDB. PMID:26657632

  13. Amino acids of the Torpedo marmorata acetylcholine receptor. cap alpha. subunit labeled by a photoaffinity ligand for the acetylcholine binding site

    SciTech Connect

    Dennis, M.; Giraudat, J.; Kotzyba-Hibert, F.; Goeldner, M.; Hirth, C.; Chang, J.Y.; Lazure, C.; Chretien, M.; Changeux, J.P.

    1988-04-05

    The acetylcholine-binding sites on the native, membrane-bound acetylcholine receptor from Torpedo marmorata were covalently labeled with the photoaffinity reagent (/sup 3/H)-p-(dimethylamino)-benzenediazonium fluoroborate (DDF) in the presence of phencyclidine by employing an energy-transfer photolysis procedure. The ..cap alpha..-chains isolated from receptor-rich membranes photolabeled in the absence or presence of carbamoylcholine were cleaved with CNBr and the radiolabeled fragments purified by high-performance liquid chromatography. Amino acid and/or sequence analysis demonstrated that the ..cap alpha..-chain residues Trp-149, Tyr-190, Cys-192, and Cys-193 and an unidentified residue(s) in the segment ..cap alpha.. 31-105 were all labeled by the photoaffinity reagent in an agonist-protectable manner. The labeled amino acids are located within three distinct regions of the large amino-terminal hydrophilic domain of the ..cap alpha..-subunit primary structure and plausibly lie in proximity to one another at the level of the acetylcholine-binding sites in the native receptor. These findings are in accord with models proposed for the transmembrane topology of the ..cap alpha..-chain that assign the amino-terminal segment ..cap alpha.. 1-210 to the synaptic cleft. Furthermore, the results suggest that the four identified (/sup 3/H)DDF-labeled resides, which are conserved in muscle and neuronal ..cap alpha..-chains but not in the other subunits, may be directly involved in agonist binding.

  14. Specific labeling and permanent activation of the retinal rod cGMP-activated channel by the photoaffinity analog 8-p-azidophenacylthio-cGMP.

    PubMed Central

    Brown, R L; Gerber, W V; Karpen, J W

    1993-01-01

    Cyclic nucleotide-gated ion channels play a key role in visual excitation and a variety of other signaling pathways. The photoaffinity probe 8-p-azidophenacylthio-cGMP (APT-cGMP) has been developed for structural and functional studies of the cGMP-activated channel of retinal rod outer segments. Using this analog, we have demonstrated both specific labeling of the channel in a partially purified biochemical preparation from bovine rod outer segments and permanent activation of the channel current in excised membrane patches from salamander outer segments. After UV illumination, a 32P-labeled version of APT-cGMP was shown by SDS/PAGE and autoradiography to be covalently attached to the 63-kDa channel subunit. This incorporation was significantly reduced by 8-Br-cGMP but was not reduced by 5'-GMP. In patch-clamp experiments APT-cGMP was a potent activator of the channel; APT-cGMP typically opened half of the channels in a patch at a 10-fold lower concentration than cGMP. Exposure of membrane patches to UV light in the presence of APT-cGMP resulted in a persistent current observed in the absence of bath-applied nucleotide. This current increased with repeated exposure of the patch to both UV light and fresh APT-cGMP, approaching the maximum current originally evoked by saturating (500 microM) cGMP. At this point, addition of 500 microM cGMP caused a negligible increase in current. The persistent current had several other properties expected of current through cGMP-activated channels: it was outwardly rectifying; outward current was blocked > 90% by 2 mM internal Mg2+, whereas inward current was blocked much less efficiently; a low concentration of cGMP caused a larger increase in current atop a half-maximal persistent current than it did originally. We conclude that the persistent current was caused by the covalent tethering of cGMP moieties to channel binding sites, resulting in irreversible channel activation. APT-cGMP should prove useful for further studies of

  15. Label-free detection of cell-contractile activity with lipid nanotubes.

    PubMed

    Sugihara, Kaori; Delai, Marco; Mahnna, Rami; Kusch, Justine; Poulikakos, Dimos; Vörös, János; Zambelli, Tomaso; Ferrari, Aldo

    2013-02-01

    Surface-bound self-assembled lipid nanotubes (LNTs) made of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) were used to visualize the contractile activity of spreading cells. The interaction of cells with LNTs resulted in the nucleation of new nanotubes, directed toward the cell center, from existing ones. This process depended on cell generated forces and required acto-myosin mediated contractility. The dynamics of de novo generation of LNTs upon cell spreading was captured using optical microscopy on fluorescently labeled nanotubes and revealed characteristic fingerprints for different cell types such as fibroblasts, endothelial and melanoma cells. Additionally, the method was applied to detect the effect of a specific inhibitor on the generation of cellular forces. The mechanism of the LNT-cell interaction and the potential applications are discussed.

  16. Multisubstrate isotope labeling and metagenomic analysis of active soil bacterial communities.

    PubMed

    Verastegui, Y; Cheng, J; Engel, K; Kolczynski, D; Mortimer, S; Lavigne, J; Montalibet, J; Romantsov, T; Hall, M; McConkey, B J; Rose, D R; Tomashek, J J; Scott, B R; Charles, T C; Neufeld, J D

    2014-07-15

    Soil microbial diversity represents the largest global reservoir of novel microorganisms and enzymes. In this study, we coupled functional metagenomics and DNA stable-isotope probing (DNA-SIP) using multiple plant-derived carbon substrates and diverse soils to characterize active soil bacterial communities and their glycoside hydrolase genes, which have value for industrial applications. We incubated samples from three disparate Canadian soils (tundra, temperate rainforest, and agricultural) with five native carbon ((12)C) or stable-isotope-labeled ((13)C) carbohydrates (glucose, cellobiose, xylose, arabinose, and cellulose). Indicator species analysis revealed high specificity and fidelity for many uncultured and unclassified bacterial taxa in the heavy DNA for all soils and substrates. Among characterized taxa, Actinomycetales (Salinibacterium), Rhizobiales (Devosia), Rhodospirillales (Telmatospirillum), and Caulobacterales (Phenylobacterium and Asticcacaulis) were bacterial indicator species for the heavy substrates and soils tested. Both Actinomycetales and Caulobacterales (Phenylobacterium) were associated with metabolism of cellulose, and Alphaproteobacteria were associated with the metabolism of arabinose; members of the order Rhizobiales were strongly associated with the metabolism of xylose. Annotated metagenomic data suggested diverse glycoside hydrolase gene representation within the pooled heavy DNA. By screening 2,876 cloned fragments derived from the (13)C-labeled DNA isolated from soils incubated with cellulose, we demonstrate the power of combining DNA-SIP, multiple-displacement amplification (MDA), and functional metagenomics by efficiently isolating multiple clones with activity on carboxymethyl cellulose and fluorogenic proxy substrates for carbohydrate-active enzymes. Importance: The ability to identify genes based on function, instead of sequence homology, allows the discovery of genes that would not be identified through sequence alone. This

  17. Architecture and active site of particulate methane monooxygenase

    PubMed Central

    Culpepper, Megen A.; Rosenzweig, Amy C.

    2012-01-01

    Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria, organisms that live on methane gas as their sole carbon source. Understanding pMMO function has important implications for bioremediation applications and for the development of new, environmentally friendly catalysts for the direct conversion of methane to methanol. Crystal structures of pMMOs from three different methanotrophs reveal a trimeric architecture, consisting of three copies each of the pmoB, pmoA, and pmoC subunits. There are three distinct metal centers in each protomer of the trimer, mononuclear and dinuclear copper sites in the periplasmic regions of pmoB and a mononuclear site within the membrane that can be occupied by copper or zinc. Various models for the pMMO active site have been proposed within these structural constraints, including dicopper, tricopper, and diiron centers. Biochemical and spectroscopic data on pMMO and recombinant soluble fragments, denoted spmoB proteins, indicate that the active site involves copper and is located at the site of the dicopper center in the pmoB subunit. Initial spectroscopic evidence for O2 binding at this site has been obtained. Despite these findings, questions remain about the active site identity and nuclearity and will be the focus of future studies. PMID:22725967

  18. Methanopyrus kandleri topoisomerase V contains three distinct AP lyase active sites in addition to the topoisomerase active site.

    PubMed

    Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

    2016-04-20

    Topoisomerase V (Topo-V) is the only topoisomerase with both topoisomerase and DNA repair activities. The topoisomerase activity is conferred by a small alpha-helical domain, whereas the AP lyase activity is found in a region formed by 12 tandem helix-hairpin-helix ((HhH)2) domains. Although it was known that Topo-V has multiple repair sites, only one had been mapped. Here, we show that Topo-V has three AP lyase sites. The atomic structure and Small Angle X-ray Scattering studies of a 97 kDa fragment spanning the topoisomerase and 10 (HhH)2 domains reveal that the (HhH)2 domains extend away from the topoisomerase domain. A combination of biochemical and structural observations allow the mapping of the second repair site to the junction of the 9th and 10th (HhH)2 domains. The second site is structurally similar to the first one and to the sites found in other AP lyases. The 3rd AP lyase site is located in the 12th (HhH)2 domain. The results show that Topo-V is an unusual protein: it is the only known protein with more than one (HhH)2 domain, the only known topoisomerase with dual activities and is also unique by having three AP lyase repair sites in the same polypeptide. PMID:26908655

  19. Methanopyrus kandleri topoisomerase V contains three distinct AP lyase active sites in addition to the topoisomerase active site

    PubMed Central

    Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

    2016-01-01

    Topoisomerase V (Topo-V) is the only topoisomerase with both topoisomerase and DNA repair activities. The topoisomerase activity is conferred by a small alpha-helical domain, whereas the AP lyase activity is found in a region formed by 12 tandem helix-hairpin-helix ((HhH)2) domains. Although it was known that Topo-V has multiple repair sites, only one had been mapped. Here, we show that Topo-V has three AP lyase sites. The atomic structure and Small Angle X-ray Scattering studies of a 97 kDa fragment spanning the topoisomerase and 10 (HhH)2 domains reveal that the (HhH)2 domains extend away from the topoisomerase domain. A combination of biochemical and structural observations allow the mapping of the second repair site to the junction of the 9th and 10th (HhH)2 domains. The second site is structurally similar to the first one and to the sites found in other AP lyases. The 3rd AP lyase site is located in the 12th (HhH)2 domain. The results show that Topo-V is an unusual protein: it is the only known protein with more than one (HhH)2 domain, the only known topoisomerase with dual activities and is also unique by having three AP lyase repair sites in the same polypeptide. PMID:26908655

  20. Fluorescence-Activated Cell Sorting of EGFP-Labeled Neural Crest Cells From Murine Embryonic Craniofacial Tissue

    PubMed Central

    2005-01-01

    During the early stages of embryogenesis, pluripotent neural crest cells (NCC) are known to migrate from the neural folds to populate multiple target sites in the embryo where they differentiate into various derivatives, including cartilage, bone, connective tissue, melanocytes, glia, and neurons of the peripheral nervous system. The ability to obtain pure NCC populations is essential to enable molecular analyses of neural crest induction, migration, and/or differentiation. Crossing Wnt1-Cre and Z/EG transgenic mouse lines resulted in offspring in which the Wnt1-Cre transgene activated permanent EGFP expression only in NCC. The present report demonstrates a flow cytometric method to sort and isolate populations of EGFP-labeled NCC. The identity of the sorted neural crest cells was confirmed by assaying expression of known marker genes by TaqMan Quantitative Real-Time Polymerase Chain Reaction (QRT-PCR). The molecular strategy described in this report provides a means to extract intact RNA from a pure population of NCC thus enabling analysis of gene expression in a defined population of embryonic precursor cells critical to development. PMID:16192680

  1. Label-free offline versus online activity methods for nucleoside diphosphate kinase b using high performance liquid chromatography.

    PubMed

    Lima, Juliana Maria; Salmazo Vieira, Plínio; Cavalcante de Oliveira, Arthur Henrique; Cardoso, Carmen Lúcia

    2016-08-01

    Nucleoside diphosphate kinase from Leishmania spp. (LmNDKb) has recently been described as a potential drug target to treat leishmaniasis disease. Therefore, screening of LmNDKb ligands requires methodologies that mimic the conditions under which LmNDKb acts in biological systems. Here, we compare two label-free methodologies that could help screen LmNDKb ligands and measure NDKb activity: an offline LC-UV assay for soluble LmNDKb and an online two-dimensional LC-UV system based on LmNDKb immobilised on a silica capillary. The target enzyme was immobilised on the silica capillary via Schiff base formation (to give LmNDKb-ICER-Schiff) or affinity attachment (to give LmNDKb-ICER-His). Several aspects of the ICERs resulting from these procedures were compared, namely kinetic parameters, stability, and procedure steps. Both the LmNDKb immobilisation routes minimised the conformational changes and preserved the substrate binding sites. However, considering the number of steps involved in the immobilisation procedure, the cost of reagents, and the stability of the immobilised enzyme, immobilisation via Schiff base formation proved to be the optimal procedure.

  2. Interaction of isocitrate dehydrogenase with (RS)-3-bromo-2-ketoglutarate. A potential affinity label for. cap alpha. -ketoglutarate binding sites

    SciTech Connect

    Hartman, F.C.

    1981-01-01

    The interaction of oxidize nicotine adenine dinucleotide phosphate dependent isocitrate dehydrogenase (from pig heart) with (RS)-3-bromo-2-ketoglutarate was investigated in an effort to evaluate the reagent's potential as a selective reagent for ..cap alpha..-ketoglutarate binding sites. The enzyme is rapidly inactivated by 0.1 mM bromoketoglutarate at pH 7.4. With increasing concentration of reagent, the reaction shows a rate saturation; the minimum inactivation half-time is 3 min and K/sub inact/ for bromoketoglutarate is 250 ..mu..M. Isocitrate and NADP/sup +/ protect against inactivation, while ketoglutarate does not. When tested in the assay that monitors isocitrate oxidation, bromoketoglutarate is a competitive inhibitor (K/sub i/=100 ..mu..M) of the dehydrogenase. As judged by oxidation of NADPH, bromoketoglutarate is also a substrate for isocitrate dehydrogenase, exhibiting a K/sub m/ of 250 ..mu..M and a V/sub max/ comparable to that for isocitrate oxidation. The reduction of bromoketoglutarate is competitively inhibited by isocitrate (K/sub i/=3 ..mu..M) and ketoglutarate (K/sub i/=50 ..mu..M). Like the enzyme-catalyzed oxidation of isocitrate, the reduction of bromoketoglutarate is stereospecific, requires divalent metal ions, and shows absolute specificity for NADPH. However, since CO/sub 2/ is not required for catalytic turnover of bromoketoglutarate, its reduction is likely comparable to that of oxalosuccinate rather than the reductive carboxylation of ketoglutarate. Although bromoketoglutarate, as a substrate for isocitrate dehydrogenase, clearly has affinity for the active site, the irreversible inactivation of the enzyme by the reagent may result from modification outside the active-site region, since inactivation during catalytic turnover of bromoketoglutarate is not observed.

  3. Labeling of active proteases in fresh-frozen tissues by topical application of quenched activity-based probes.

    PubMed

    Withana, Nimali P; Garland, Megan; Verdoes, Martijn; Ofori, Leslie O; Segal, Ehud; Bogyo, Matthew

    2016-01-01

    Active enzymes, such as proteases, often serve as valuable biomarkers for various disease pathologies. Therefore, methods to detect specific enzyme activities in biological samples can provide information to guide disease detection and diagnosis and to increase our understanding of the biological roles of specific enzyme targets. In this protocol, we outline methods for the topical application of fluorescently quenched activity-based probes (qABPs) to fresh-frozen tissue samples. This technique enables rapid imaging of enzyme activity at cellular resolution, and it can be combined with antibody labeling for immunodiagnosis. In this method, fresh-frozen tissue sections are fixed, incubated with the probe and imaged using fluorescence microscopy. This provides an advance over classical immunohistochemistry (IHC) in that it is rapid (4-8 h) and inexpensive, and it provides information on enzyme activity. Furthermore, it can be used with any of the growing number of fluorescent ABPs to provide data for more effective disease monitoring and diagnosis. PMID:26716706

  4. An integrative framework of the skin receptors activation: mechanoreceptors activity patterns versus "labeled lines".

    PubMed

    Zeveke, Alexander V; Efes, Ekaterina D; Polevaya, Sofia A

    2013-03-01

    The paper presents a review of electrophysiological data which indicate the integrative mechanisms of information coded in the human and animal peripheral skin receptors. The activity of the skin sensory receptors was examined by applying various natural stimuli. It was revealed that numerous identical receptors respond to various stimuli (mechanical, temperature, and pain ones), but the spike patterns of these receptors were found to be specific for each stimulus. The description of characteristic structures of spike patterns in the cutaneous nerve fibers in response to five major modalities, namely: "touch", "pain", "vibration/breath", "cold", and "heat", is being presented. The recordings of the cutaneous physical state revealed a correlation between the patterns of spatiotemporal skin deformation and the receptors activity. A rheological state of the skin can be changed either in response to external temperature variation or by the sympathetic pilomotor activation. These results indicate that the skin sensory receptors activity may be considered as an integrative process. It depends not only on the receptors themselves, but also on the changes in the surrounding tissue and on the adaptive influence of the central nervous system. A new framework for the sensory channel system related to the skin is proposed on the basis of experimental results.

  5. Lymphokine-activated killer (LAK) cells can be focused at sites of tumor growth by products of macrophage activation

    SciTech Connect

    Migliori, R.J.; Gruber, S.A.; Sawyer, M.D.; Hoffman, R.; Ochoa, A.; Bach, F.H.; Simmons, R.L.

    1987-08-01

    Successful adoptive cancer immunotherapy presumably depends on the accumulation of tumoricidal leukocytes at the sites of tumor growth. Large numbers of lymphokine-activated killer (LAK) cells can be generated in vitro by growth in high concentrations of interleukin-2 (IL-2), but relatively few arrive at the tumor site after intravenous injection. We hypothesize that the delivery of LAK cells to tumor sites may be augmented by previously demonstrated lymphocyte-recruiting factors, including activated macrophage products such as interleukin-1 (IL-1) and tumor necrosis factor. /sup 111/Indium-labeled LAK cells were injected intravenously into syngeneic mice bearing the macrophage activator endotoxin (LPS) in one hind footpad, and saline solution was injected into the contralateral footpad. Significantly more activity was recovered from the LPS-bearing footpad at all times during a 96-hour period. Recombinant IL-1 also attracted more LAK cells after injection into tumor-free hind footpads. Furthermore, LAK cells preferentially homed to hind footpads that were bearing 3-day established sarcomas after intralesional injections of LPS, IL-1, or tumor necrosis factor when compared with contralateral tumor-bearing footpads injected with saline solution alone. In preliminary experiments, mice with hind-footpad tumors appeared to survive longer after combined systemic IL-2 and LAK therapy if intralesional LPS was administered. These studies demonstrate that macrophage activation factors that have been shown capable of attracting circulating normal lymphocytes can also effectively attract LAK cells from the circulation. By the stimulation of macrophages at the sites of tumor growth, more LAK cells can be attracted. It is hoped that by focusing the migration of LAK cells to tumors, LAK cells and IL-2 would effect tumor regression more efficiently and with less toxicity.

  6. Photoaffinity labeling of human serum vitamin D binding protein and chemical cleavage of the labeled protein: Identification of an 11. 5-kDa peptide containing the putative 25-hydroxyvitamin D sub 3 binding site

    SciTech Connect

    Ray, R.; Holick, M.F. ); Bouillon, R.; Baelen, H.V. )

    1991-07-30

    In this paper, the authors describe photoaffinity labeling and related studies of human serum vitamin D binding protein (hDBP) with 25-hydroxyvitamin D{sub 3} 3{beta}-3{prime}-(N-(4-azido-2-nitrophenyl)amino)propyl ether (25-ANE) and its radiolabeled counterpart, i.e., 25-hydroxyvitamin D{sub 3} 3{beta}-3{prime}-(N-(4-azido-2-nitro-(3,5-{sup 3}H)phenyl)amino)propyl ether ({sup 3}H-25-ANE). They have carried out studies to demonstrate that (1) 25-ANE competes with 25-OH-D{sub 3} for the binding site of the latter in hDBP and (2) {sup 3}H-25-ANE is capable of covalently labeling the hDBP molecule when exposed ot UV light. Treatment of a sample of purified hDBP, labeled with {sup 3}H-25-ANE, with BNPS-skatole produced two Coomassie Blue stained peptide fragments, and the majority of the radioactivity was assoicated with the smaller of the two peptide fragments (16.5 kDa). On the other hand, cleavage of the labeled protein with cyanogen bromide produced a peptide (11.5 kDa) containing most of the covalently attached radioactivity. Considering the primary amino acid structure of hDBP, this peptide fragment (11.5 kDa) represents the N-terminus through residue 108 of the intact protein. Thus, the results tentatively identify this segment of the protein containing the binding pocket for 25-OH-D{sub 3}.

  7. Potential effect of physical activity based menu labels on the calorie content of selected fast food meals.

    PubMed

    Dowray, Sunaina; Swartz, Jonas J; Braxton, Danielle; Viera, Anthony J

    2013-03-01

    In this study we examined the effect of physical activity based labels on the calorie content of meals selected from a sample fast food menu. Using a web-based survey, participants were randomly assigned to one of four menus which differed only in their labeling schemes (n=802): (1) a menu with no nutritional information, (2) a menu with calorie information, (3) a menu with calorie information and minutes to walk to burn those calories, or (4) a menu with calorie information and miles to walk to burn those calories. There was a significant difference in the mean number of calories ordered based on menu type (p=0.02), with an average of 1020 calories ordered from a menu with no nutritional information, 927 calories ordered from a menu with only calorie information, 916 calories ordered from a menu with both calorie information and minutes to walk to burn those calories, and 826 calories ordered from the menu with calorie information and the number of miles to walk to burn those calories. The menu with calories and the number of miles to walk to burn those calories appeared the most effective in influencing the selection of lower calorie meals (p=0.0007) when compared to the menu with no nutritional information provided. The majority of participants (82%) reported a preference for physical activity based menu labels over labels with calorie information alone and no nutritional information. Whether these labels are effective in real-life scenarios remains to be tested.

  8. Active Sites Environmental Monitoring Program: Mid-FY 1991 report

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1991-10-01

    This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) from October 1990 through March 1991. The ASEMP was established in 1989 by Solid Waste Operations and the Environmental Sciences Division to provide early detection and performance monitoring at active low-level radioactive waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 as required by chapters II and III of US Department of Energy Order 5820.2A. Monitoring results continue to demonstrate the no LLW is being leached from the storage vaults on the tumulus pads. Loading of vaults on Tumulus II began during this reporting period and 115 vaults had been loaded by the end of March 1991.

  9. Active chemisorption sites in functionalized ionic liquids for carbon capture.

    PubMed

    Cui, Guokai; Wang, Jianji; Zhang, Suojiang

    2016-07-25

    Development of novel technologies for the efficient and reversible capture of CO2 is highly desired. In the last decade, CO2 capture using ionic liquids has attracted intensive attention from both academia and industry, and has been recognized as a very promising technology. Recently, a new approach has been developed for highly efficient capture of CO2 by site-containing ionic liquids through chemical interaction. This perspective review focuses on the recent advances in the chemical absorption of CO2 using site-containing ionic liquids, such as amino-based ionic liquids, azolate ionic liquids, phenolate ionic liquids, dual-functionalized ionic liquids, pyridine-containing ionic liquids and so on. Other site-containing liquid absorbents such as amine-based solutions, switchable solvents, and functionalized ionic liquid-amine blends are also investigated. Strategies have been discussed for how to activate the existent reactive sites and develop novel reactive sites by physical and chemical methods to enhance CO2 absorption capacity and reduce absorption enthalpy. The carbon capture mechanisms of these site-containing liquid absorbents are also presented. Particular attention has been paid to the latest progress in CO2 capture in multiple-site interactions by amino-free anion-functionalized ionic liquids. In the last section, future directions and prospects for carbon capture by site-containing ionic liquids are outlined.

  10. Active chemisorption sites in functionalized ionic liquids for carbon capture.

    PubMed

    Cui, Guokai; Wang, Jianji; Zhang, Suojiang

    2016-07-25

    Development of novel technologies for the efficient and reversible capture of CO2 is highly desired. In the last decade, CO2 capture using ionic liquids has attracted intensive attention from both academia and industry, and has been recognized as a very promising technology. Recently, a new approach has been developed for highly efficient capture of CO2 by site-containing ionic liquids through chemical interaction. This perspective review focuses on the recent advances in the chemical absorption of CO2 using site-containing ionic liquids, such as amino-based ionic liquids, azolate ionic liquids, phenolate ionic liquids, dual-functionalized ionic liquids, pyridine-containing ionic liquids and so on. Other site-containing liquid absorbents such as amine-based solutions, switchable solvents, and functionalized ionic liquid-amine blends are also investigated. Strategies have been discussed for how to activate the existent reactive sites and develop novel reactive sites by physical and chemical methods to enhance CO2 absorption capacity and reduce absorption enthalpy. The carbon capture mechanisms of these site-containing liquid absorbents are also presented. Particular attention has been paid to the latest progress in CO2 capture in multiple-site interactions by amino-free anion-functionalized ionic liquids. In the last section, future directions and prospects for carbon capture by site-containing ionic liquids are outlined. PMID:27243042

  11. Studies on the active site of pig plasma amine oxidase.

    PubMed Central

    Collison, D; Knowles, P F; Mabbs, F E; Rius, F X; Singh, I; Dooley, D M; Cote, C E; McGuirl, M

    1989-01-01

    Amine oxidase from pig plasma (PPAO) has two bound Cu2+ ions and at least one pyrroloquinoline quinone (PQQ) moiety as cofactors. It is shown that recovery of activity by copper-depleted PPAO is linear with respect to added Cu2+ ions. Recovery of e.s.r. and optical spectral characteristics of active-site copper parallel the recovery of catalytic activity. These results are consistent with both Cu2+ ions contributing to catalysis. Further e.s.r. studies indicate that the two copper sites in PPAO, unlike those in amine oxidases from other sources, are chemically distinct. These comparative studies establish that non-identity of the Cu2+ ions in PPAO is not a requirement for amine oxidase activity. It is shown through the use of a new assay procedure that there are two molecules of PQQ bound per molecule of protein in PPAO; only the more reactive of these PQQ moieties is required for activity. PMID:2559715

  12. 21 CFR 341.85 - Labeling of permitted combinations of active ingredients.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... “pain reliever” or “analgesic (pain reliever).” If the product is also labeled to relieve fever, then the analgesic-antipyretic component is identified as a “pain reliever-fever reducer” or “analgesic (pain reliever)-antipyretic (fever reducer).” (2) (b) Indications. The labeling of the product...

  13. 21 CFR 341.85 - Labeling of permitted combinations of active ingredients.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... “pain reliever” or “analgesic (pain reliever).” If the product is also labeled to relieve fever, then the analgesic-antipyretic component is identified as a “pain reliever-fever reducer” or “analgesic (pain reliever)-antipyretic (fever reducer).” (2) (b) Indications. The labeling of the product...

  14. 21 CFR 341.85 - Labeling of permitted combinations of active ingredients.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... SERVICES (CONTINUED) DRUGS FOR HUMAN USE COLD, COUGH, ALLERGY, BRONCHODILATOR, AND ANTIASTHMATIC DRUG...), (o), (q), and (r) when labeled for relief of general cough-cold symptoms and/or the common cold. (i... pains headache” and “ temporarily reduces fever”. (ii) The labeling for the cough-cold ingredient(s)...

  15. Purification and sequencing of the active site tryptic peptide from penicillin-binding protein 1b of Escherichia coli

    SciTech Connect

    Nicholas, R.A.; Suzuki, H.; Hirota, Y.; Strominger, J.L.

    1985-07-02

    This paper reports the sequence of the active site peptide of penicillin-binding protein 1b from Escherichia coli. Purified penicillin-binding protein 1b was labeled with (/sup 14/C)penicillin G, digested with trypsin, and partially purified by gel filtration. Upon further purification by high-pressure liquid chromatography, two radioactive peaks were observed, and the major peak, representing over 75% of the applied radioactivity, was submitted to amino acid analysis and sequencing. The sequence Ser-Ile-Gly-Ser-Leu-Ala-Lys was obtained. The active site nucleophile was identified by digesting the purified peptide with aminopeptidase M and separating the radioactive products on high-pressure liquid chromatography. Amino acid analysis confirmed that the serine residue in the middle of the sequence was covalently bonded to the (/sup 14/C)penicilloyl moiety. A comparison of this sequence to active site sequences of other penicillin-binding proteins and beta-lactamases is presented.

  16. 21 CFR 358.760 - Labeling of permitted combinations of active ingredients for the control of dandruff.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... ingredients for the control of dandruff. 358.760 Section 358.760 Food and Drugs FOOD AND DRUG ADMINISTRATION... FOR OVER-THE-COUNTER HUMAN USE Drug Products for the Control of Dandruff, Seborrheic Dermatitis, and Psoriasis § 358.760 Labeling of permitted combinations of active ingredients for the control of...

  17. Computer simulation of the active site of human serum cholinesterase

    SciTech Connect

    Kefang Jiao; Song Li; Zhengzheng Lu

    1996-12-31

    The first 3D-structure of acetylchelinesterase from Torpedo California electric organ (T.AChE) was published by JL. Sussman in 1991. We have simulated 3D-structure of human serum cholinesterase (H.BuChE) and the active site of H.BuChE. It is discovered by experiment that the residue of H.BuChE is still active site after a part of H.BuChE is cut. For example, the part of 21KD + 20KD is active site of H.BuChE. The 20KD as it is. Studies on these peptides by Hemelogy indicate that two active peptides have same negative electrostatic potential maps diagram. These negative electrostatic areas attached by acetyl choline with positive electrostatic potency. We predict that 147...236 peptide of AChE could be active site because it was as 20KD as with negative electrostatic potential maps. We look forward to proving from other ones.

  18. Observation of the Fe—CN and Fe—CO Vibrations in the Active Site of [NiFe] Hydrogenase by Nuclear Resonance Vibrational Spectroscopy**

    PubMed Central

    Kamali, Saeed; Wang, Hongxin; Mitra, Devrani; Ogata, Hideaki; Manor, Brian C.; Rauchfuss, Thomas B.; Byrne, Deborah; Bonnefoy, Violaine; Jenney, Francis E.; Adams, Michael W. W.; Yoda, Yoshitaka; Alp, Ercan; Zhao, Jiyong; Cramer, Stephen P.

    2012-01-01

    Nuclear inelastic scattering of 57Fe labeled [NiFe] hydrogenase is shown to give information on different states of the enzyme. It was thus possible to detect and assign Fe–CO and Fe–CN bending and stretching vibrations of the active site outside the spectral range of the Fe–S cluster normal modes. PMID:23136119

  19. Resonant active sites in catalytic ammonia synthesis: A structural model

    NASA Astrophysics Data System (ADS)

    Cholach, Alexander R.; Bryliakova, Anna A.; Matveev, Andrey V.; Bulgakov, Nikolai N.

    2016-03-01

    Adsorption sites Mn consisted of n adjacent atoms M, each bound to the adsorbed species, are considered within a realistic model. The sum of bonds Σ lost by atoms in a site in comparison with the bulk atoms was used for evaluation of the local surface imperfection, while the reaction enthalpy at that site was used as a measure of activity. The comparative study of Mn sites (n = 1-5) at basal planes of Pt, Rh, Ir, Fe, Re and Ru with respect to heat of N2 dissociative adsorption QN and heat of Nad + Had → NHad reaction QNH was performed using semi-empirical calculations. Linear QN(Σ) increase and QNH(Σ) decrease allowed to specify the resonant Σ for each surface in catalytic ammonia synthesis at equilibrium Nad coverage. Optimal Σ are realizable for Ru2, Re2 and Ir4 only, whereas other centers meet steric inhibition or unreal crystal structure. Relative activity of the most active sites in proportion 5.0 × 10- 5: 4.5 × 10- 3: 1: 2.5: 3.0: 1080: 2270 for a sequence of Pt4, Rh4, Fe4(fcc), Ir4, Fe2-5(bcc), Ru2, Re2, respectively, is in agreement with relevant experimental data. Similar approach can be applied to other adsorption or catalytic processes exhibiting structure sensitivity.

  20. Multi-site Phosphorylation Regulates Bim Stability and Apoptotic Activity

    PubMed Central

    Hübner, Anette; Barrett, Tamera; Flavell, Richard A.; Davis, Roger J.

    2008-01-01

    The pro-apoptotic BH3-only protein Bim is established to be an important mediator of signaling pathways that induce cell death. Multi-site phosphorylation of Bim by several members of the MAP kinase group is implicated as a regulatory mechanism that controls the apoptotic activity of Bim. To test the role of Bim phosphorylation in vivo, we constructed mice with a series of mutant alleles that express phosphorylation-defective Bim proteins. We show that mutation of the phosphorylation site Thr-112 causes decreased binding of Bim to the anti-apoptotic protein Bcl2 and can increase cell survival. In contrast, mutation of the phosphorylation sites Ser-55, Ser-65, and Ser-73 can cause increased apoptosis because of reduced proteasomal degradation of Bim. Together, these data indicate that phosphorylation can regulate Bim by multiple mechanisms and that the phosphorylation of Bim on different sites can contribute to the sensitivity of cellular apoptotic responses. PMID:18498746

  1. Validation of Web-Based Physical Activity Measurement Systems Using Doubly Labeled Water

    PubMed Central

    Yamaguchi, Yukio; Yamada, Yosuke; Tokushima, Satoru; Hatamoto, Yoichi; Sagayama, Hiroyuki; Kimura, Misaka; Higaki, Yasuki; Tanaka, Hiroaki

    2012-01-01

    Background Online or Web-based measurement systems have been proposed as convenient methods for collecting physical activity data. We developed two Web-based physical activity systems—the 24-hour Physical Activity Record Web (24hPAR WEB) and 7 days Recall Web (7daysRecall WEB). Objective To examine the validity of two Web-based physical activity measurement systems using the doubly labeled water (DLW) method. Methods We assessed the validity of the 24hPAR WEB and 7daysRecall WEB in 20 individuals, aged 25 to 61 years. The order of email distribution and subsequent completion of the two Web-based measurements systems was randomized. Each measurement tool was used for a week. The participants’ activity energy expenditure (AEE) and total energy expenditure (TEE) were assessed over each week using the DLW method and compared with the respective energy expenditures estimated using the Web-based systems. Results The mean AEE was 3.90 (SD 1.43) MJ estimated using the 24hPAR WEB and 3.67 (SD 1.48) MJ measured by the DLW method. The Pearson correlation for AEE between the two methods was r = .679 (P < .001). The Bland-Altman 95% limits of agreement ranged from –2.10 to 2.57 MJ between the two methods. The Pearson correlation for TEE between the two methods was r = .874 (P < .001). The mean AEE was 4.29 (SD 1.94) MJ using the 7daysRecall WEB and 3.80 (SD 1.36) MJ by the DLW method. The Pearson correlation for AEE between the two methods was r = .144 (P = .54). The Bland-Altman 95% limits of agreement ranged from –3.83 to 4.81 MJ between the two methods. The Pearson correlation for TEE between the two methods was r = .590 (P = .006). The average input times using terminal devices were 8 minutes and 10 seconds for the 24hPAR WEB and 6 minutes and 38 seconds for the 7daysRecall WEB. Conclusions Both Web-based systems were found to be effective methods for collecting physical activity data and are appropriate for use in epidemiological studies. Because the measurement

  2. Mitochondrial nicotinamide nucleotide transhydrogenase: active site modification by 5'-(p-(fluorosulfonyl)benzoyl)adenosine

    SciTech Connect

    Phelps, D.C.; Hatefi, Y.

    1985-07-02

    Membrane-bound and purified mitochondrial energy-linked nicotinamide nucleotide transhydrogenase (TH) was inhibited by incubation with 5'-(p-(fluorosulfonyl)benzoyl)adenosine (FSBA), which is an analogue of TH substrates and their competitive inhibitors, namely, 5'-, 2'-, or 3'-AMP. NAD(H) and analogues, NADP, 5'-AMP, 5'-ADP, and 2'-AMP/3'-AMP mixed isomers protected TH against inhibition by FSBA, but NADPH accelerated the inhibition rate. In the absence of protective ligands or in the presence of NADP, FSBA appeared to modify the NAD(H) binding site of TH, because, unlike unmodified TH, the enzyme modified by FSBA under these conditions did not bind to an NAD-affinity column (NAD-agarose). However, when the NAD(H) binding site of TH was protected in the presence of 5'-AMP or NAD, then FSBA modification resulted in an inhibited enzyme that did bind to NAD-agarose, suggesting FSBA modification of the NADP(H) binding site or an essential residue outside the active site. (/sup 3/H)FSBA was covalently bound to TH, and complete inhibition corresponded to the binding of about 0.5 mol of (3H)FSBA/mol of TH. Since purified TH is known to be dimeric in the isolated state, this binding stoichiometry suggests half-of-the-sites reactivity. A similar binding stoichiometry was found earlier for complete inhibition of TH by (/sup 14/C)DCCD. The active site directed labeling of TH by radioactive FSBA should allow isolation of appropriate peptides for sequence analysis of the NAD(H) and possibly the NADP(H) binding domains.

  3. Water in the Active Site of Ketosteroid Isomerase

    PubMed Central

    Hanoian, Philip; Hammes-Schiffer, Sharon

    2011-01-01

    Classical molecular dynamics simulations were utilized to investigate the structural and dynamical properties of water in the active site of ketosteroid isomerase (KSI) to provide insight into the role of these water molecules in the enzyme-catalyzed reaction. This reaction is thought to proceed via a dienolate intermediate that is stabilized by hydrogen bonding with residues Tyr16 and Asp103. A comparative study was performed for the wild-type (WT) KSI and the Y16F, Y16S, and Y16F/Y32F/Y57F (FFF) mutants. These systems were studied with three different bound ligands: equilenin, which is an intermediate analog, and the intermediate states of two steroid substrates. Several distinct water occupation sites were identified in the active site of KSI for the WT and mutant systems. Three additional sites were identified in the Y16S mutant that were not occupied in WT KSI or the other mutants studied. The number of water molecules directly hydrogen bonded to the ligand oxygen was approximately two waters in the Y16S mutant, one water in the Y16F and FFF mutants, and intermittent hydrogen bonding of one water molecule in WT KSI. The molecular dynamics trajectories of the Y16F and FFF mutants reproduced the small conformational changes of residue 16 observed in the crystal structures of these two mutants. Quantum mechanical/molecular mechanical calculations of 1H NMR chemical shifts of the protons in the active site hydrogen-bonding network suggest that the presence of water in the active site does not prevent the formation of short hydrogen bonds with far-downfield chemical shifts. The molecular dynamics simulations indicate that the active site water molecules exchange much more frequently for WT KSI and the FFF mutant than for the Y16F and Y16S mutants. This difference is most likely due to the hydrogen-bonding interaction between Tyr57 and an active site water molecule that is persistent in the Y16F and Y16S mutants but absent in the FFF mutant and significantly less

  4. Activation of phenylalanine hydroxylase by phenylalanine does not require binding in the active site.

    PubMed

    Roberts, Kenneth M; Khan, Crystal A; Hinck, Cynthia S; Fitzpatrick, Paul F

    2014-12-16

    Phenylalanine hydroxylase (PheH), a liver enzyme that catalyzes the hydroxylation of excess phenylalanine in the diet to tyrosine, is activated by phenylalanine. The lack of activity at low levels of phenylalanine has been attributed to the N-terminus of the protein's regulatory domain acting as an inhibitory peptide by blocking substrate access to the active site. The location of the site at which phenylalanine binds to activate the enzyme is unknown, and both the active site in the catalytic domain and a separate site in the N-terminal regulatory domain have been proposed. Binding of catecholamines to the active-site iron was used to probe the accessibility of the active site. Removal of the regulatory domain increases the rate constants for association of several catecholamines with the wild-type enzyme by ∼2-fold. Binding of phenylalanine in the active site is effectively abolished by mutating the active-site residue Arg270 to lysine. The k(cat)/K(phe) value is down 10⁴ for the mutant enzyme, and the K(m) value for phenylalanine for the mutant enzyme is >0.5 M. Incubation of the R270K enzyme with phenylalanine also results in a 2-fold increase in the rate constants for catecholamine binding. The change in the tryptophan fluorescence emission spectrum seen in the wild-type enzyme upon activation by phenylalanine is also seen with the R270K mutant enzyme in the presence of phenylalanine. Both results establish that activation of PheH by phenylalanine does not require binding of the amino acid in the active site. This is consistent with a separate allosteric site, likely in the regulatory domain.

  5. Chemical Modification of Papain and Subtilisin: An Active Site Comparison

    ERIC Educational Resources Information Center

    St-Vincent, Mireille; Dickman, Michael

    2004-01-01

    An experiment using methyle methanethiosulfonate (MMTS) and phenylmethylsulfonyl flouride (PMSF) to specifically modify the cysteine and serine residues in the active sites of papain and subtilism respectively is demonstrated. The covalent modification of these enzymes and subsequent rescue of papain shows the beginning biochemist that proteins…

  6. Energy transfer at the active sites of heme proteins

    SciTech Connect

    Dlott, D.D.; Hill, J.R.

    1995-12-31

    Experiments using a picosecond pump-probe apparatus at the Picosecond Free-electron Laser Center at Stanford University, were performed to investigate the relaxation of carbon monoxide bound to the active sites of heme proteins. The significance of these experiments is two-fold: (1) they provide detailed information about molecular dynamics occurring at the active sites of proteins; and (2) they provide insight into the nature of vibrational relaxation processes in condensed matter. Molecular engineering is used to construct various molecular systems which are studied with the FEL. We have studied native proteins, mainly myoglobin obtained from different species, mutant proteins produced by genetic engineering using recombinant DNA techniques, and a variety of model systems which mimic the structures of the active sites of native proteins, which are produced using molecular synthesis. Use of these different systems permits us to investigate how specific molecular structural changes affect dynamical processes occurring at the active sites. This research provides insight into the problems of how different species needs are fulfilled by heme proteins which have greatly different functionality, which is induced by rather small structural changes.

  7. Gastrointestinal tract radionuclide activity on In-111 labeled leukocyte imaging: clinical significance in patients with fever of unknown origin

    SciTech Connect

    Datz, F.L.; Thorne, D.A.

    1986-09-01

    To determine the frequency and clinical significance of indium-111 labeled leukocyte activity in the gastrointestinal (GI) tract of patients with fever of unknown origin, we reviewed 312 leukocyte studies involving 271 patients. Radionuclide activity was noted in the bowel in 59 cases. Of these, only 27 were due to the infection or inflammatory disease that caused the patient's fever. The 32 false-positive results were due primarily to swallowed leukocytes or bleeding. In two cases, no explanation was found for the activity in the GI tract. We conclude that bowel activity on In-111 labeled leukocyte scans in patients with fever of unknown origin often does not correlate with the true cause of the patient's fever.

  8. Quantitative analysis of spin exchange interactions to identify β strand and turn regions in Ure2 prion domain fibrils with site-directed spin labeling.

    PubMed

    Ngo, Sam; Chiang, Vicky; Guo, Zhefeng

    2012-11-01

    Amyloid formation is associated with a range of debilitating human disorders including Alzheimer's and prion diseases. The amyloid structure is essential for understanding the role of amyloids in these diseases. Amyloid formation of Ure2 protein underlies the yeast prion [URE3]. Here we use site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy to investigate the structure of amyloid fibrils formed by the Ure2 prion domain. The Ure2 prion domain under study contains a Sup35M domain at C-terminus as a solubilization element. We introduced spin labels at every residue from positions 2-15, and every 5th residue from positions 20-80 in Ure2 prion domain. EPR spectra at most labeling sites show strong spin exchange interactions, suggesting a parallel in-register β structure. With quantitative analysis of spin exchange interactions, we show that residues 8-12 form the first β strand, followed by a turn at residues 13-14, and then the second β strand from residue 15 to at least residue 20. Comparison of the spin exchange frequency for the fibrils formed under quiescent and agitated conditions also revealed differences in the fibril structures. Currently there is a lack of techniques for in-depth structural studies of amyloid fibrils. Detailed structural information is obtained almost exclusively from solid-state NMR. The identification of β-strand and turn regions in this work suggests that quantitative analysis of spin exchange interactions in spin-labeled amyloid fibrils is a powerful approach for identifying the β-strand and turn/loop residues and for studying structural differences of different fibril polymorphs.

  9. Changes in active site histidine hydrogen bonding trigger cryptochrome activation.

    PubMed

    Ganguly, Abir; Manahan, Craig C; Top, Deniz; Yee, Estella F; Lin, Changfan; Young, Michael W; Thiel, Walter; Crane, Brian R

    2016-09-01

    Cryptochrome (CRY) is the principal light sensor of the insect circadian clock. Photoreduction of the Drosophila CRY (dCRY) flavin cofactor to the anionic semiquinone (ASQ) restructures a C-terminal tail helix (CTT) that otherwise inhibits interactions with targets that include the clock protein Timeless (TIM). All-atom molecular dynamics (MD) simulations indicate that flavin reduction destabilizes the CTT, which undergoes large-scale conformational changes (the CTT release) on short (25 ns) timescales. The CTT release correlates with the conformation and protonation state of conserved His378, which resides between the CTT and the flavin cofactor. Poisson-Boltzmann calculations indicate that flavin reduction substantially increases the His378 pKa Consistent with coupling between ASQ formation and His378 protonation, dCRY displays reduced photoreduction rates with increasing pH; however, His378Asn/Arg variants show no such pH dependence. Replica-exchange MD simulations also support CTT release mediated by changes in His378 hydrogen bonding and verify other responsive regions of the protein previously identified by proteolytic sensitivity assays. His378 dCRY variants show varying abilities to light-activate TIM and undergo self-degradation in cellular assays. Surprisingly, His378Arg/Lys variants do not degrade in light despite maintaining reactivity toward TIM, thereby implicating different conformational responses in these two functions. Thus, the dCRY photosensory mechanism involves flavin photoreduction coupled to protonation of His378, whose perturbed hydrogen-bonding pattern alters the CTT and surrounding regions. PMID:27551082

  10. Azido Auxins: Synthesis and Biological Activity of Fluorescent Photoaffinity Labeling Agents 12

    PubMed Central

    Melhado, L. Lee; Jones, Alan M.; Leonard, Nelson J.; Vanderhoef, Larry N.

    1981-01-01

    Three auxin analogs, 4−, 5−, and 6-azido-3-indoleacetic acid (4-N3-IAA, 5-N3-IAA, and 6-N3-IAA) have been synthesized for use as fluorescent photoaffinity labeling agents. The pKa values of these compounds (4-N3-IAA, 4.67; 5-N3-IAA, 4.65; 6-N3-IAA, 4.66; all ± 0.04) are experimentally indistinguishable from the pKa of 3-indoleacetic acid (IAA, 4.69 ± 0.04). The auxin activity of these IAA derivatives has been determined in several systems. In soybean, pea, and corn straight growth assays, all three analogs induce growth comparable to that caused by IAA. In the tobacco pith assay, all three analogs elicit a maximum increase in fresh weight at least 40 to 50% of that caused by IAA. Optimal growth is attained in the tobacco pith assay at slightly higher concentrations of 4-N3-IAA and 6-N3-IAA (30 micromolar) than required for IAA (10 micromolar); however, maximal growth is achieved at a slightly lower concentration of 5-N3-IAA (3 micromolar). The N3-IAAs, like IAA, are transported basipetally through tobacco pith tissue. PMID:16661939

  11. Diffusional dynamics of an active rhodamine-labeled 1,4-dihydropyridine in sarcolemmal lipid multibilayers.

    PubMed Central

    Mason, R P; Chester, D W

    1989-01-01

    A "membrane bilayer pathway" model, involving ligand partition into the bilayer, lateral diffusion, and receptor binding has been invoked to describe the 1,4-dihydropyridine (DHP) calcium channel antagonist receptor binding mechanism. In an earlier study (Chester et al. 1987. Biophys. J. 52:1021-1030), the diffusional component of this model was examined using an active fluorescence labeled DHP calcium channel antagonist, nisoldipine-lissamine rhodamine B (Ns-R), in purified cardiac sarcolemmal (CSL) lipid multibilayers. Diffusion coefficient measurements on membrane-bound drug and phospholipid at maximum bilayer hydration yielded similar values (3.8 x 10(-8) cm2/s). However, decreases in bilayer hydration resulted in dramatically reduced diffusion coefficient values for both probes with substantially greater impact on Ns-R diffusion. These data suggested that hydration dependent diffusional differences could be a function of relative probe location along the bilayer normal. In this communication, we have addressed the relative effect of the rhodamine substituent on Ns-R diffusion complex by examining the diffusional dynamics of free rhodamine B under the same conditions used to evaluate Ns-R complex and phospholipid diffusion. X-ray diffraction studies were performed to determine the Ns-R location in the membrane and model the CSL lipid bilayer profile structure to give a rationale for the differences in probe diffusional dynamics as a function of interbilayer water space. PMID:2611332

  12. Conformational Transitions in Human AP Endonuclease 1 and Its Active Site Mutant during Abasic Site Repair†

    PubMed Central

    Kanazhevskaya, Lyubov Yu.; Koval, Vladimir V.; Zharkov, Dmitry O.; Strauss, Phyllis R.; Fedorova, Olga S.

    2010-01-01

    AP endonuclease 1 (APE 1) is a crucial enzyme of the base excision repair pathway (BER) in human cells. APE1 recognizes apurinic/apyrimidinic (AP) sites and makes a nick in the phosphodiester backbone 5′ to them. The conformational dynamics and presteady-state kinetics of wild-type APE1 and its active site mutant, Y171F-P173L-N174K, have been studied. To observe conformational transitions occurring in the APE1 molecule during the catalytic cycle, we detected intrinsic tryptophan fluorescence of the enzyme under single turnover conditions. DNA duplexes containing a natural AP site, its tetrahydrofuran analogue, or a 2′-deoxyguanosine residue in the same position were used as specific substrates or ligands. The stopped-flow experiments have revealed high flexibility of the APE1 molecule and the complexity of the catalytic process. The fluorescent traces indicate that wild-type APE1 undergoes at least four conformational transitions during the processing of abasic sites in DNA. In contrast, nonspecific interactions of APE1 with undamaged DNA can be described by a two-step kinetic scheme. Rate and equilibrium constants were extracted from the stopped-flow and fluorescence titration data for all substrates, ligands, and products. A replacement of three residues at the enzymatic active site including the replacement of tyrosine 171 with phenylalanine in the enzyme active site resulted in a 2 × 104-fold decrease in the reaction rate and reduced binding affinity. Our data indicate the important role of conformational changes in APE1 for substrate recognition and catalysis. PMID:20575528

  13. N-methyl-D-aspartate recognition site ligands modulate activity at the coupled glycine recognition site.

    PubMed

    Hood, W F; Compton, R P; Monahan, J B

    1990-03-01

    In synaptic plasma membranes from rat forebrain, the potencies of glycine recognition site agonists and antagonists for modulating [3H]1-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) binding and for displacing strychnine-insensitive [3H]glycine binding are altered in the presence of N-methyl-D-aspartate (NMDA) recognition site ligands. The NMDA competitive antagonist, cis-4-phosphonomethyl-2-piperidine carboxylate (CGS 19755), reduces [3H]glycine binding, and the reduction can be fully reversed by the NMDA recognition site agonist, L-glutamate. Scatchard analysis of [3H]glycine binding shows that in the presence of CGS 19755 there is no change in Bmax (8.81 vs. 8.79 pmol/mg of protein), but rather a decrease in the affinity of glycine (KD of 0.202 microM vs. 0.129 microM). Similar decreases in affinity are observed for the glycine site agonists, D-serine and 1-aminocyclopropane-1-carboxylate, in the presence of CGS 19755. In contrast, the affinity of glycine antagonists, 1-hydroxy-3-amino-2-pyrrolidone and 1-aminocyclobutane-1-carboxylate, at this [3H]glycine recognition site increases in the presence of CGS 19755. The functional consequence of this change in affinity was addressed using the modulation of [3H]TCP binding. In the presence of L-glutamate, the potency of glycine agonists for the stimulation of [3H]TCP binding increases, whereas the potency of glycine antagonists decreases. These data are consistent with NMDA recognition site ligands, through their interactions at the NMDA recognition site, modulating activity at the associated glycine recognition site.

  14. Changes in diacylglycerol labeling, cell shape, and protein phosphorylation distinguish triggering from activation of human neutrophils

    SciTech Connect

    Reibman, J.; Korchak, H.M.; Vosshall, L.B.; Haines, K.A.; Rich, A.M.; Weissmann, G.

    1988-05-05

    Upon activation neutrophils release reactive oxygen intermediates such as superoxide anion (O/sub 2//sup -/) which are potent mediators of inflammation. Various agents elicit different responses. In contrast, phorbol myristate acetate (PMA, 1.6 ..mu..M) acting directly via protein kinase C is a potent stimulus for O/sub 2//sup -/. The authors compared the kinetics of appearance of various second messengers with the capacity of these ligands to elicit O/sub 2//sup -/ generation. Kinetic analysis showed a two-phase response to membrane ligands; both an early (greater than or equal to 15 s) and a late (>15 s) increase in (/sup 3/H)- and (/sup 14/C)diacylglycerol (DG) was noted in response to fMLP. In contrast, LTB/sub 4/ elicited only a rapid early increase in DG. The rise in DG evoked by PMA was late. Moreover, comparison of increases in (/sup 3/H)DG versus those of (/sup 14/C)DG at early and late time points suggested that DG was not formed exclusively from the hydrolysis of polyphosphoinositides. Kinetic analysis of protein phosphorylation was compared to the early and late increments of DG labeling. A 47,000 M/sub r/ protein was phosphorylated with kinetics consistent with the production of O/sub 2//sup -/ and DG in response to fMLP and PMA. The temporal pattern of the formation of diacylglycerol and the phosphorylation of proteins describe a dual signal. The data suggest that neutrophils require not only triggering (the rapid generation of a signal) but also activation (the maintenance of a signal) to sustain responses.

  15. Nutritional supplement products: does the label information influence purchasing decisions for the physically active?

    PubMed Central

    2013-01-01

    Background The increase in sales of nutritional supplement globally can be attributed, in part, to aggressive marketing by manufacturers, rather than because the nutritional supplements have become more effective. Furthermore, the accuracy of the labelling often goes unchallenged. Therefore, any effects of the supplement, may be due to contaminants or adulterants in these products not reflected on the label. Methods A self-administered questionnaire was used to determine how consumers of nutritional supplements acquired information to assist their decision-making processes, when purchasing a product. The study was approved by the University of Cape Town, Faculty of Health Sciences Human Research Ethics Committee. The questionnaire consisted of seven, closed and open-ended questions. The participants were asked to respond to the questions according to a defined list of statements. A total of 259 participants completed and returned questionnaires. The data and processing of the returned questionnaires was captured using Windows-based Microsoft® Office Excel 2003 SP 1 (Excel © 1985–2003 Microsoft Corporation). Statistica Version 10 (copyright © Stat Soft, Inc. 1984–2011) was used to calculate the descriptive statistics. Results The main finding of the study was that nearly 70% of the respondents who purchased supplements were strongly influenced by container label information that stipulated that the nutritional supplement product is free of banned substances. The second finding was that just over 50% of the respondents attached importance to the quality of the nutritional supplement product information on the container label. The third finding was that about 40% of the respondents were strongly influenced by the ingredients on the labels when they purchased nutritional supplements. Conclusion This study, (i) identifies short-comings in current labelling information practices, (ii) provides opportunities to improve label and non-label information and

  16. Control of active sites in selective flocculation: I -- Mathematical model

    SciTech Connect

    Behl, S.; Moudgil, B.M.; Prakash, T.S. . Dept. of Materials Science and Engineering)

    1993-12-01

    Heteroflocculation has been determined to be another major reason for loss in selectivity for flocculation process. In a mathematical model developed earlier, conditions for controlling heteroflocculation were discussed. Blocking active sites to control selective adsorption of a flocculant oil a desirable solid surface is discussed. It has been demonstrated that the lower molecular weight fraction of a flocculant which is incapable of flocculating the particles is an efficient site blocking agent. The major application of selective flocculation has been in mineral processing but many potential uses exist in biological and other colloidal systems. These include purification of ceramic powders, separating hazardous solids from chemical waste, and removal of deleterious components from paper pulp.

  17. Site-specific Orientation of an α-helical Peptide Ovispirin-1 from Isotope Labeled SFG Spectroscopy

    PubMed Central

    Ding, Bei; Laaser, Jennifer E.; Liu, Yuwei; Wang, Pengrui; Zanni, Martin T.; Chen, Zhan

    2013-01-01

    Sum-frequency generation (SFG) vibrational spectroscopy is often used to probe the backbone structures and orientations of polypeptides at surfaces. Using the ovispirin-1 polypeptide at the solid/liquid interface of polystyrene, we demonstrate for the first time that SFG can probe the polarization response of a single isotope labeled residue. To interpret the spectral intensities, we simulated the spectra using an excitonic Hamiltonian approach. We show that the polarization dependence of either the label or the unlabeled amide I band alone does not provide sufficient structural constraints to obtain both the tilt and the twist of the ovispirin helix at a solid/liquid interface, but that both can be determined from the polarization dependence of the complete spectrum. For ovispirin, the detailed analysis of the polarized SFG experimental data shows that the helix axis is tilted at roughly 138 degrees from the surface normal, and the transition dipole of the isotope labeled C=O group is tilted at 23 degrees from the surface normal, with the hydrophobic region facing the polystyrene surface. We further demonstrated that the Hamiltonian approach is able to address the coupling effect and the structural disorder. For comparison, we also collected the FTIR spectrum of ovispirin under similar conditions, which reveals the enhanced sensitivity of SFG for structural studies of single monolayer peptide surfaces. Our study provides insight into how structural and environmental effects appear in SFG spectra of the amide I band and establishes that SFG of isotope labeled peptides will be a powerful technique for elucidating secondary structures with residue-by-residue resolution. PMID:24228619

  18. Structural difference at the active site of dibucaine resistant variant of human plasma cholinesterase.

    PubMed Central

    Muensch, H; Yoshida, A; Altland, K; Jensen, W; Goedde, H W

    1978-01-01

    Human plasma cholinesterase from five different genotypes -- E1U E1U, E1U E1A, E1A E1A, E1U E1S, E1A E1S, and E1U E1U C5+ -- was purified 8,000 fold from serum by a two-step procedure involving chromatography on DEAE-cellulose and preparative disc electrophoresis. The esterases were labeled with diisopropyl-1, 3-C14-fluorophosphate (DFP) aminoethylated, and digested by trypsin. The trytic digests were subjected to high voltage electrophoresis, and the radioactive peptides were detected by radioautography. Comparison of the peptides revealed different electrophoretic mobilities of the usual and atypical (dibucaine resistant) plasma cholinesterase peptides. The results are consistent with a structural abnormality of the active center in the variant enzyme. No difference was observed an the esteratic site of the enzyme with C5 component. Images Fig. 1 PMID:677127

  19. The site of activation of factor X by cancer procoagulant.

    PubMed

    Gordon, S G; Mourad, A M

    1991-12-01

    Cancer procoagulant (CP) is a cysteine proteinase found in a variety of malignant cells and tissues and in human amnion-chorion tissue. It initiates coagulation by activating factor X. However, the amino acid sequence of the substrate protein that determines the cleavage site of cysteine proteinases is different from that of the serine proteinases that normally activate factor X, such as factor IXa, VIIa and Russell's Viper Venom (RVV). Therefore, it was of interest to determine the site of cleavage of human factor X by CP. Purified CP was incubated with purified factor X and the reaction mixture was electrophoresed on a 10% Tris-tricine SDS-PAGE gel. The proteins were electroeluted on to a polyvinylidene difluoride (PVDF) membrane, and stained with Coomassie blue. The heavy chain of activated factor X was cut out of the PVDF membrane and sequenced with an Applied Biosystems 477A with on-line HPLC. The primary cleavage sequence was Asp-Ala-Ala-Asp-Leu-Asp-Pro-; two other secondary sequences Ser-Ile-Thr-Trp-Lys-Pro- and Glu-Asn-Pro-Phe-Asp-Leu were found. The penultimate amino acid on the carbonyl side of the hydrolysed amide bond plays a critical role for the recognition of the cleavage site of cysteine proteinases. These data indicate that the penultimate amino acid for the primary cleavage site of factor X by CP is proline-20 and for the secondary sites, proline-13 and proline-28. This is in contrast to arginine-52 that determines the specificity of the cleavage by normal serine proteinase activation.(ABSTRACT TRUNCATED AT 250 WORDS)

  20. The impact of aminopyrene trisulfonate (APTS) label in acceptor glycan substrates for profiling plant pectin β-galactosyltransferase activities.

    PubMed

    Goetz, Stephan; Rejzek, Martin; Nepogodiev, Sergey A; Field, Robert A

    2016-10-01

    Aminopyrene trisulfonate (APTS)-labelled disaccharides are demonstrated to serve as readily accessible acceptor substrates for galactosyltransferase activities present in Arabidopsis microsome preparations. The reductive amination procedure used to install the fluorophore results in loss of the ring structure of the reducing terminal sugar unit, such that a single intact sugar ring is present, attached via an alditol tether to the aminopyrene fluorophore. The configuration of the alditol portion of the labelled acceptor, as well as the position of alditol galactosylation, substantially influence the ability of compounds to serve as Arabidopsis galactosyltransferase acceptor substrates. The APTS label exhibits an unexpected reaction-promoting effect that is not evident for structurally similar sulfonated aromatic fluorophores ANDS and ANTS. When APTS-labelled β-(1 → 4)-Gal3 was employed as an acceptor substrate with Arabidopsis microsomes, glycan extension generated β-(1 → 4)-galactan chains running to beyond 60 galactose residues. These studies demonstrate the potential of even very short glycan-APTS probes for assessing plant galactosyltransferase activities and the suitability CE-LIF for CAZyme profiling. PMID:27479753

  1. UDP-( sup 14 C)glucose-labelable polypeptides from pea: Possible components of glucan synthase I activity

    SciTech Connect

    Ray, P.M.; Dhugga, K.S.; Gallaghar, S.R. )

    1989-04-01

    A membrane-bound polypeptide doublet of about 40 kD can be rapidly labeled with UDP-({sup 14}C)glucose under the assay conditions for glucan synthase I (GS-I). Label seems covalently bound, and chases when unlabeled UDPG is added; it might represent a covalent intermediate in polysaccharide synthesis. Labeling and GS-I activity show several common features: they co-sediment with Golgi membranes in sucrose gradients; they depend similarly on Mg{sup 2+} or Mn{sup 2+} (not Ca{sup 2+}); they decrease dramatically from stem apex to base, and are higher in epidermis than internal tissue; they show similar sensitivities to several inhibitors. But the doublet still labels after polysaccharide-synthesizing activity has been destroyed by Triton X-100. The doublet polypeptides might be glucosyl tranferases whose ability to transfer glucose units to a glucan chain is detergent-sensitive, but to accept glucose from UDPG is not; or they might be detergent-insensitive primary glucose acceptors, from which a distinct, detergent-sensitive transferase(s) move(s) these units to glucan chains.

  2. Ultrastructural localization of wheat germ agglutinin binding sites on the sperm surface of water buffalo (Bubalus bubalis). A fracture label study.

    PubMed

    Bains, H K; Pabst, M A; Werner, G; Bawa, S R

    1993-10-01

    In the present study we have examined the plasma membrane surface organization employing fluorescein isothiocyanate linked wheat germ agglutinin (WGA) of the cauda epididymal and ejaculated spermatozoa of water buffalo. Intramembrane particle distribution pattern in the various segments of the spermatozoa has also been observed. WGA-ovomucoid gold has been used to study the distribution of sialoproteins on the sperm surface. With fracture label, WGA receptor sites have been identified on the fractured membrane halves of the sperm plasma membrane overlying the acrosome as well as the middle piece and the principle piece.

  3. Preparation and immunoreactivity of high specific activity indium-111-DTPA labeled monoclonal antibody (MoAb) using ultrapure indium-111

    SciTech Connect

    Zoghbi, S.S.; Neumann, R.D.; Gottschalk, A.

    1986-10-01

    The preparation of high-specific activity /sup 111/In-DTPA-MoAb without increasing the number of DTPA molecules per Ab was investigated. Instant thin layer chromatography was used to assay the relationship between labeling efficiencies and specific activities. With ultrapurified /sup 111/In, the specific activity of the radiolabeled MoAb approached the expected theoretic maximum of 100 muCi/microgram. The bioactivity of such high-specific activity preparation showed no degradation as measured by in vitro cell binding assay.

  4. Active-Site-Accessible, Porphyrinic Metal;#8722;Organic Framework Materials

    SciTech Connect

    Farha, Omar K.; Shultz, Abraham M.; Sarjeant, Amy A.; Nguyen, SonBinh T.; Hupp, Joseph T.

    2012-02-06

    On account of their structural similarity to cofactors found in many metallo-enzymes, metalloporphyrins are obvious potential building blocks for catalytically active, metal-organic framework (MOF) materials. While numerous porphyrin-based MOFs have already been described, versions featuring highly accessible active sites and permanent microporosity are remarkably scarce. Indeed, of the more than 70 previously reported porphyrinic MOFs, only one has been shown to be both permanently microporous and contain internally accessible active sites for chemical catalysis. Attempts to generalize the design approach used in this single successful case have failed. Reported here, however, is the synthesis of an extended family of MOFs that directly incorporate a variety of metalloporphyrins (specifically Al{sup 3+}, Zn{sup 2+}, Pd{sup 2+}, Mn{sup 3+}, and Fe{sup 3+} complexes). These robust porphyrinic materials (RPMs) feature large channels and readily accessible active sites. As an illustrative example, one of the manganese-containing RPMs is shown to be catalytically competent for the oxidation of alkenes and alkanes.

  5. Functional constituents of the active site of human neutrophil collagenase.

    PubMed

    Mookhtiar, K A; Wang, F; Van Wart, H E

    1986-05-01

    A series of chemical modification reactions has been carried out to identify functional constituents of the active site of human neutrophil collagenase. The enzyme is reversibly inhibited by the transition metal chelating agent 1,10-phenanthroline, and inhibition is fully reversed by zinc. Removal of weakly bound metal ions by gel filtration inactivates collagenase, and activity is fully restored on immediate readdition of calcium. The enzyme is unaffected by reagents that modify serine, cysteine, and arginine residues. However, reaction with the carboxyl reagents cyclohexylmorpholinocarbodiimide and Woodward's Reagent K lowers the activity of the enzyme substantially. Acetylimidazole inactivates the enzyme, but activity is completely restored on addition of hydroxylamine. The enzyme is also inactivated by tetranitromethane, indicating that it contains an essential tyrosine residue. Acylation of collagenase with diethyl pyrocarbonate, diketene, acetic anhydride, or trinitrobenzenesulfonate inactivates the enzyme, and activity is not restored on addition of hydroxylamine, indicating the presence of an essential lysine residue.

  6. Site-specific φ- and ψ-torsion angle determination in a uniformly/extensively 13C- and 15N-labeled peptide

    NASA Astrophysics Data System (ADS)

    Wi, Sungsool; Spano, Justin

    2011-10-01

    A solid-state rotational-echo double resonance (REDOR) NMR method was introduced to identify the ϕ- and ψ-torsion angle from a 1H- 15N or 1H- 13C' spin system of alanine-like residues in a selectively, uniformly, or extensively 15N-/ 13C-labeled peptide. When a C α( i) or a 15N peak is site-specifically obtainable in the NMR spectrum of a uniformly 15N/ 13C-labeled sample system, the ψ- or ϕ-torsion angle specified by the conformational structure of peptide geometry involving 15N( i)- 1H αi - 15N( i + 1) or 13C'( i - 1)- 1H Ni- 13C'( i) spin system can be identified based on 13C α- or 15N-detected 1H α- 15N or 1H N- 13C REDOR experiment. This method will conveniently be utilized to identify major secondary motifs, such as α-helix, β-sheet, and β-turn, from a uniformly 15N-/ 13C-labled peptide sample system. When tested on a 13C-/ 15N-labeled model system of a three amino acid peptide Gly-[U- 13C, 15N]Ala-[U- 13C, 15N]Leu, the ψ-angle of alanine obtained experimentally, ψ = -40 ± 30°, agreed reasonably well with the X-ray determined angle, ψ = -39°.

  7. Nest predation increases with parental activity: Separating nest site and parental activity effects

    USGS Publications Warehouse

    Martin, T.E.; Scott, J.; Menge, C.

    2000-01-01

    Alexander Skutch hypothesized that increased parental activity can increase the risk of nest predation. We tested this hypothesis using ten open-nesting bird species in Arizona, USA. Parental activity was greater during the nestling than incubation stage because parents visited the nest frequently to feed their young during the nestling stage. However, nest predation did not generally increase with parental activity between nesting stages across the ten study species. Previous investigators have found similar results. We tested whether nest site effects might yield higher predation during incubation because the most obvious sites are depredated most rapidly. We conducted experiments using nest sites from the previous year to remove parental activity. Our results showed that nest sites have highly repeatable effects on nest predation risk; poor nest sites incurred rapid predation and caused predation rates to be greater during the incubation than nestling stage. This pattern also was exhibited in a bird species with similar (i.e. controlled) parental activity between nesting stages. Once nest site effects are taken into account, nest predation shows a strong proximate increase with parental activity during the nestling stage within and across species. Parental activity and nest sites exert antagonistic influences on current estimates of nest predation between nesting stages and both must be considered in order to understand current patterns of nest predation, which is an important source of natural selection.

  8. Evaluation of a method for nitrotyrosine site identification and relative quantitation using a stable isotope-labeled nitrated spike-in standard and high resolution fourier transform MS and MS/MS analysis.

    PubMed

    Seeley, Kent W; Fertig, Alison R; Dufresne, Craig P; Pinho, Joao P C; Stevens, Stanley M

    2014-04-14

    The overproduction of reactive oxygen and nitrogen species (ROS and RNS) can have deleterious effects in the cell, including structural and possible activity-altering modifications to proteins. Peroxynitrite is one such RNS that can result in a specific protein modification, nitration of tyrosine residues to form nitrotyrosine, and to date, the identification of nitrotyrosine sites in proteins continues to be a major analytical challenge. We have developed a method by which 15N-labeled nitrotyrosine groups are generated on peptide or protein standards using stable isotope-labeled peroxynitrite (O15NOO-), and the resulting standard is mixed with representative samples in which nitrotyrosine formation is to be measured by mass spectrometry (MS). Nitropeptide MS/MS spectra are filtered using high mass accuracy Fourier transform MS (FTMS) detection of the nitrotyrosine immonium ion. Given that the nitropeptide pair is co-isolated for MS/MS fragmentation, the nitrotyrosine immonium ions (at m/z=181 or 182) can be used for relative quantitation with negligible isotopic interference at a mass resolution of greater than 50,000 (FWHM, full width at half-maximum). Furthermore, the standard potentially allows for the increased signal of nitrotyrosine-containing peptides, thus facilitating selection for MS/MS in a data-dependent mode of acquisition. We have evaluated the methodology in terms of nitrotyrosine site identification and relative quantitation using nitrated peptide and protein standards.

  9. A 1-methyl-4-piperidinyl cytectrene carboxylate labeled by the technetium 99m, a radiotracer for rat brain acetylcholinesterase activity.

    PubMed

    Mejri, Najoua; Barhoumi, Chokri; Trabelsi, Moez; Mekni, Abdelkader; Said, Nadia Malek; Saidi, Mouldi

    2010-02-01

    Alzheimer's disease (AD) is a degenerative neurological disorder that causes progressive and irreversible loss of connections between brain cells and loss of mental functions. Clinical and postmortem studies show that the biochemical changes in brains of AD patients include decrease in acetylcholinesterase (AChE) activity. Our aim was to study AChE activity using piperidinyl ester labelled with technetium-99m. In vivo and in vitro studies demonstrated that labelled piperidinyl ester was a substrate for AChE. The hydrolytic rate of this substrate was measured and the specificity was evaluated using the inhibitor BW284c51. The rhenium analogues of the technetium-labelled substrate were used to determine the affinity constant (K(m)) and the maximum reaction velocity (V(max)) because of the high specific activity of technetium. The high hydrolytic rate and high specificity of the substrate for AChE make it suitable as an in vivo radiotracer for studying AChE activity in the brain.

  10. Active sites in char gasification: Final technical report

    SciTech Connect

    Wojtowicz, M.; Lilly, W.D.; Perkins, M.T.; Hradil, G.; Calo, J.M.; Suuberg, E.M.

    1987-09-01

    Among the key variables in the design of gasifiers and combustors is the reactivity of the chars which must be gasified or combusted. Significant loss of unburned char is unacceptable in virtually any process; the provision of sufficient residence time for complete conversion is essential. A very wide range of reactivities are observed, depending upon the nature of the char in a process. The current work focuses on furthering the understanding of gasification reactivities of chars. It has been well established that the reactivity of char to gasification generally depends upon three principal factors: (1) the concentration of ''active sites'' in the char; (2) mass transfer within the char; and (3) the type and concentration of catalytic impurities in the char. The present study primarily addresses the first factor. The subject of this research is the origin, nature, and fate of active sites in chars derived from parent hydrocarbons with coal-like structure. The nature and number of the active sites and their reactivity towards oxygen are examined in ''model'' chars derived from phenol-formaldehyde type resins. How the active sites are lost by the process of thermal annealing during heat treatment of chars are studied, and actual rate for the annealing process is derived. Since intrinsic char reactivities are of primary interest in the present study, a fair amount of attention was given to the model char synthesis and handling so that the effect of catalytic impurities and oxygen-containing functional groups in the chemical structure of the material were minimized, if not completely eliminated. The project would not be considered complete without comparing characteristic features of synthetic chars with kinetic behavior exhibited by natural chars, including coal chars.

  11. Brownian aggregation rate of colloid particles with several active sites

    SciTech Connect

    Nekrasov, Vyacheslav M.; Yurkin, Maxim A.; Chernyshev, Andrei V.; Polshchitsin, Alexey A.; Yakovleva, Galina E.; Maltsev, Valeri P.

    2014-08-14

    We theoretically analyze the aggregation kinetics of colloid particles with several active sites. Such particles (so-called “patchy particles”) are well known as chemically anisotropic reactants, but the corresponding rate constant of their aggregation has not yet been established in a convenient analytical form. Using kinematic approximation for the diffusion problem, we derived an analytical formula for the diffusion-controlled reaction rate constant between two colloid particles (or clusters) with several small active sites under the following assumptions: the relative translational motion is Brownian diffusion, and the isotropic stochastic reorientation of each particle is Markovian and arbitrarily correlated. This formula was shown to produce accurate results in comparison with more sophisticated approaches. Also, to account for the case of a low number of active sites per particle we used Monte Carlo stochastic algorithm based on Gillespie method. Simulations showed that such discrete model is required when this number is less than 10. Finally, we applied the developed approach to the simulation of immunoagglutination, assuming that the formed clusters have fractal structure.

  12. Active Sites Environmental Monitoring Program: FY 1991 report

    SciTech Connect

    Ashwood, T.L.; Hicks, D.S.; Morrissey, C.M.

    1992-11-01

    This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) from April 1991 through September 1991. The ASEMP was established in 1989 by Solid Waste Operations (SWO) and the Environmental Sciences Division, both of Oak Ridge National Laboratory, to provide early detection and performance monitoring at active low-level (radioactive) waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 as required by chapters II and III of US Department of Energy Order 5820.2A. A new set of action levels was developed on the basis of a statistical analysis of background contamination. These new action levels have been used to evaluate results in this report. Results of ASEMP monitoring continue to demonstrate that no LLW (except [sup 3]H) is being leached from the storage vaults on the tumulus pads. Loading of vaults on Tumulus II, which began in early FY 1991, was >90% complete at the end of September 1991. Results of sampling of groundwater and surface waters is presented.

  13. Inhibition and active-site modelling of prolidase.

    PubMed

    King, G F; Crossley, M J; Kuchel, P W

    1989-03-15

    Consideration of the active-site model of prolidase led us to examine azetidine, pyrrolidine and piperidine substrate analogs as potential in vivo inhibitors of the enzyme. One of these, N-benzyloxycarbonyl-L-proline, was shown to be a potent competitive inhibitor of porcine kidney prolidase (Ki = 90 microM); its rapid protein-mediated permeation of human and sheep erythrocytes suggests that it may be effective in vivo. The higher homolog, N-benzyloxycarbonyl-L-pipecolic acid, was also a potent inhibitor of the enzyme while the antihypertensive drugs, captopril and enalaprilat, were shown to have mild and no inhibitory effects, respectively. Analysis of inhibitor action and consideration of X-ray crystallographic data of relevant Mn2+ complexes allowed the active-site model of prolidase to be further refined; a new model is presented in which the substrate acts as a bidentate ligand towards the active-site manganous ion. Various aspects of the new model help to explain why Mn2+ has been 'chosen' by the enzyme in preference to other biologically available metal ions. PMID:2924773

  14. Interactions of dopaminergic agonists and antagonists with dopaminergic D3 binding sites in rat striatum. Evidence that (/sup 3/H)dopamine can label a high affinity agonist-binding state of the D1 dopamine receptor

    SciTech Connect

    Leff, S.E.; Creese, I.

    1985-02-01

    The interactions of dopaminergic agonists and antagonists with /sup 3/H-agonist labeled D3 dopaminergic binding sites of rat striatum have been characterized by radioligand-binding techniques. When the binding of (/sup 3/H)dopamine and (/sup 3/H)apomorphine to D2 dopamine receptors is blocked by the inclusion of D2 selective concentrations of unlabeled spiroperidol or domperidone, these ligands appear to label selectively the previously termed D3 binding site. Antagonist/(/sup 3/H)dopamine competition curves are of uniformly steep slope (nH . 1.0), suggesting the presence of a single D3 binding site. The relative potencies of antagonists to inhibit D3 specific (/sup 3/H)dopamine binding are significantly correlated with their potencies to block D1 dopamine receptors as measured by the inhibition of both dopamine-stimulated adenylate cyclase and (/sup 3/H)flupentixol-binding activities. The affinities of agonists to inhibit D3 specific (/sup 3/H)dopamine binding are also correlated with estimates of these agonists affinities for the high affinity binding component of agonist/(/sup 3/H)flupentixol competition curves. Both D3 specific (/sup 3/H) dopamine binding and the high affinity agonist-binding component of dopamine/(/sup 3/H)flupentixol competition curves show a similar sensitivity to guanine nucleotides. Taken together, these data strongly suggest that the D3 binding site is related to a high affinity agonist-binding state of the D1 dopamine receptor.

  15. Sulfhydryl site-specific cross-linking and labeling of monoclonal antibodies by a fluorescent equilibrium transfer alkylation cross-link reagent.

    PubMed

    del Rosario, R B; Wahl, R L; Brocchini, S J; Lawton, R G; Smith, R H

    1990-01-01

    The site-specific intramolecular cross-linking of sulfhydryls of monoclonal antibodies via a new class of "equilibrium transfer alkylation cross-link (ETAC) reagents" is described. Following complete or partial reduction of interchain disulfides with dithiothreitol (DTT), two murine IgG2a monoclonal antibodies, 225.28S and 5G6.4, were reacted with alpha,alpha-bis[(p-tolylsulfonyl)methyl]-m-aminoacetophenone (ETAC 1a) and a fluorescent conjugated derivative, sulforhodamine B m-(alpha,alpha-bis(p-tolysulfonylmethyl)acetyl)anilide derivative (ETAC 1b). Reducing SDS-polyacrylamide gel electrophoresis analysis of the products from 1b indicated the formation of S-ETAC-S interchain heavy and light chain cross-links (approximately 23-34% overall yield by video-camera densitometry) which do not undergo disulfide-thiol exchange with DTT at 100 degrees C. In contrast, no interchain cross-links were observed upon reaction of unreduced or reduced antibody wherein the thiols have been previously alkylated with iodoacetamide. These results indicated site-specific cross-linking of interchain sulfhydryls and places their distance within 3-4 A. Flow cytometry of the ETAC 1b 5G6.4 cross-linked product using 77 IP3 human ovarian carcinoma target cells showed positive binding and retention of immunoreactivity. The in vivo biodistributions of 131I-labeled intact 5G6.4 and 125I-labeled reduced 5G6.4 + ETAC 1a product in rats were essentially identical over a period of 24 h. The present study illustrates the potential applications of labelable ETAC reagents as thiol-specific probes for a wide variety of immunological studies. PMID:2128870

  16. Target-controlled formation of silver nanoclusters in abasic site-incorporated duplex DNA for label-free fluorescence detection of theophylline

    NASA Astrophysics Data System (ADS)

    Park, Ki Soo; Oh, Seung Soo; Soh, H. Tom; Park, Hyun Gyu

    2014-08-01

    A novel, label-free, fluorescence based sensor for theophylline has been developed. In the new sensor system, an abasic site-incorporated duplex DNA probe serves as both a pocket for recognition of theophylline and a template for the preparation of fluorescent silver nanoclusters. The strategy relies on theophylline-controlled formation of fluorescent silver nanoclusters from abasic site-incorporated duplex DNA. When theophylline is not present, silver ions interact with the cytosine groups opposite to the abasic site in duplex DNA. This interaction leads to efficient formation of intensely red fluorescent silver nanoclusters. In contrast, when theophylline is bound at the abasic site through pseudo base-pairing with appropriately positioned cytosines, silver ion binding to the cytosine nucleobase is prevented. Consequently, fluorescent silver nanoclusters are not formed causing a significant reduction of the fluorescence signal. By employing this new sensor, theophylline can be highly selectively detected at a concentration as low as 1.8 μM. Finally, the diagnostic capability and practical application of this sensor were demonstrated by its use in detecting theophylline in human blood serum.A novel, label-free, fluorescence based sensor for theophylline has been developed. In the new sensor system, an abasic site-incorporated duplex DNA probe serves as both a pocket for recognition of theophylline and a template for the preparation of fluorescent silver nanoclusters. The strategy relies on theophylline-controlled formation of fluorescent silver nanoclusters from abasic site-incorporated duplex DNA. When theophylline is not present, silver ions interact with the cytosine groups opposite to the abasic site in duplex DNA. This interaction leads to efficient formation of intensely red fluorescent silver nanoclusters. In contrast, when theophylline is bound at the abasic site through pseudo base-pairing with appropriately positioned cytosines, silver ion binding to

  17. Physical location of the site for N-acetyl-L-glutamate, the allosteric activator of carbamoyl phosphate synthetase, in the 20-kilodalton COOH-terminal domain.

    PubMed

    Rodriguez-Aparicio, L B; Guadalajara, A M; Rubio, V

    1989-04-01

    Mammalian liver mitochondrial carbamoyl phosphate synthetase, a polypeptide of 160 kDa, is activated allosterically by N-acetyl-L-glutamate. The analogue of this activator N-(chloroacetyl)-L-[14C]glutamate has been found to serve as a photoaffinity label for this enzyme. The specificity was demonstrated by the drastic reduction in the radioactivity bound to the protein when (a) an excess of unlabeled acetylglutamate was present during the irradiation and (b) the enzyme was replaced by pyruvate kinase, an enzyme that is not affected by acetylglutamate. The labeling was due to the photoactivation of the chloroacetyl group since there was no labeling under equal conditions with acetyl[14C]glutamate. To localize the binding site, limited proteolysis was used. Trypsin cleaves carbamoyl phosphate synthetase into complementary NH2- and COOH-terminal fragments of about 140 and 20 kDa, respectively [Powers-Lee, S. G., & Corina, K. (1986) J. Biol. Chem. 261, 15349-15352], but only the latter was found to be labeled. Similarly, of the various fragments generated by elastase, only two, of 20 and 120 kDa, contain the COOH terminus [see Powers-Lee and Corina (1986) above] and were found to be labeled. Thus, the binding site for acetylglutamate is within 20 kDa from the COOH terminus. This excludes the possibility that the acetylglutamate binding site evolved from an ancestral substrate site for glutamine: this substrate binds to the small subunit of the Escherichia coli enzyme, which is homologous to the NH2-terminal domain of the rat liver enzyme. Exhaustive tryptic digestion of photolabeled carbamoyl phosphate synthetase yielded a single radioactive peak, suggesting that the labeling is restricted to a single minimal tryptic peptide. PMID:2742825

  18. In vivo, large-scale preparation of uniformly (15)N- and site-specifically (13)C-labeled homogeneous, recombinant RNA for NMR studies.

    PubMed

    Le, My T; Brown, Rachel E; Simon, Anne E; Dayie, T Kwaku

    2015-01-01

    Knowledge of how ribonucleic acid (RNA) structures fold to form intricate, three-dimensional structures has provided fundamental insights into understanding the biological functions of RNA. Nuclear magnetic resonance (NMR) spectroscopy is a particularly useful high-resolution technique to investigate the dynamic structure of RNA. Effective study of RNA by NMR requires enrichment with isotopes of (13)C or (15)N or both. Here, we present a method to produce milligram quantities of uniformly (15)N- and site-specifically (13)C-labeled RNAs using wild-type K12 and mutant tktA Escherichia coli in combination with a tRNA-scaffold approach. The method includes a double selection protocol to obtain an E. coli clone with consistently high expression of the recombinant tRNA-scaffold. We also present protocols for the purification of the tRNA-scaffold from a total cellular RNA extract and the excision of the RNA of interest from the tRNA-scaffold using DNAzymes. Finally, we showcase NMR applications to demonstrate the benefit of using in vivo site-specifically (13)C-labeled RNA. PMID:26577743

  19. Label-free fluorescence strategy for sensitive detection of exonuclease activity using SYBR Green I as probe.

    PubMed

    Xu, Min; Li, Baoxin

    2015-01-01

    A label-free and sensitive fluorescence assay for exonuclease activity is developed using commercially available SYBR Green I (SG) dye as signal probe. A proof-of-concept of this assay has been demonstrated by using exonuclease III (Exo III) as a model enzyme. In this assay, double-stranded DNA (dsDNA) can bind SG, resulting in a strong fluorescence signal of SG. Upon the addition of Exo III, dsDNA would be digested, and SG emits very weak fluorescence. Thus, Exo III activity can be facilely measured with a simple fluorescence reader. This method has a linear detection range from 1 U/mL to 200 U/mL with a detection limit of 0.7 U/mL. This label-free approach is selective, simple, convenient and cost-efficient without any complex DNA sequence design or fluorescence dye label. The method not only provides a platform for monitoring activity and inhibition of exonuclease but also shows great potential in biological process researches, drug discovery, and clinic diagnostics.

  20. Druggability analysis and classification of protein tyrosine phosphatase active sites

    PubMed Central

    Ghattas, Mohammad A; Raslan, Noor; Sadeq, Asil; Al Sorkhy, Mohammad; Atatreh, Noor

    2016-01-01

    Protein tyrosine phosphatases (PTP) play important roles in the pathogenesis of many diseases. The fact that no PTP inhibitors have reached the market so far has raised many questions about their druggability. In this study, the active sites of 17 PTPs were characterized and assessed for its ability to bind drug-like molecules. Consequently, PTPs were classified according to their druggability scores into four main categories. Only four members showed intermediate to very druggable pocket; interestingly, the rest of them exhibited poor druggability. Particularly focusing on PTP1B, we also demonstrated the influence of several factors on the druggability of PTP active site. For instance, the open conformation showed better druggability than the closed conformation, while the tight-bound water molecules appeared to have minimal effect on the PTP1B druggability. Finally, the allosteric site of PTP1B was found to exhibit superior druggability compared to the catalytic pocket. This analysis can prove useful in the discovery of new PTP inhibitors by assisting researchers in predicting hit rates from high throughput or virtual screening and saving unnecessary cost, time, and efforts via prioritizing PTP targets according to their predicted druggability. PMID:27757011

  1. 21 CFR 333.160 - Labeling of permitted combinations of active ingredients.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... the temporary relief of” (select one of the following: “pain,” “discomfort,” “pain or discomfort” or “pain and itching”) “in minor cuts, scrapes, and burns.” (c) Warnings. The labeling of the...

  2. 21 CFR 333.160 - Labeling of permitted combinations of active ingredients.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... the temporary relief of” (select one of the following: “pain,” “discomfort,” “pain or discomfort” or “pain and itching”) “in minor cuts, scrapes, and burns.” (c) Warnings. The labeling of the...

  3. 21 CFR 352.60 - Labeling of permitted combinations of active ingredients.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... SERVICES (CONTINUED) DRUGS FOR HUMAN USE SUNSCREEN DRUG PRODUCTS FOR OVER-THE-COUNTER HUMAN USE Labeling... identified in the applicable monographs. (2) For permitted combinations containing a sunscreen and a skin protectant identified in § 352.20(b), any or all of the applicable indications for sunscreens in §...

  4. 21 CFR 352.60 - Labeling of permitted combinations of active ingredients.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... SERVICES (CONTINUED) DRUGS FOR HUMAN USE SUNSCREEN DRUG PRODUCTS FOR OVER-THE-COUNTER HUMAN USE Labeling... identified in the applicable monographs. (2) For permitted combinations containing a sunscreen and a skin protectant identified in § 352.20(b), any or all of the applicable indications for sunscreens in §...

  5. 21 CFR 352.60 - Labeling of permitted combinations of active ingredients.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... SERVICES (CONTINUED) DRUGS FOR HUMAN USE SUNSCREEN DRUG PRODUCTS FOR OVER-THE-COUNTER HUMAN USE Labeling... identified in the applicable monographs. (2) For permitted combinations containing a sunscreen and a skin protectant identified in § 352.20(b), any or all of the applicable indications for sunscreens in §...

  6. 21 CFR 352.60 - Labeling of permitted combinations of active ingredients.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... SERVICES (CONTINUED) DRUGS FOR HUMAN USE SUNSCREEN DRUG PRODUCTS FOR OVER-THE-COUNTER HUMAN USE Labeling... identified in the applicable monographs. (2) For permitted combinations containing a sunscreen and a skin protectant identified in § 352.20(b), any or all of the applicable indications for sunscreens in §...

  7. 21 CFR 352.60 - Labeling of permitted combinations of active ingredients.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... SERVICES (CONTINUED) DRUGS FOR HUMAN USE SUNSCREEN DRUG PRODUCTS FOR OVER-THE-COUNTER HUMAN USE Labeling... identified in the applicable monographs. (2) For permitted combinations containing a sunscreen and a skin protectant identified in § 352.20(b), any or all of the applicable indications for sunscreens in §...

  8. 21 CFR 341.85 - Labeling of permitted combinations of active ingredients.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...), (o), (q), and (r) when labeled for relief of general cough-cold symptoms and/or the common cold. (i... general cough-cold symptoms and/or the common cold and for relief of hay fever/allergic rhinitis and/or... SERVICES (CONTINUED) DRUGS FOR HUMAN USE COLD, COUGH, ALLERGY, BRONCHODILATOR, AND ANTIASTHMATIC...

  9. 21 CFR 333.160 - Labeling of permitted combinations of active ingredients.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... combination, as established in the statement of identity sections of the applicable OTC drug monographs. For a... sections of the applicable OTC drug monographs. (b) Indications. The labeling of the product states, under... the “Indications” sections of the applicable OTC drug monographs, unless otherwise stated in...

  10. 21 CFR 333.160 - Labeling of permitted combinations of active ingredients.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... combination, as established in the statement of identity sections of the applicable OTC drug monographs. For a... sections of the applicable OTC drug monographs. (b) Indications. The labeling of the product states, under... the “Indications” sections of the applicable OTC drug monographs, unless otherwise stated in...

  11. 77 FR 49818 - Agency Information Collection Activities; Proposed Collection; Comment Request; Bar Code Label...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-08-17

    ... (69 FR 9120), we issued regulations that required human drug product and biological product labels to...-counter (OTC) drug products that are dispensed under an order and commonly used in health care facilities... prescription drug products and OTC drug products that are dispensed under an order and commonly used in...

  12. Sulfated Low Molecular Weight Lignins, Allosteric Inhibitors of Coagulation Proteinases via the Heparin Binding Site, Significantly Alter the Active Site of Thrombin and Factor Xa Compared to Heparin

    PubMed Central

    Henry, Brian L.; Desai, Umesh R.

    2014-01-01

    Sulfated low molecular weight lignins (LMWLs) have been found to bind in the heparin binding sites of coagulation proteinases. LMWLs represent a library of diverse non-carbohydrate, aromatic molecules which are structures different from heparin, but still potently inhibit thrombin and factor Xa. To better understand their mechanism of action, we studied the effects of three sulfated LMWLs (CDSO3, FDSO3, and SDSO3) on the active sites of thrombin and factor Xa. LMWLs were found to uniformly inhibit the catalytic activity of thrombin and factor Xa, regardless of the substrate used. Michaelis-Menten kinetic studies indicate that maximal velocity of hydrolysis of each chromogenic substrate decreases significantly in the presence of sulfated LMWLs, while the effect on Michaelis constant is dependent on the nature of the substrate. These studies indicate that LMWLs inhibit thrombin and factor Xa through allosteric disruption of the catalytic apparatus, specifically through the catalytic step. As opposed to heparin, LMWLs significantly alter the binding of the active site fluorescent ligand p-aminobenzamidine. LMWLs also had a greater effect on the molecular orientation of fluorescein-labeled His 57 than heparin. The molecular geometry surrounding the most important catalytic amino acid, Ser 195, was significantly altered by the binding of LMWLs while heparin had no measurable effect on Ser 195. These results further advance the concept of sulfated LMWLs as heparin mimics and will aid the design of anticoagulants based on their novel scaffold. PMID:25242245

  13. Sulfated low molecular weight lignins, allosteric inhibitors of coagulation proteinases via the heparin binding site, significantly alter the active site of thrombin and factor xa compared to heparin.

    PubMed

    Henry, Brian L; Desai, Umesh R

    2014-11-01

    Sulfated low molecular weight lignins (LMWLs) have been found to bind in the heparin binding sites of coagulation proteinases. LMWLs represent a library of diverse non-carbohydrate, aromatic molecules which are structures different from heparin, but still potently inhibit thrombin and factor Xa. To better understand their mechanism of action, we studied the effects of three sulfated LMWLs (CDSO3, FDSO3, and SDSO3) on the active sites of thrombin and factor Xa. LMWLs were found to uniformly inhibit the catalytic activity of thrombin and factor Xa, regardless of the substrate used. Michaelis-Menten kinetic studies indicate that maximal velocity of hydrolysis of each chromogenic substrate decreases significantly in the presence of sulfated LMWLs, while the effect on Michaelis constant is dependent on the nature of the substrate. These studies indicate that LMWLs inhibit thrombin and factor Xa through allosteric disruption of the catalytic apparatus, specifically through the catalytic step. As opposed to heparin, LMWLs significantly alter the binding of the active site fluorescent ligand p-aminobenzamidine. LMWLs also had a greater effect on the molecular orientation of fluorescein-labeled His 57 than heparin. The molecular geometry surrounding the most important catalytic amino acid, Ser 195, was significantly altered by the binding of LMWLs while heparin had no measurable effect on Ser 195. These results further advance the concept of sulfated LMWLs as heparin mimics and will aid the design of anticoagulants based on their novel scaffold. PMID:25242245

  14. Effect of exercise on erythrocyte count and blood activity concentration after technetium-99m in vivo red blood cell labeling

    SciTech Connect

    Konstom, M.A.; Tu'meh, S.; Wynne, J.; Beck, J.R.; Kozlowski, J.; Holman, B.L.

    1982-09-01

    The effects of exercise on blood radiotracer concentration after technetium-99m in vivo red blood cell labeling was studied. After red blood cell labeling, 13 subjects underwent maximal supine bicycle exercise. Radioactivity, analyzed with a well counter, was measured in heparinized venous blood samples drawn at rest and during peak exercise. Changes in activity were compared with changes in erythrocyte count. Activity and erythrocyte counts increased in erythrocyte count (r=0.78), but did not correlate with either duration of exercise or maximal heart rate. Twenty minutes after termination of exercise, activity and erythrocyte count had decreased from peak exercise values but remained higher than preexercise values. In nine nonexercised control subjects, samples drawn 20 minutes apart showed no change in activity or in erythrocyte count. It was concluded that exercise increases blood activity, primarily because of an increase in erythrocyte count. During radionuclide ventriculography, blood activity must be measured before and after any intervention, particularly exercise, before a change in left ventricular activity can be attributed to a change in left ventricular volume.

  15. Neurospora tryptophan synthase: N-terminal analysis and the sequence of the pyridoxal phosphate active site peptide

    SciTech Connect

    Pratt, M.L.; Hsu, P.Y.; DeMoss, J.A.

    1986-05-01

    Tryptophan synthase (TS), which catalyzes the final step of tryptophan biosynthesis, is a multifunctional protein requiring pyridoxal phosphate (B6P) for two of its three distinct enzyme activities. TS from Neurospora has a blocked N-terminal, is a homodimer of 150 KDa and binds one mole of B6P per mole of subunit. The authors shown the N-terminal residue to be acyl-serine. The B6P-active site of holoenzyme was labelled by reduction of the B6P-Schiff base with (/sup 3/H)-NaBH/sub 4/, and resulted in a proportionate loss of activity in the two B6P-requiring reactions. SDS-polyacrylamide gel electrophoresis of CNBr-generated peptides showed the labelled, active site peptide to be 6 KDa. The sequence of this peptide, purified to apparent homogeneity by a combination of C-18 reversed phase and TSK gel filtration HPLC is: gly-arg-pro-gly-gln-leu-his-lys-ala-glu-arg-leu-thr-glu-tyr-ala-gly-gly-ala-gln-ile-xxx-leu-lys-arg-glu-asp-leu-asn-his-xxx-gly-xxx-his-/sub ***/-ile-asn-asn-ala-leu. Although four residues (xxx, /sub ***/) are unidentified, this peptide is minimally 78% homologous with the corresponding peptide from yeast TS, in which residue (/sub ***/) is the lysine that binds B6P.

  16. Cloning and characterization of a novel nuclease from shrimp hepatopancreas, and prediction of its active site.

    PubMed

    Wang, W Y; Liaw, S H; Liao, T H

    2000-03-15

    Approximately 95% of the amino acid sequence of a shrimp (Penaeus japonicus) nuclease was derived from protease-digested peptides. A 1461-base cDNA for the nuclease was amplified and sequenced with degenerate primers based on the amino acid sequence and then specific primers by 3' and 5' RACE (rapid amplification of cDNA ends). It contains an open reading frame encoding a putative 21-residue signal peptide and a 381-residue mature protein. The N-terminus of the enzyme is pyroglutamate, deduced from composition and matrix-assisted laser desorption ionization-time-of-flight MS analyses, and confirmed by a glutamine residue in the cDNA sequence. The enzyme has 11 Cys residues, forming five intramolecular disulphides. The eleventh Cys residue was linked to a thiol compound with an estimated molecular mass of between 500 and 700 Da. A sequence similarity search revealed no homologous proteins but residues 205-255 shared a conserved active-site motif within a distinct group of nucleases. His(211) in this conserved motif was shown to be very important in catalysis by site-specific modification with (14)C-labelled iodoacetate. The shrimp nuclease, previously designated DNase I, does indeed possess a low level of hydrolytic activity towards RNA in the presence of Mg(2+) and Ca(2+). The conservation of functionally important residues during distant evolution might imply that the catalytic mechanisms are similar in these nucleases, which should be classified in one subfamily. Finally, an active-site structure for shrimp nuclease was proposed on the basis of published structural data and the results of mutational and biochemical analyses of Serratia nuclease.

  17. Comparison of two site-specifically (18)F-labeled affibodies for PET imaging of EGFR positive tumors.

    PubMed

    Su, Xinhui; Cheng, Kai; Jeon, Jongho; Shen, Bin; Venturin, Gianina Teribele; Hu, Xiang; Rao, Jianghong; Chin, Frederick T; Wu, Hua; Cheng, Zhen

    2014-11-01

    The epidermal growth factor receptor (EGFR) serves as an attractive target for cancer molecular imaging and therapy. Our previous positron emission tomography (PET) studies showed that the EGFR-targeting affibody molecules (64)Cu-DOTA-ZEGFR:1907 and (18)F-FBEM-ZEGFR:1907 can discriminate between high and low EGFR-expression tumors and have the potential for patient selection for EGFR-targeted therapy. Compared with (64)Cu, (18)F may improve imaging of EGFR-expression and is more suitable for clinical application, but the labeling reaction of (18)F-FBEM-ZEGFR:1907 requires a long synthesis time. The aim of the present study is to develop a new generation of (18)F labeled affibody probes (Al(18)F-NOTA-ZEGFR:1907 and (18)F-CBT-ZEGFR:1907) and to determine whether they are suitable agents for imaging of EGFR expression. The first approach consisted of conjugating ZEGFR:1907 with NOTA and radiolabeling with Al(18)F to produce Al(18)F-NOTA-ZEGFR:1907. In a second approach the prosthetic group (18)F-labeled-2-cyanobenzothiazole ((18)F-CBT) was conjugated to Cys-ZEGFR:1907 to produce (18)F-CBT-ZEGFR:1907. Binding affinity and specificity of Al(18)F-NOTA-ZEGFR:1907 and (18)F-CBT-ZEGFR:1907 to EGFR were evaluated using A431 cells. Biodistribution and PET studies were conducted on mice bearing A431 xenografts after injection of Al(18)F-NOTA-ZEGFR:1907 or (18)F-CBT-ZEGFR:1907 with or without coinjection of unlabeled affibody proteins. The radiosyntheses of Al(18)F-NOTA-ZEGFR:1907 and (18)F-CBT-ZEGFR:1907 were completed successfully within 40 and 120 min with a decay-corrected yield of 15% and 41% using a 2-step, 1-pot reaction and 2-step, 2-pot reaction, respectively. Both probes bound to EGFR with low nanomolar affinity in A431 cells. Although (18)F-CBT-ZEGFR:1907 showed instability in vivo, biodistribution studies revealed rapid and high tumor accumulation and quick clearance from normal tissues except the bones. In contrast, Al(18)F-NOTA-ZEGFR:1907 demonstrated high in

  18. Current activities handbook: formerly utilized sites remedial action program

    SciTech Connect

    1981-02-27

    This volume is one of a series produced under contract with the DOE, by Politech Corporation to develop a legislative and regulatory data base to assist the FUSRAP management in addressing the institutional and socioeconomic issues involved in carrying out the Formerly Utilized Sites Remedial Action Program. This Information Handbook series contains information about all relevant government agencies at the Federal and state levels, the pertinent programs they administer, each affected state legislature, and current Federal and state legislative and regulatory initiatives. This volume is a compilation of information about the activities each of the thirteen state legislatures potentially affected by the Formerly Utilized Sites Remedial Action Program. It contains a description of the state legislative procedural rules and a schedule of each legislative session; a summary of pending relevant legislation; the name and telephone number of legislative and state agency contacts; and the full text of all bills identified.

  19. Parallel assessment of nutrition and activity in athletes: validation against doubly labelled water, 24-h urea excretion, and indirect calorimetry.

    PubMed

    Koehler, Karsten; Braun, Hans; De Marees, Markus; Fusch, Gerhard; Fusch, Christoph; Mester, Joachim; Schaenzer, Wilhelm

    2010-11-01

    The assessment of nutrition and activity in athletes requires accurate and precise methods. The aim of this study was to validate a protocol for parallel assessment of diet and exercise against doubly labelled water, 24-h urea excretion, and respiratory gas exchange. The participants were 14 male triathletes under normal training conditions. Energy intake and doubly labelled water were weakly associated with each other (r = 0.69, standard error of estimate [SEE] = 304 kcal x day(-1)). Protein intake was strongly correlated with 24-h urea (r = 0.89) but showed considerable individual variation (SEE = 0.34 g kg(-1) x day(-1)). Total energy expenditure based on recorded activities was highly correlated with doubly labelled water (r = 0.95, SEE = 195 kcal x day(-1)) but was proportionally biased. During running and cycling, estimated exercise energy expenditure was highly correlated with gas exchange (running: r = 0.89, SEE = 1.6 kcal x min(-1); cycling: r = 0.95, SEE = 1.4 kcal x min(-1)). High exercise energy expenditure was slightly underestimated during running. For nutrition data, variations appear too large for precise measurements in individual athletes, which is a common problem of dietary assessment methods. Despite the high correlations of total energy expenditure and exercise energy expenditure with reference methods, a correction for systematic errors is necessary for the valid estimation of energetic requirements in individual athletes. PMID:20967672

  20. Target-controlled formation of silver nanoclusters in abasic site-incorporated duplex DNA for label-free fluorescence detection of theophylline.

    PubMed

    Park, Ki Soo; Oh, Seung Soo; Soh, H Tom; Park, Hyun Gyu

    2014-09-01

    A novel, label-free, fluorescence based sensor for theophylline has been developed. In the new sensor system, an abasic site-incorporated duplex DNA probe serves as both a pocket for recognition of theophylline and a template for the preparation of fluorescent silver nanoclusters. The strategy relies on theophylline-controlled formation of fluorescent silver nanoclusters from abasic site-incorporated duplex DNA. When theophylline is not present, silver ions interact with the cytosine groups opposite to the abasic site in duplex DNA. This interaction leads to efficient formation of intensely red fluorescent silver nanoclusters. In contrast, when theophylline is bound at the abasic site through pseudo base-pairing with appropriately positioned cytosines, silver ion binding to the cytosine nucleobase is prevented. Consequently, fluorescent silver nanoclusters are not formed causing a significant reduction of the fluorescence signal. By employing this new sensor, theophylline can be highly selectively detected at a concentration as low as 1.8 μM. Finally, the diagnostic capability and practical application of this sensor were demonstrated by its use in detecting theophylline in human blood serum.

  1. Electrostatic fields in the active sites of lysozymes.

    PubMed

    Sun, D P; Liao, D I; Remington, S J

    1989-07-01

    Considerable experimental evidence is in support of several aspects of the mechanism that has been proposed for the catalytic activity of lysozyme. However, the enzymatically catalyzed hydrolysis of polysaccharides proceeds over 5 orders of magnitude faster than that of model compounds that mimic the configuration of the substrate in the active site of the enzyme. Although several possible explanations for this rate enhancement have been discussed elsewhere, a definitive mechanism has not emerged. Here we report striking results obtained by classical electrodynamics, which suggest that bond breakage and the consequent separation of charge in lysozyme is promoted by a large electrostatic field across the active site cleft, produced in part by a very asymmetric distribution of charged residues on the enzyme surface. Lysozymes unrelated in amino acid sequence have similar distributions of charged residues and electric fields. The results reported here suggest that the electrostatic component of the rate enhancement is greater than 9 kcal.mol-1. Thus, electrostatic interactions may play a more important role in the enzymatic mechanism than has generally been appreciated.

  2. Histidine at the active site of Neurospora tyrosinase.

    PubMed

    Pfiffner, E; Lerch, K

    1981-10-13

    The involvement of histidyl residues as potential ligands to the binuclear active-site copper of Neurospora tyrosinase was explored by dye-sensitized photooxidation. The enzymatic activity of the holoenzyme was shown to be unaffected by exposure to light in the presence of methylene blue; however, irradiation of the apoenzyme under the same conditions led to a progressive loss of its ability to be reactivated with Cu2+. This photoinactivation was paralleled by a decrease in the histidine content whereas the number of histidyl residues in the holoenzyme remained constant. Copper measurements of photooxidized, reconstituted apoenzyme demonstrated the loss of binding of one copper atom per mole of enzyme as a consequence of photosensitized oxidation of three out of nine histidine residues. Their sequence positions were determined by a comparison of the relative yields of the histidine containing peptides of photooxidized holo- and apotyrosinases. The data obtained show the preferential modification of histidyl residues 188, 193, and 289 and suggest that they constitute metal ligands to one of the two active-site copper atoms. Substitution of copper by cobalt was found to afford complete protection of the histidyl residues from being modified by dye-sensitized photooxidation. PMID:6458322

  3. Global Structure of a Three-Way Junction in a Phi29 Packaging RNA Dimer Determined Using Site-Directed Spin Labeling

    SciTech Connect

    Zhang, Xiaojun; Tung, Chang-Shung; Sowa, Glenna; Hatmal, Ma'mon M.; Haworth, Ian S.; Qin, Peter Z.

    2012-02-08

    The condensation of bacteriophage phi29 genomic DNA into its preformed procapsid requires the DNA packaging motor, which is the strongest known biological motor. The packaging motor is an intricate ring-shaped protein/RNA complex, and its function requires an RNA component called packaging RNA (pRNA). Current structural information on pRNA is limited, which hinders studies of motor function. Here, we used site-directed spin labeling to map the conformation of a pRNA three-way junction that bridges binding sites for the motor ATPase and the procapsid. The studies were carried out on a pRNA dimer, which is the simplest ring-shaped pRNA complex and serves as a functional intermediate during motor assembly. Using a nucleotide-independent labeling scheme, stable nitroxide radicals were attached to eight specific pRNA sites without perturbing RNA folding and dimer formation, and a total of 17 internitroxide distances spanning the three-way junction were measured using Double Electron-Electron Resonance spectroscopy. The measured distances, together with steric chemical constraints, were used to select 3662 viable three-way junction models from a pool of 65 billion. The results reveal a similar conformation among the viable models, with two of the helices (HT and HL) adopting an acute bend. This is in contrast to a recently reported pRNA tetramer crystal structure, in which HT and HL stack onto each other linearly. The studies establish a new method for mapping global structures of complex RNA molecules, and provide information on pRNA conformation that aids investigations of phi29 packaging motor and developments of pRNA-based nanomedicine and nanomaterial.

  4. New Synthesis and Tritium Labeling of a Selective Ligand for Studying High-affinity γ-Hydroxybutyrate (GHB) Binding Sites

    PubMed Central

    Vogensen, Stine B.; Marek, Aleš; Bay, Tina; Wellendorph, Petrine; Kehler, Jan; Bundgaard, Christoffer; Frølund, Bente; Pedersen, Martin H.F.; Clausen, Rasmus P.

    2013-01-01

    3-Hydroxycyclopent-1-enecarboxylic acid (HOCPCA, 1) is a potent ligand for the high-affinity GHB binding sites in the CNS. An improved synthesis of 1 together with a very efficient synthesis of [3H]-1 is described. The radiosynthesis employs in situ generated lithium trimethoxyborotritide. Screening of 1 against different CNS targets establishes a high selectivity and we demonstrate in vivo brain penetration. In vitro characterization of [3H]-1 binding shows high specificity to the high-affinity GHB binding sites. PMID:24053696

  5. Trichodiene synthase. Identification of active site residues by site-directed mutagenesis.

    PubMed

    Cane, D E; Shim, J H; Xue, Q; Fitzsimons, B C; Hohn, T M

    1995-02-28

    Derivatization of 5,5'-dithiobis(2-nitrobenzoic acid)-treated trichodiene synthase with [methyl-14C]methyl methanethiosulfonate and analysis of the derived tryptic peptides suggested the presence of two cysteine residues at the active site. The corresponding C146A and C190A mutants were constructed by site-directed mutagenesis. The C190A mutant displayed partial but significantly reduced activity, with a reduction in kcat/Km of 3000 compared to the wild-type trichodiene synthase, while the C146A mutant was essentially inactive. A hybrid trichodiene synthase, constructed from amino acids 1-309 of the Fusarium sporotrichioides enzyme and amino acids 310-383 of the Gibberella pulicaris cyclase, had steady state kinetic parameters nearly identical to those of the wild-type F. sporotrichioides enzyme. From this parent hybrid, a series of mutants was constructed by site-directed mutagenesis in which the amino acids in the base-rich region, 302-306 (DRRYR), were systematically modified. Three of these mutants were overexpressed and purified to homogeneity. The importance of Arg304 for catalysis was established by the observation that the R304K mutant showed a more than 25-fold increase in Km, as well as a 200-fold reduction in kcat. In addition, analysis of the incubation products of the R304K mutant by gas chromatography-mass spectrometry (GC-MS) indicated that farnesyl diphosphate was converted not only to trichodiene but to at least two additional C15H24 hydrocarbons, mle 204. Replacement of the Tyr305 residue of trichodiene synthase with Phe had little effect on kcat, while increasing the Km by a factor of ca. 7-8.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7873527

  6. Membrane Docking Geometry of GRP1 PH Domain Bound to a Target Lipid Bilayer: An EPR Site-Directed Spin-Labeling and Relaxation Study

    PubMed Central

    Landgraf, Kyle E.; Corbin, John A.; Falke, Joseph J.

    2012-01-01

    The second messenger lipid PIP3 (phosphatidylinositol-3,4,5-trisphosphate) is generated by the lipid kinase PI3K (phosphoinositide-3-kinase) in the inner leaflet of the plasma membrane, where it regulates a broad array of cell processes by recruiting multiple signaling proteins containing PIP3-specific pleckstrin homology (PH) domains to the membrane surface. Despite the broad importance of PIP3-specific PH domains, the membrane docking geometry of a PH domain bound to its target PIP3 lipid on a bilayer surface has not yet been experimentally determined. The present study employs EPR site-directed spin labeling and relaxation methods to elucidate the membrane docking geometry of GRP1 PH domain bound to bilayer-embedded PIP3. The model target bilayer contains the neutral background lipid PC and both essential targeting lipids: (i) PIP3 target lipid that provides specificity and affinity, and (ii) PS facilitator lipid that enhances the PIP3 on-rate via an electrostatic search mechanism. The EPR approach measures membrane depth parameters for 18 function-retaining spin labels coupled to the PH domain, and for calibration spin labels coupled to phospholipids. The resulting depth parameters, together with the known high resolution structure of the co-complex between GRP1 PH domain and the PIP3 headgroup, provide sufficient constraints to define an optimized, self-consistent membrane docking geometry. In this optimized geometry the PH domain engulfs the PIP3 headgroup with minimal bilayer penetration, yielding the shallowest membrane position yet described for a lipid binding domain. This binding interaction displaces the PIP3 headgroup from its lowest energy position and orientation in the bilayer, but the headgroup remains within its energetically accessible depth and angular ranges. Finally, the optimized docking geometry explains previous biophysical findings including mutations observed to disrupt membrane binding, and the rapid lateral diffusion observed for PIP3

  7. Coordination of the Filament Stabilizing Versus Destabilizing Activities of Cofilin Through its Secondary Binding Site on Actin

    PubMed Central

    Aggeli, Dimitra; Kish-Trier, Erik; Lin, Meng Chi; Haarer, Brian; Cingolani, Gino; Cooper, John A.; Wilkens, Stephan; Amberg, David C.

    2014-01-01

    Cofilin is a ubiquitous modulator of actin cytoskeleton dynamics that can both stabilize and destabilize actin filaments depending on its concentration and/or the presence of regulatory co-factors. Three charge-reversal mutants of yeast cofilin, located in cofilin’s filament-specific secondary binding site, were characterized in order to understand why disruption of this site leads to enhanced filament disassembly. Crystal structures of the mutants showed that the mutations specifically affect the secondary actin-binding interface, leaving the primary binding site unaltered. The mutant cofilins show enhanced activity compared to wild-type cofilin in severing and disassembling actin filaments. Electron microscopy and image analysis revealed long actin filaments in the presence of wild-type cofilin, while the mutants induced many short filaments, consistent with enhanced severing. Real-time fluorescence microscopy of labeled actin filaments confirmed that the mutants, unlike wild-type cofilin, were functioning as constitutively active severing proteins. In cells, the mutant cofilins delayed endocytosis, which depends on rapid actin turnover. We conclude that mutating cofilin’s secondary actin-binding site increases cofilin’s ability to sever and depolymerize actin filaments. We hypothesize that activators of cofilin severing, like Aip1p, may act by disrupting the interface between cofilin’s secondary actin-binding site and the actin filament. PMID:24943913

  8. The copper active site of CBM33 polysaccharide oxygenases.

    PubMed

    Hemsworth, Glyn R; Taylor, Edward J; Kim, Robbert Q; Gregory, Rebecca C; Lewis, Sally J; Turkenburg, Johan P; Parkin, Alison; Davies, Gideon J; Walton, Paul H

    2013-04-24

    The capacity of metal-dependent fungal and bacterial polysaccharide oxygenases, termed GH61 and CBM33, respectively, to potentiate the enzymatic degradation of cellulose opens new possibilities for the conversion of recalcitrant biomass to biofuels. GH61s have already been shown to be unique metalloenzymes containing an active site with a mononuclear copper ion coordinated by two histidines, one of which is an unusual τ-N-methylated N-terminal histidine. We now report the structural and spectroscopic characterization of the corresponding copper CBM33 enzymes. CBM33 binds copper with high affinity at a mononuclear site, significantly stabilizing the enzyme. X-band EPR spectroscopy of Cu(II)-CBM33 shows a mononuclear type 2 copper site with the copper ion in a distorted axial coordination sphere, into which azide will coordinate as evidenced by the concomitant formation of a new absorption band in the UV/vis spectrum at 390 nm. The enzyme's three-dimensional structure contains copper, which has been photoreduced to Cu(I) by the incident X-rays, confirmed by X-ray absorption/fluorescence studies of both aqueous solution and intact crystals of Cu-CBM33. The single copper(I) ion is ligated in a T-shaped configuration by three nitrogen atoms from two histidine side chains and the amino terminus, similar to the endogenous copper coordination geometry found in fungal GH61. PMID:23540833

  9. Activation of muscarinic acetylcholine receptors via their allosteric binding sites.

    PubMed Central

    Jakubík, J; Bacáková, L; Lisá, V; el-Fakahany, E E; Tucek, S

    1996-01-01

    Ligands that bind to the allosteric-binding sites on muscarinic acetylcholine receptors alter the conformation of the classical-binding sites of these receptors and either diminish or increase their affinity for muscarinic agonists and classical antagonists. It is not known whether the resulting conformational change also affects the interaction between the receptors and the G proteins. We have now found that the muscarinic receptor allosteric modulators alcuronium, gallamine, and strychnine (acting in the absence of an agonist) alter the synthesis of cAMP in Chinese hamster ovary (CHO) cells expressing the M2 or the M4 subtype of muscarinic receptors in the same direction as the agonist carbachol. In addition, most of their effects on the production of inositol phosphates in CHO cells expressing the M1 or the M3 muscarinic receptor subtypes are also similar to (although much weaker than) those of carbachol. The agonist-like effects of the allosteric modulators are not observed in CHO cells that have not been transfected with the gene for any of the subtypes of muscarinic receptors. The effects of alcuronium on the formation of cAMP and inositol phosphates are not prevented by the classical muscarinic antagonist quinuclidinyl benzilate. These observations demonstrate for the first time that the G protein-mediated functional responses of muscarinic receptors can be evoked not only from their classical, but also from their allosteric, binding sites. This represents a new mechanism of receptor activation. PMID:8710935

  10. Highly sensitive immunoassay based on Raman reporter-labeled immuno-Au aggregates and SERS-active immune substrate.

    PubMed

    Song, Chunyuan; Wang, Zhuyuan; Zhang, Ruohu; Yang, Jing; Tan, Xuebin; Cui, Yiping

    2009-12-15

    A highly sensitive immunoassay based on surface enhanced Raman scattering (SERS) has been developed with a novel immune marker named as Raman reporter-labeled immuno-Au aggregates on a SERS-active immune substrate. The features of those immune aggregates were characterized by UV-vis extinction spectra, TEM images, SEM pictures and SERS spectra. It is found that stable gold aggregates in appropriate morphologies can be induced by mixing proper amounts of reporter molecules with gold nanoparticles. Based on those reporter-labeled Au aggregates, the immune aggregates with high stability can be prepared successfully by immobilizing antibody to the surface of the aggregates. Using those easily prepared aggregates showing strong SERS activity and high bio-specificity, a highly effective SERS-based immunoassay has been performed. Moreover, a SERS-active immune substrate has been introduced to replace a typical immune substrate without any SERS activity. Our experimental results indicate that the SERS-active immune substrate makes a certain contribution to the highly sensitive immunoassay. As a result, with this proposed immunoassay structure, the concentration detection of human-IgG was performed and a calibration curve was obtained in the range from 100ngmL(-1) to 100fgmL(-1). This work opens a new avenue for sensitive immunoassay and other biochemical analysis based on SERS.

  11. Radiation inactivation study of aminopeptidase: probing the active site

    NASA Astrophysics Data System (ADS)

    Jamadar, V. K.; Jamdar, S. N.; Mohan, Hari; Dandekar, S. P.; Harikumar, P.

    2004-04-01

    Ionizing radiation inactivated purified chicken intestinal aminopeptidase in media saturated with gases in the order N 2O>N 2>air. The D 37 values in the above conditions were 281, 210 and 198 Gy, respectively. OH radical scavengers such as t-butanol and isopropanol effectively nullified the radiation-induced damage in N 2O. The radicals (SCN) 2•-, Br 2•- and I 2•- inactivated the enzyme, pointing to the involvement of aromatic amino acids and cysteine in its catalytic activity. The enzyme exhibited fluorescence emission at 340 nm which is characteristic of tryptophan. The radiation-induced loss of activity was accompanied by a decrease in the fluorescence of the enzyme suggesting a predominant influence on tryptophan residues. The enzyme inhibition was associated with a marked increase in the Km and a decrease in the Vmax and kcat values, suggesting an irreversible alteration in the catalytic site. The above observations were confirmed by pulse radiolysis studies.

  12. Mimicking enzymatic active sites on surfaces for energy conversion chemistry.

    PubMed

    Gutzler, Rico; Stepanow, Sebastian; Grumelli, Doris; Lingenfelder, Magalí; Kern, Klaus

    2015-07-21

    Metal-organic supramolecular chemistry on surfaces has matured to a point where its underlying growth mechanisms are well understood and structures of defined coordination environments of metal atoms can be synthesized in a controlled and reproducible procedure. With surface-confined molecular self-assembly, scientists have a tool box at hand which can be used to prepare structures with desired properties, as for example a defined oxidation number and spin state of the transition metal atoms within the organic matrix. From a structural point of view, these coordination sites in the supramolecular structure resemble the catalytically active sites of metallo-enzymes, both characterized by metal centers coordinated to organic ligands. Several chemical reactions take place at these embedded metal ions in enzymes and the question arises whether these reactions also take place using metal-organic networks as catalysts. Mimicking the active site of metal atoms and organic ligands of enzymes in artificial systems is the key to understanding the selectivity and efficiency of enzymatic reactions. Their catalytic activity depends on various parameters including the charge and spin configuration in the metal ion, but also on the organic environment, which can stabilize intermediate reaction products, inhibits catalytic deactivation, and serves mostly as a transport channel for the reactants and products and therefore ensures the selectivity of the enzyme. Charge and spin on the transition metal in enzymes depend on the one hand on the specific metal element, and on the other hand on its organic coordination environment. These two parameters can carefully be adjusted in surface confined metal-organic networks, which can be synthesized by virtue of combinatorial mixing of building synthons. Different organic ligands with varying functional groups can be combined with several transition metals and spontaneously assemble into ordered networks. The catalytically active metal

  13. Polarizability of the active site of cytochrome c reduces the activation barrier for electron transfer

    NASA Astrophysics Data System (ADS)

    Dinpajooh, Mohammadhasan; Martin, Daniel R.; Matyushov, Dmitry V.

    2016-06-01

    Enzymes in biology’s energy chains operate with low energy input distributed through multiple electron transfer steps between protein active sites. The general challenge of biological design is how to lower the activation barrier without sacrificing a large negative reaction free energy. We show that this goal is achieved through a large polarizability of the active site. It is polarized by allowing a large number of excited states, which are populated quantum mechanically by electrostatic fluctuations of the protein and hydration water shells. This perspective is achieved by extensive mixed quantum mechanical/molecular dynamics simulations of the half reaction of reduction of cytochrome c. The barrier for electron transfer is consistently lowered by increasing the number of excited states included in the Hamiltonian of the active site diagonalized along the classical trajectory. We suggest that molecular polarizability, in addition to much studied electrostatics of permanent charges, is a key parameter to consider in order to understand how enzymes work.

  14. Polarizability of the active site of cytochrome c reduces the activation barrier for electron transfer

    PubMed Central

    Dinpajooh, Mohammadhasan; Martin, Daniel R.; Matyushov, Dmitry V.

    2016-01-01

    Enzymes in biology’s energy chains operate with low energy input distributed through multiple electron transfer steps between protein active sites. The general challenge of biological design is how to lower the activation barrier without sacrificing a large negative reaction free energy. We show that this goal is achieved through a large polarizability of the active site. It is polarized by allowing a large number of excited states, which are populated quantum mechanically by electrostatic fluctuations of the protein and hydration water shells. This perspective is achieved by extensive mixed quantum mechanical/molecular dynamics simulations of the half reaction of reduction of cytochrome c. The barrier for electron transfer is consistently lowered by increasing the number of excited states included in the Hamiltonian of the active site diagonalized along the classical trajectory. We suggest that molecular polarizability, in addition to much studied electrostatics of permanent charges, is a key parameter to consider in order to understand how enzymes work. PMID:27306204

  15. Spectroscopic Definition of the Ferroxidase Site in M Ferritin: Comparison of Binuclear Substrate vs. Cofactor Active Sites

    PubMed Central

    Schwartz, Jennifer K.; Liu, Xiaofeng S.; Tosha, Takehiko; Theil, Elizabeth C.; Solomon, Edward I.

    2008-01-01

    Maxi ferritins, 24 subunit protein nanocages, are essential in humans, plants, bacteria, and other animals for the concentration and storage of iron as hydrated ferric oxide, while minimizing free radical generation or use by pathogens. Formation of the precursors to these ferric oxides is catalyzed at a non-heme biferrous substrate site, which has some parallels with the cofactor sites in other biferrous enzymes. A combination of circular dichroism (CD), magnetic circular dichroism (MCD), and variable-temperature, variable-field MCD (VTVH MCD) has been used to probe Fe(II) binding to the substrate active site in frog M ferritin. These data determined that the active site within each subunit consists of two inequivalent five-coordinate (5C) ferrous centers that are weakly anti-ferromagnetically coupled, consistent with a μ-1,3 carboxylate bridge. The active site ligand set is unusual and likely includes a terminal water bound to each Fe(II) center. The Fe(II) ions bind to the active sites in a concerted manner, and cooperativity among the sites in each subunit is observed, potentially providing a mechanism for the control of ferritin iron loading. Differences in geometric and electronic structure – including a weak ligand field, availability of two water ligands at the biferrous substrate site, and the single carboxylate bridge in ferritin – coincide with the divergent reaction pathways observed between this substrate site and the previously studied cofactor active sites. PMID:18576633

  16. Movements of Individual BKCa Channels in Live Cell Membrane Monitored by Site-Specific Labeling Using Quantum Dots

    PubMed Central

    Won, Sehoon; Kim, Hae-Deun; Kim, Ji-Yeon; Lee, Byoung-Cheol; Chang, Sunghoe; Park, Chul-Seung

    2010-01-01

    The movements of BKCa channels were investigated in live cells using quantum dots (QDs). The extracellular N-terminus was metabolically tagged with biotin, labeled with streptavidin-conjugated QDs and then monitored using real-time time-lapse imaging in COS-7 cells and cultured neurons. By tracking hundreds of channels, we were able to determine the characteristics of channel movements quantitatively. Channels in COS-7 cells exhibited a confined diffusion in an area of 1.915 μm2, with an initial diffusion coefficient of 0.033 μm2/s. In neurons, the channel movements were more heterogeneous and highly dependent on subcellular location. While the channels in soma diffused slowly without clear confinement, axodendritic channels showed more rapid and pseudo-one-dimensional movements. Intriguingly, the channel movement in somata was drastically increased by the neuronal β4 subunit, in contrast to the channels in the axodendritic area where the mobility were significantly decreased. Thus, our results demonstrate that the membrane mobility of BKCa channels can be greatly influenced by the expression system used, subunit composition, and subcellular location. This QD-based, single-molecule tracking technique can be utilized to investigate the cellular mechanisms that determine the mobility as well as the localization of various membrane proteins in live cells. PMID:21044582

  17. An active-site lysine in avian liver phosphoenolpyruvate carboxykinase

    SciTech Connect

    Guidinger, P.F.; Nowak, T. )

    1991-09-10

    The participation of lysine in the catalysis by avian liver phosphoenolpyruvate carboxykinase was studied by chemical modification and by a characterization of the modified enzyme. The rate of inactivation by 2,4-pentanedione is pseudo-first-order and linearly dependent on reagent concentration with a second-order rate constant of 0.36 {plus minus} 0.025 M{sup {minus}1} min{sup {minus}1}. Inactivation by pyridoxal 5{prime}-phosphate of the reversible reaction catalyzed by phosphoenolpyruvate carboxykinase follows bimolecular kinetics with a second-order rate constant of 7,700 {plus minus} 860 m{sup {minus}1} min{sup {minus}1}. Treatment of the enzyme or one lysine residue modified concomitant with 100% loss in activity. A stoichiometry of 1:1 is observed when either the reversible or the irreversible reactions catalyzed by the enzyme are monitored. A study of k{sub obs} vs pH suggests this active-site lysine has a pK{sub a} of 8.1 and a pH-independent rate constant of inactivation of 47,700 m{sup {minus}1} min{sup {minus}1}. Proton relaxation rate measurements suggest that pyridoxal 5{prime}-phosphate modification alters binding of the phosphate-containing substrates. {sup 31}P NMR relaxation rate measurements show altered binding of the substrates in the ternary enzyme {center dot}Mn{sup 2+}{center dot}substrate complex. Circular dichroism studies show little change in secondary structure of pyridoxal 5{prime}-phosphate modified phosphoenolpyruvate carboxykinase. These results indicate that avian liver phosphoenolpyruvate carboxykinase has one reactive lysine at the active site and it is involved in the binding and activation of the phosphate-containing substrates.

  18. Perchlorate Reductase Is Distinguished by Active Site Aromatic Gate Residues.

    PubMed

    Youngblut, Matthew D; Tsai, Chi-Lin; Clark, Iain C; Carlson, Hans K; Maglaqui, Adrian P; Gau-Pan, Phonchien S; Redford, Steven A; Wong, Alan; Tainer, John A; Coates, John D

    2016-04-22

    Perchlorate is an important ion on both Earth and Mars. Perchlorate reductase (PcrAB), a specialized member of the dimethylsulfoxide reductase superfamily, catalyzes the first step of microbial perchlorate respiration, but little is known about the biochemistry, specificity, structure, and mechanism of PcrAB. Here we characterize the biophysics and phylogeny of this enzyme and report the 1.86-Å resolution PcrAB complex crystal structure. Biochemical analysis revealed a relatively high perchlorate affinity (Km = 6 μm) and a characteristic substrate inhibition compared with the highly similar respiratory nitrate reductase NarGHI, which has a relatively much lower affinity for perchlorate (Km = 1.1 mm) and no substrate inhibition. Structural analysis of oxidized and reduced PcrAB with and without the substrate analog SeO3 (2-) bound to the active site identified key residues in the positively charged and funnel-shaped substrate access tunnel that gated substrate entrance and product release while trapping transiently produced chlorate. The structures suggest gating was associated with shifts of a Phe residue between open and closed conformations plus an Asp residue carboxylate shift between monodentate and bidentate coordination to the active site molybdenum atom. Taken together, structural and mutational analyses of gate residues suggest key roles of these gate residues for substrate entrance and product release. Our combined results provide the first detailed structural insight into the mechanism of biological perchlorate reduction, a critical component of the chlorine redox cycle on Earth.

  19. Eel calcitonin binding site distribution and antinociceptive activity in rats

    SciTech Connect

    Guidobono, F.; Netti, C.; Sibilia, V.; Villa, I.; Zamboni, A.; Pecile, A.

    1986-03-01

    The distribution of binding site for (/sup 125/I)-eel-calcitonin (ECT) to rat central nervous system, studied by an autoradiographic technique, showed concentrations of binding in the diencephalon, the brain stem and the spinal cord. Large accumulations of grains were seen in the hypothalamus, the amygdala, in the fasciculus medialis prosencephali, in the fasciculus longitudinalis medialis, in the ventrolateral part of the periventricular gray matter, in the lemniscus medialis and in the raphe nuclei. The density of grains in the reticular formation and in the nucleus tractus spinalis nervi trigemini was more moderate. In the spinal cord, grains were scattered throughout the dorsal horns. Binding of the ligand was displaced equally by cold ECT and by salmon CT(sCT), indicating that both peptides bind to the same receptors. Human CT was much weaker than sCT in displacing (/sup 125/I)-ECT binding. The administration of ECT into the brain ventricles of rats dose-dependently induced a significant and long-lasting enhancement of hot-plate latencies comparable with that obtained with sCT. The antinociceptive activity induced by ECT is compatible with the topographical distribution of binding sites for the peptide and is a further indication that fish CTs are active in the mammalian brain.

  20. Perchlorate Reductase Is Distinguished by Active Site Aromatic Gate Residues.

    PubMed

    Youngblut, Matthew D; Tsai, Chi-Lin; Clark, Iain C; Carlson, Hans K; Maglaqui, Adrian P; Gau-Pan, Phonchien S; Redford, Steven A; Wong, Alan; Tainer, John A; Coates, John D

    2016-04-22

    Perchlorate is an important ion on both Earth and Mars. Perchlorate reductase (PcrAB), a specialized member of the dimethylsulfoxide reductase superfamily, catalyzes the first step of microbial perchlorate respiration, but little is known about the biochemistry, specificity, structure, and mechanism of PcrAB. Here we characterize the biophysics and phylogeny of this enzyme and report the 1.86-Å resolution PcrAB complex crystal structure. Biochemical analysis revealed a relatively high perchlorate affinity (Km = 6 μm) and a characteristic substrate inhibition compared with the highly similar respiratory nitrate reductase NarGHI, which has a relatively much lower affinity for perchlorate (Km = 1.1 mm) and no substrate inhibition. Structural analysis of oxidized and reduced PcrAB with and without the substrate analog SeO3 (2-) bound to the active site identified key residues in the positively charged and funnel-shaped substrate access tunnel that gated substrate entrance and product release while trapping transiently produced chlorate. The structures suggest gating was associated with shifts of a Phe residue between open and closed conformations plus an Asp residue carboxylate shift between monodentate and bidentate coordination to the active site molybdenum atom. Taken together, structural and mutational analyses of gate residues suggest key roles of these gate residues for substrate entrance and product release. Our combined results provide the first detailed structural insight into the mechanism of biological perchlorate reduction, a critical component of the chlorine redox cycle on Earth. PMID:26940877

  1. Label-free molecular beacon-based quadratic isothermal exponential amplification: a simple and sensitive one-pot method to detect DNA methyltransferase activity.

    PubMed

    Xue, Qingwang; Wang, Lei; Jiang, Wei

    2015-09-11

    We developed a one-pot label-free molecular beacon-mediated quadratic isothermal exponential amplification strategy (LFMB-QIEA) for simple, rapid and sensitive DNA methyltransferase (MTase) activity detection.

  2. An Active Site Water Network in the Plasminogen Activator Pla from Yersinia pestis

    SciTech Connect

    Eren, Elif; Murphy, Megan; Goguen, Jon; van den Berg, Bert

    2010-08-13

    The plasminogen activator Pla from Yersinia pestis is an outer membrane protease (omptin) that is important for the virulence of plague. Here, we present the high-resolution crystal structure of wild-type, enzymatically active Pla at 1.9 {angstrom}. The structure shows a water molecule located between active site residues D84 and H208, which likely corresponds to the nucleophilic water. A number of other water molecules are present in the active site, linking residues important for enzymatic activity. The R211 sidechain in loop L4 is close to the nucleophilic water and possibly involved in the stabilization of the oxyanion intermediate. Subtle conformational changes of H208 result from the binding of lipopolysaccharide to the outside of the barrel, explaining the unusual dependence of omptins on lipopolysaccharide for activity. The Pla structure suggests a model for the interaction with plasminogen substrate and provides a more detailed understanding of the catalytic mechanism of omptin proteases.

  3. Characterization and sequencing of an active-site cysteine-containing peptide from the xylanase of a thermotolerant Streptomyces.

    PubMed

    Keskar, S S; Rao, M B; Deshpande, V V

    1992-02-01

    The kinetics of chemical modification of the xylanase from a thermotolerant Streptomyces T7 indicated the involvement of 1 mol of cysteine residue/mol of enzyme [Keskar, Srinivasan & Deshpande (1989) Biochem. J. 261, 49-55]. The chromophoric reagent N-(2,4-dinitroanilino)maleimide (DAM) reacts covalently with thiol groups of xylanase with complete inactivation. Protection against inactivation was provided by the substrate (xylan). The purified xylanase that had been modified with DAM was digested with pepsin and the peptides were purified by gel filtration followed by peptide mapping. The active-site peptide was distinguished from the other thiol-containing peptides by comparison of the peptides generated by labelling the enzyme in the presence and in the absence of the substrate. The peptide mapping of the modified enzyme in the absence of xylan showed three yellow peptides, whereas in the presence of xylan only two yellow peptides were detected. The active-site peptide protected by the substrate failed to form the complex with DAM. The modified active-site peptide was isolated and sequenced. Gas-phase sequencing provided the following sequence: Ser-Val-Ile-Met-Xaa-Ile-Asp-His-Ile-Arg-Phe. This is the first report on the isolation and sequencing of the active-site peptide from a xylanase. The comparison of reactive cysteine-containing peptide sequence with the catalytic regions of other glucanases revealed the presence of a conserved aspartic acid residue.

  4. Metal active site elasticity linked to activation of homocysteine in methionine synthases

    SciTech Connect

    Koutmos, Markos; Pejchal, Robert; Bomer, Theresa M.; Matthews, Rowena G.; Smith, Janet L.; Ludwig, Martha L.

    2008-04-02

    Enzymes possessing catalytic zinc centers perform a variety of fundamental processes in nature, including methyl transfer to thiols. Cobalamin-independent (MetE) and cobalamin-dependent (MetH) methionine synthases are two such enzyme families. Although they perform the same net reaction, transfer of a methyl group from methyltetrahydrofolate to homocysteine (Hcy) to form methionine, they display markedly different catalytic strategies, modular organization, and active site zinc centers. Here we report crystal structures of zinc-replete MetE and MetH, both in the presence and absence of Hcy. Structural investigation of the catalytic zinc sites of these two methyltransferases reveals an unexpected inversion of zinc geometry upon binding of Hcy and displacement of an endogenous ligand in both enzymes. In both cases a significant movement of the zinc relative to the protein scaffold accompanies inversion. These structures provide new information on the activation of thiols by zinc-containing enzymes and have led us to propose a paradigm for the mechanism of action of the catalytic zinc sites in these and related methyltransferases. Specifically, zinc is mobile in the active sites of MetE and MetH, and its dynamic nature helps facilitate the active site conformational changes necessary for thiol activation and methyl transfer.

  5. Anaerobic decomposition of benzoic acid during methane fermentation: Specific activity of fatty acid intermediates and postion of radioactive label

    SciTech Connect

    Bridges, R.L.

    1990-01-01

    A study of the pathway of anaerobic decomposition of benzoic acid by a mixed methanogenic culture of bacteria was conducted. Specific activities of the possible fatty acid intermediates cyclohexanecarboxylic acid, propanoic acid, and acetic acid were determined. In the case of propanoic acid, the position of the radioactive label was also determined by isotropic trapping and Phares-Schmidt degradation of the intermediate. The specific activities of cyclohexanecarboxylic acid and propanoic acid are the same as the benzoate substrate fed to the mixed methanogenic cultures. These fatty acids must be direct breakdown products from the aromatic ring. When (4{minus}{sup 14}C) benzoate is the substrate, the propanoic acid produced is labeled exclusively in the carboxyl position. This supports the pathway proposed by Keith et al. (1978), but would be unlikely for the pathway proposed by Evans (1977). The specific activity of the acetic acid isolated from a culture fed (4{minus}{sup 14}C) benzoate is 42% of the specific activity of the substrate. This is possible only if the methylmalonyl-CoA pathway for the conversion of propanoate to acetate is not being utilized. The amount of various intermediates found indicates that at least three syntrophically linked organisms are present in the mixed methanogenic culture. One is responsible for the production of cyclohexanecarboxylic acid, one for the production of acetate from propanoate, and one for the production of methane.

  6. Active Sites Environmental Monitoring Program: Program plan. Revision 1

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1992-02-01

    The Active Sites Environmental Monitoring Program (ASEMP), initiated in 1989, provides early detection and performance monitoring of transuranic (TRU) waste and active low-level waste (LLW) facilities at Oak Ridge National Laboratory (ORNL) in accordance with US Department of Energy (DOE) Order 5820.2A. Active LLW facilities in Solid Waste Storage Area (SWSA) 6 include Tumulus I and Tumulus II, the Interim Waste Management Facility (IWMF), LLW silos, high-range wells, asbestos silos, and fissile wells. The tumulus pads and IWMF are aboveground, high-strength concrete pads on which concrete vaults containing metal boxes of LLW are placed; the void space between the boxes and vaults is filled with grout. Eventually, these pads and vaults will be covered by an engineered multilayered cap. All other LLW facilities in SWSA 6 are below ground. In addition, this plan includes monitoring of the Hillcut Disposal Test Facility (HDTF) in SWSA 6, even though this facility was completed prior to the data of the DOE order. In SWSA 5 North, the TRU facilities include below-grade engineered caves, high-range wells, and unlined trenches. All samples from SWSA 6 are screened for alpha and beta activity, counted for gamma-emitting isotopes, and analyzed for tritium. In addition to these analytes, samples from SWSA 5 North are analyzed for specific transuranic elements.

  7. Growth activity in human septal cartilage: age-dependent incorporation of labeled sulfate in different anatomic locations

    SciTech Connect

    Vetter, U.; Pirsig, W.; Heinze, E.

    1983-02-01

    Growth activity in different areas of human septal cartilage was measured by the in vitro incorporation of /sup 35/S-labeled NaSO/sub 4/ into chondroitin sulfate. Septal cartilage without perichondrium was obtained during rhinoplasty from 36 patients aged 6 to 35 years. It could be shown that the anterior free end of the septum displays high growth activity in all age groups. The supra-premaxillary area displayed its highest growth activity during prepuberty, showing thereafter a continuous decline during puberty and adulthood. A similar age-dependent pattern in growth activity was found in the caudal prolongation of the septal cartilage. No age-dependent variations could be detected in the posterior area of the septal cartilage.

  8. Topology of AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, determined by site-directed fluorescence labeling.

    PubMed

    Nanatani, Kei; Fujiki, Takashi; Kanou, Kazuhiko; Takeda-Shitaka, Mayuko; Umeyama, Hideaki; Ye, Liwen; Wang, Xicheng; Nakajima, Tasuku; Uchida, Takafumi; Maloney, Peter C; Abe, Keietsu

    2007-10-01

    The gram-positive lactic acid bacterium Tetragenococcus halophilus catalyzes the decarboxylation of L-aspartate (Asp) with release of L-alanine (Ala) and CO(2). The decarboxylation reaction consists of two steps: electrogenic exchange of Asp for Ala catalyzed by an aspartate:alanine antiporter (AspT) and intracellular decarboxylation of the transported Asp catalyzed by an L-aspartate-beta-decarboxylase (AspD). AspT belongs to the newly classified aspartate:alanine exchanger family (transporter classification no. 2.A.81) of transporters. In this study, we were interested in the relationship between the structure and function of AspT and thus analyzed the topology by means of the substituted-cysteine accessibility method using the impermeant, fluorescent, thiol-specific probe Oregon Green 488 maleimide (OGM) and the impermeant, nonfluorescent, thiol-specific probe [2-(trimethylammonium)ethyl]methanethiosulfonate bromide. We generated 23 single-cysteine variants from a six-histidine-tagged cysteineless AspT template. A cysteine position was assigned an external location if the corresponding single-cysteine variant reacted with OGM added to intact cells, and a position was assigned an internal location if OGM labeling required cell lysis. The topology analyses revealed that AspT has a unique topology; the protein has 10 transmembrane helices (TMs), a large hydrophilic cytoplasmic loop (about 180 amino acids) between TM5 and TM6, N and C termini that face the periplasm, and a positively charged residue (arginine 76) within TM3. Moreover, the three-dimensional structure constructed by means of the full automatic modeling system indicates that the large hydrophilic cytoplasmic loop of AspT possesses a TrkA_C domain and a TrkA_C-like domain and that the three-dimensional structures of these domains are similar to each other even though their amino acid sequences show low similarity.

  9. Active Site and Laminarin Binding in Glycoside Hydrolase Family 55*

    PubMed Central

    Bianchetti, Christopher M.; Takasuka, Taichi E.; Deutsch, Sam; Udell, Hannah S.; Yik, Eric J.; Bergeman, Lai F.; Fox, Brian G.

    2015-01-01

    The Carbohydrate Active Enzyme (CAZy) database indicates that glycoside hydrolase family 55 (GH55) contains both endo- and exo-β-1,3-glucanases. The founding structure in the GH55 is PcLam55A from the white rot fungus Phanerochaete chrysosporium (Ishida, T., Fushinobu, S., Kawai, R., Kitaoka, M., Igarashi, K., and Samejima, M. (2009) Crystal structure of glycoside hydrolase family 55 β-1,3-glucanase from the basidiomycete Phanerochaete chrysosporium. J. Biol. Chem. 284, 10100–10109). Here, we present high resolution crystal structures of bacterial SacteLam55A from the highly cellulolytic Streptomyces sp. SirexAA-E with bound substrates and product. These structures, along with mutagenesis and kinetic studies, implicate Glu-502 as the catalytic acid (as proposed earlier for Glu-663 in PcLam55A) and a proton relay network of four residues in activating water as the nucleophile. Further, a set of conserved aromatic residues that define the active site apparently enforce an exo-glucanase reactivity as demonstrated by exhaustive hydrolysis reactions with purified laminarioligosaccharides. Two additional aromatic residues that line the substrate-binding channel show substrate-dependent conformational flexibility that may promote processive reactivity of the bound oligosaccharide in the bacterial enzymes. Gene synthesis carried out on ∼30% of the GH55 family gave 34 active enzymes (19% functional coverage of the nonredundant members of GH55). These active enzymes reacted with only laminarin from a panel of 10 different soluble and insoluble polysaccharides and displayed a broad range of specific activities and optima for pH and temperature. Application of this experimental method provides a new, systematic way to annotate glycoside hydrolase phylogenetic space for functional properties. PMID:25752603

  10. Human uroporphyrinogen III synthase: NMR-based mapping of the active site.

    PubMed

    Cunha, Luis; Kuti, Miklos; Bishop, David F; Mezei, Mihaly; Zeng, Lei; Zhou, Ming-Ming; Desnick, Robert J

    2008-05-01

    Uroporphyrinogen III synthase (URO-synthase) catalyzes the cyclization and D-ring isomerization of hydroxymethylbilane (HMB) to uroporphyrinogen (URO'gen) III, the cyclic tetrapyrrole and physiologic precursor of heme, chlorophyl, and corrin. The deficient activity of human URO-synthase results in the autosomal recessive cutaneous disorder, congenital erythropoietic porphyria. Mapping of the structural determinants that specify catalysis and, potentially, protein-protein interactions is lacking. To map the active site and assess the enzyme's possible interaction in a complex with hydroxymethylbilane-synthase (HMB-synthase) and/or uroporphyrinogen-decarboxylase (URO-decarboxylase) by NMR, an efficient expression and purification procedure was developed for these cytosolic enzymes of heme biosynthesis that enabled preparation of special isotopically-labeled protein samples for NMR characterization. Using an 800 MHz instrument, assignment of the URO-synthase backbone (13)C(alpha) (100%), (1)H(alpha) (99.6%), and nonproline (1)H(N) and (15)N resonances (94%) was achieved as well as 85% of the side-chain (13)C and (1)H resonances. NMR analyses of URO-synthase titrated with competitive inhibitors N(D)-methyl-1-formylbilane (NMF-bilane) or URO'gen III, revealed resonance perturbations of specific residues lining the cleft between the two major domains of URO synthase that mapped the enzyme's active site. In silico docking of the URO-synthase crystal structure with NMF-bilane and URO'gen III was consistent with the perturbation results and provided a 3D model of the enzyme-inhibitor complex. The absence of chemical shift changes in the (15)N spectrum of URO-synthase mixed with the homogeneous HMB-synthase holoenzyme or URO-decarboxylase precluded occurrence of a stable cytosolic enzyme complex. PMID:18004775

  11. Active site loop conformation regulates promiscuous activity in a lactonase from Geobacillus kaustophilus HTA426.

    PubMed

    Zhang, Yu; An, Jiao; Yang, Guang-Yu; Bai, Aixi; Zheng, Baisong; Lou, Zhiyong; Wu, Geng; Ye, Wei; Chen, Hai-Feng; Feng, Yan; Manco, Giuseppe

    2015-01-01

    Enzyme promiscuity is a prerequisite for fast divergent evolution of biocatalysts. A phosphotriesterase-like lactonase (PLL) from Geobacillus kaustophilus HTA426 (GkaP) exhibits main lactonase and promiscuous phosphotriesterase activities. To understand its catalytic and evolutionary mechanisms, we investigated a "hot spot" in the active site by saturation mutagenesis as well as X-ray crystallographic analyses. We found that position 99 in the active site was involved in substrate discrimination. One mutant, Y99L, exhibited 11-fold improvement over wild-type in reactivity (kcat/Km) toward the phosphotriesterase substrate ethyl-paraoxon, but showed 15-fold decrease toward the lactonase substrate δ-decanolactone, resulting in a 157-fold inversion of the substrate specificity. Structural analysis of Y99L revealed that the mutation causes a ∼6.6 Å outward shift of adjacent loop 7, which may cause increased flexibility of the active site and facilitate accommodation and/or catalysis of organophosphate substrate. This study provides for the PLL family an example of how the evolutionary route from promiscuity to specificity can derive from very few mutations, which promotes alteration in the conformational adjustment of the active site loops, in turn draws the capacity of substrate binding and activity.

  12. Active Site Loop Conformation Regulates Promiscuous Activity in a Lactonase from Geobacillus kaustophilus HTA426

    PubMed Central

    Zhang, Yu; An, Jiao; Yang, Guang-Yu; Bai, Aixi; Zheng, Baisong; Lou, Zhiyong; Wu, Geng; Ye, Wei; Chen, Hai-Feng; Feng, Yan; Manco, Giuseppe

    2015-01-01

    Enzyme promiscuity is a prerequisite for fast divergent evolution of biocatalysts. A phosphotriesterase-like lactonase (PLL) from Geobacillus kaustophilus HTA426 (GkaP) exhibits main lactonase and promiscuous phosphotriesterase activities. To understand its catalytic and evolutionary mechanisms, we investigated a “hot spot” in the active site by saturation mutagenesis as well as X-ray crystallographic analyses. We found that position 99 in the active site was involved in substrate discrimination. One mutant, Y99L, exhibited 11-fold improvement over wild-type in reactivity (kcat/Km) toward the phosphotriesterase substrate ethyl-paraoxon, but showed 15-fold decrease toward the lactonase substrate δ-decanolactone, resulting in a 157-fold inversion of the substrate specificity. Structural analysis of Y99L revealed that the mutation causes a ∼6.6 Å outward shift of adjacent loop 7, which may cause increased flexibility of the active site and facilitate accommodation and/or catalysis of organophosphate substrate. This study provides for the PLL family an example of how the evolutionary route from promiscuity to specificity can derive from very few mutations, which promotes alteration in the conformational adjustment of the active site loops, in turn draws the capacity of substrate binding and activity. PMID:25706379

  13. Extensive site-directed mutagenesis reveals interconnected functional units in the alkaline phosphatase active site.

    PubMed

    Sunden, Fanny; Peck, Ariana; Salzman, Julia; Ressl, Susanne; Herschlag, Daniel

    2015-01-01

    Enzymes enable life by accelerating reaction rates to biological timescales. Conventional studies have focused on identifying the residues that have a direct involvement in an enzymatic reaction, but these so-called 'catalytic residues' are embedded in extensive interaction networks. Although fundamental to our understanding of enzyme function, evolution, and engineering, the properties of these networks have yet to be quantitatively and systematically explored. We dissected an interaction network of five residues in the active site of Escherichia coli alkaline phosphatase. Analysis of the complex catalytic interdependence of specific residues identified three energetically independent but structurally interconnected functional units with distinct modes of cooperativity. From an evolutionary perspective, this network is orders of magnitude more probable to arise than a fully cooperative network. From a functional perspective, new catalytic insights emerge. Further, such comprehensive energetic characterization will be necessary to benchmark the algorithms required to rationally engineer highly efficient enzymes. PMID:25902402

  14. Extensive site-directed mutagenesis reveals interconnected functional units in the alkaline phosphatase active site.

    PubMed

    Sunden, Fanny; Peck, Ariana; Salzman, Julia; Ressl, Susanne; Herschlag, Daniel

    2015-01-01

    Enzymes enable life by accelerating reaction rates to biological timescales. Conventional studies have focused on identifying the residues that have a direct involvement in an enzymatic reaction, but these so-called 'catalytic residues' are embedded in extensive interaction networks. Although fundamental to our understanding of enzyme function, evolution, and engineering, the properties of these networks have yet to be quantitatively and systematically explored. We dissected an interaction network of five residues in the active site of Escherichia coli alkaline phosphatase. Analysis of the complex catalytic interdependence of specific residues identified three energetically independent but structurally interconnected functional units with distinct modes of cooperativity. From an evolutionary perspective, this network is orders of magnitude more probable to arise than a fully cooperative network. From a functional perspective, new catalytic insights emerge. Further, such comprehensive energetic characterization will be necessary to benchmark the algorithms required to rationally engineer highly efficient enzymes.

  15. Desformylflustrabromine (dFBr) and [3H]dFBr-Labeled Binding Sites in a Nicotinic Acetylcholine Receptor.

    PubMed

    Hamouda, Ayman K; Wang, Ze-Jun; Stewart, Deirdre S; Jain, Atul D; Glennon, Richard A; Cohen, Jonathan B

    2015-07-01

    Desformylflustrabromine (dFBr) is a positive allosteric modulator (PAM) of α4β2 and α2β2 nAChRs that, at concentrations >1 µM, also inhibits these receptors and α7 nAChRs. However, its interactions with muscle-type nAChRs have not been characterized, and the locations of its binding site(s) in any nAChR are not known. We report here that dFBr inhibits human muscle (αβεδ) and Torpedo (αβγδ) nAChR expressed in Xenopus oocytes with IC50 values of ∼ 1 μM. dFBr also inhibited the equilibrium binding of ion channel blockers to Torpedo nAChRs with higher affinity in the nAChR desensitized state ([(3)H]phencyclidine; IC50 = 4 μM) than in the resting state ([(3)H]tetracaine; IC50 = 60 μM), whereas it bound with only very low affinity to the ACh binding sites ([(3)H]ACh, IC50 = 1 mM). Upon irradiation at 312 nm, [(3)H]dFBr photoincorporated into amino acids within the Torpedo nAChR ion channel with the efficiency of photoincorporation enhanced in the presence of agonist and the agonist-enhanced photolabeling inhibitable by phencyclidine. In the presence of agonist, [(3)H]dFBr also photolabeled amino acids in the nAChR extracellular domain within binding pockets identified previously for the nonselective nAChR PAMs galantamine and physostigmine at the canonical α-γ interface containing the transmitter binding sites and at the noncanonical δ-β subunit interface. These results establish that dFBr inhibits muscle-type nAChR by binding in the ion channel and that [(3)H]dFBr is a photoaffinity probe with broad amino acid side chain reactivity. PMID:25870334

  16. Site-specific labeling of the type 1 ryanodine receptor using biarsenical fluorophores targeted to engineered tetracysteine motifs.

    PubMed

    Fessenden, James D; Mahalingam, Mohana

    2013-01-01

    The type 1 ryanodine receptor (RyR1) is an intracellular Ca(2+) release channel that mediates skeletal muscle excitation contraction coupling. While the overall shape of RyR1 has been elucidated using cryo electron microscopic reconstructions, fine structural details remain elusive. To better understand the structure of RyR1, we have previously used a cell-based fluorescence resonance energy transfer (FRET) method using a fused green fluorescent protein (GFP) donor and a fluorescent acceptor, Cy3NTA that binds specifically to short poly-histidine 'tags' engineered into RyR1. However, the need to permeabilize cells to allow Cy3NTA entry as well as the noncovalent binding of Cy3NTA to the His tag limits future applications of this technique for studying conformational changes of the RyR. To overcome these problems, we used a dodecapeptide sequence containing a tetracysteine (Tc) motif to target the biarsenical fluorophores, FlAsH and ReAsH to RyR1. These compounds freely cross intact cell membranes where they then bind covalently to the tetracysteine motif. First, we used this system to conduct FRET measurements in intact cells by fusing a yellow fluorescent protein (YFP) FRET donor to the N-terminus of RyR1 and then targeting the FRET acceptor, ReAsH to an adjacent Tc tag. Moderate energy transfer (∼33%) was observed whereas ReAsH incubation of a YFPRyR1 fusion protein lacking the Tc tag resulted in no detectable FRET. We also developed a FRET-based system that did not require RyR fluorescent protein fusions by labeling N-terminal Tc-tagged RyR1 with FlAsH, a FRET donor and then targeting the FRET acceptor Cy3NTA to an adjacent decahistidine (His10) tag. A high degree of energy transfer (∼66%) indicated proper binding of both compounds to these unique recognition sequences in RyR1. Thus, these two systems should provide unprecedented flexibility in future FRET-based structural determinations of RyR1.

  17. Identification of a Tumor Specific, Active-Site Mutation in Casein Kinase 1α by Chemical Proteomics.

    PubMed

    Okerberg, Eric S; Hainley, Anna; Brown, Heidi; Aban, Arwin; Alemayehu, Senait; Shih, Ann; Wu, Jane; Patricelli, Matthew P; Kozarich, John W; Nomanbhoy, Tyzoon; Rosenblum, Jonathan S

    2016-01-01

    We describe the identification of a novel, tumor-specific missense mutation in the active site of casein kinase 1α (CSNK1A1) using activity-based proteomics. Matched normal and tumor colon samples were analyzed using an ATP acyl phosphate probe in a kinase-targeted LC-MS2 platform. An anomaly in the active-site peptide from CSNK1A1 was observed in a tumor sample that was consistent with an altered catalytic aspartic acid. Expression and analysis of the suspected mutant verified the presence of asparagine in the probe-labeled, active-site peptide for CSNK1A1. Genomic sequencing of the colon tumor samples confirmed the presence of a missense mutation in the catalytic aspartic acid of CSNK1A1 (GAC→AAC). To our knowledge, the D163N mutation in CSNK1A1 is a newly defined mutation to the conserved, catalytic aspartic acid of a protein kinase and the first missense mutation identified using activity-based proteomics. The tumorigenic potential of this mutation remains to be determined. PMID:27031502

  18. Identification of a Tumor Specific, Active-Site Mutation in Casein Kinase 1α by Chemical Proteomics

    PubMed Central

    Okerberg, Eric S.; Hainley, Anna; Brown, Heidi; Aban, Arwin; Alemayehu, Senait; Shih, Ann; Wu, Jane; Patricelli, Matthew P.; Kozarich, John W.; Nomanbhoy, Tyzoon; Rosenblum, Jonathan S.

    2016-01-01

    We describe the identification of a novel, tumor-specific missense mutation in the active site of casein kinase 1α (CSNK1A1) using activity-based proteomics. Matched normal and tumor colon samples were analyzed using an ATP acyl phosphate probe in a kinase-targeted LC-MS2 platform. An anomaly in the active-site peptide from CSNK1A1 was observed in a tumor sample that was consistent with an altered catalytic aspartic acid. Expression and analysis of the suspected mutant verified the presence of asparagine in the probe-labeled, active-site peptide for CSNK1A1. Genomic sequencing of the colon tumor samples confirmed the presence of a missense mutation in the catalytic aspartic acid of CSNK1A1 (GAC→AAC). To our knowledge, the D163N mutation in CSNK1A1 is a newly defined mutation to the conserved, catalytic aspartic acid of a protein kinase and the first missense mutation identified using activity-based proteomics. The tumorigenic potential of this mutation remains to be determined. PMID:27031502

  19. Cp*Co(III) Catalyzed Site-Selective C-H Activation of Unsymmetrical O-Acyl Oximes: Synthesis of Multisubstituted Isoquinolines from Terminal and Internal Alkynes.

    PubMed

    Sun, Bo; Yoshino, Tatsuhiko; Kanai, Motomu; Matsunaga, Shigeki

    2015-10-26

    The synthesis of isoquinolines by site-selective C-H activation of O-acyl oximes with a Cp*Co(III) catalyst is described. In the presence of this catalyst, the C-H activation of various unsymmetrically substituted O-acyl oximes selectively occurred at the sterically less hindered site, and reactions with terminal as well as internal alkynes afforded the corresponding products in up to 98 % yield. Whereas the reactions catalyzed by the Cp*Co(III) system proceeded with high site selectivity (15:1 to 20:1), use of the corresponding Cp*Rh(III) catalysts led to low selectivities and/or yields when unsymmetrical O-acyl oximes and terminal alkynes were used. Deuterium labeling studies indicate a clear difference in the site selectivity of the C-H activation step under Cp*Co(III) and Cp*Rh(III) catalysis.

  20. Metals in the active site of native protein phosphatase-1.

    PubMed

    Heroes, Ewald; Rip, Jens; Beullens, Monique; Van Meervelt, Luc; De Gendt, Stefan; Bollen, Mathieu

    2015-08-01

    Protein phosphatase-1 (PP1) is a major protein Ser/Thr phosphatase in eukaryotic cells. Its activity depends on two metal ions in the catalytic site, which were identified as manganese in the bacterially expressed phosphatase. However, the identity of the metal ions in native PP1 is unknown. In this study, total reflection X-ray fluorescence (TXRF) was used to detect iron and zinc in PP1 that was purified from rabbit skeletal muscle. Metal exchange experiments confirmed that the distinct substrate specificity of recombinant and native PP1 is determined by the nature of their associated metals. We also found that the iron level associated with native PP1 is decreased by incubation with inhibitor-2, consistent with a function of inhibitor-2 as a PP1 chaperone. PMID:25890482

  1. Metavanadate at the active site of the phosphatase VHZ.

    PubMed

    Kuznetsov, Vyacheslav I; Alexandrova, Anastassia N; Hengge, Alvan C

    2012-09-01

    Vanadate is a potent modulator of a number of biological processes and has been shown by crystal structures and NMR spectroscopy to interact with numerous enzymes. Although these effects often occur under conditions where oligomeric forms dominate, the crystal structures and NMR data suggest that the inhibitory form is usually monomeric orthovanadate, a particularly good inhibitor of phosphatases because of its ability to form stable trigonal-bipyramidal complexes. We performed a computational analysis of a 1.14 Å structure of the phosphatase VHZ in complex with an unusual metavanadate species and compared it with two classical trigonal-bipyramidal vanadate-phosphatase complexes. The results support extensive delocalized bonding to the apical ligands in the classical structures. In contrast, in the VHZ metavanadate complex, the central, planar VO(3)(-) moiety has only one apical ligand, the nucleophilic Cys95, and a gap in electron density between V and S. A computational analysis showed that the V-S interaction is primarily ionic. A mechanism is proposed to explain the formation of metavanadate in the active site from a dimeric vanadate species that previous crystallographic evidence has shown to be able to bind to the active sites of phosphatases related to VHZ. Together, the results show that the interaction of vanadate with biological systems is not solely reliant upon the prior formation of a particular inhibitory form in solution. The catalytic properties of an enzyme may act upon the oligomeric forms primarily present in solution to generate species such as the metavanadate ion observed in the VHZ structure. PMID:22876963

  2. Zymogen Activation and Subcellular Activity of Subtilisin Kexin Isozyme 1/Site 1 Protease*

    PubMed Central

    da Palma, Joel Ramos; Burri, Dominique Julien; Oppliger, Joël; Salamina, Marco; Cendron, Laura; de Laureto, Patrizia Polverino; Seidah, Nabil Georges; Kunz, Stefan; Pasquato, Antonella

    2014-01-01

    The proprotein convertase subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P) plays crucial roles in cellular homeostatic functions and is hijacked by pathogenic viruses for the processing of their envelope glycoproteins. Zymogen activation of SKI-1/S1P involves sequential autocatalytic processing of its N-terminal prodomain at sites B′/B followed by the herein newly identified C′/C sites. We found that SKI-1/S1P autoprocessing results in intermediates whose catalytic domain remains associated with prodomain fragments of different lengths. In contrast to other zymogen proprotein convertases, all incompletely matured intermediates of SKI-1/S1P showed full catalytic activity toward cellular substrates, whereas optimal cleavage of viral glycoproteins depended on B′/B processing. Incompletely matured forms of SKI-1/S1P further process cellular and viral substrates in distinct subcellular compartments. Using a cell-based sensor for SKI-1/S1P activity, we found that 9 amino acid residues at the cleavage site (P1–P8) and P1′ are necessary and sufficient to define the subcellular location of processing and to determine to what extent processing of a substrate depends on SKI-1/S1P maturation. In sum, our study reveals novel and unexpected features of SKI-1/S1P zymogen activation and subcellular specificity of activity toward cellular and pathogen-derived substrates. PMID:25378398

  3. Up-regulation of serotonergic binding sites labeled by (/sup 3/H) WB4101 following fimbrial transection and 5,7-dihydroxytryptamine-induced lesions

    SciTech Connect

    Morrow, A.L.; Norman, A.B.; Battaglia, G.; Loy, R.; Creese, I.

    1985-11-18

    Lesions of the serotonergic afferents to the hippocampus, by fimbrial transection or by 5,7-dihydroxytryptamine treatment, produce an increase in the Bmax of (/sup 3/H)WB4101 to its nanomolar affinity binding site, with no effect on its picomolar affinity binding site or on (/sup 3/H)prazosin binding. The nanomolar site is serotonergic as the serotonergic agonists, serotonin and 8-hydroxy-dipropylaminotetraline (8-OH-DPAT) have nanomolar affinity for (/sup 3/H)WB4101 binding when studied in the presence of a prazosin mask (30nM) of the alpha-1 component of (/sup 3/H)WB4101 binding. The serotonin receptor antagonists metergoline, lysergic acid diethylamide and lisuride also have high nanomolar affinities while ketanserin, yohimbine, prazosin and noradrenergic agonists have affinities in the micromolar range. Fimbrial transection or 5,7-dihydroxytryptamine injections produced 32% and 44% increases in the Bmax of (/sup 3/H)WB4101 binding in the presence of a prazosin mask. Serotonin competition for (/sup 3/H)WB4101 binding was identical in control and experimental tissues from each lesion experiment. Although specific binding of (/sup 3/H)WB4101 was increased, there was no change in the affinities or the percentages of the two binding components for serotonin competition with (/sup 3/H)WB4101. These data suggest that removal of the serotonergic input to the hippocampus produces an increase in the Bmax of serotonin receptor binding sites labeled by (/sup 3/H)WB4101. 33 references, 3 figures, 3 tables.

  4. Label-free electrochemical detection of methyltransferase activity and inhibitor screening based on endonuclease HpaII and the deposition of polyaniline.

    PubMed

    Zhang, Linqun; Wei, Min; Gao, Chunyan; Wei, Wei; Zhang, Yuanjian; Liu, Songqin

    2015-11-15

    Detection of DNA methylation and methyltransferase (MTase) activity are important in determining human cancer because aberrant methylation was linked to cancer initiation and progression. In this work, we proposed an electrochemical method for sensitive detection of DNA methylation and MTase activity based on methylation sensitive restriction endonuclease HpaII and the deposition of polyaniline (PANI) catalyzed by HRP-mimicking DNAzyme. In the presence of methylated DNA, HRP-mimicking DNAzyme catalyzed the polymerization of aniline on the dsDNA template, producing huge DPV current. In the presence of non-methylated DNA, dsDNA are cleaved and digested by HpaII and exonuclease III, as a result, no PANI are deposited. This method can be used to determine DNA methylation at the site of CpG. It exhibits a wide linear response toward M.SssI MTase activity in the range of 0.5-0.6 U mL(-1) with the detection limit of 0.12 U mL(-1). G-rich DNA forms HRP mimicking DNAzyme, which avoids complex labeling procedures and is robust. The method is simple, reliable, sensitive and specific, which has been successfully applied in human serum samples and been used to screen the inhibitors. Thus, the proposed method may be a potential and powerful tool for clinical diagnosis and drug development in the future.

  5. Site-specific PEGylation of lidamycin and its antitumor activity.

    PubMed

    Li, Liang; Shang, Boyang; Hu, Lei; Shao, Rongguang; Zhen, Yongsu

    2015-05-01

    In this study, N-terminal site-specific mono-PEGylation of the recombinant lidamycin apoprotein (rLDP) of lidamycin (LDM) was prepared using a polyethyleneglycol (PEG) derivative (M w 20 kDa) through a reactive terminal aldehyde group under weak acidic conditions (pH 5.5). The biochemical properties of mPEG-rLDP-AE, an enediyne-integrated conjugate, were analyzed by SDS-PAGE, RP-HPLC, SEC-HPLC and MALDI-TOF. Meanwhile, in vitro and in vivo antitumor activity of mPEG-rLDP-AE was evaluated by MTT assays and in xenograft model. The results indicated that mPEG-rLDP-AE showed significant antitumor activity both in vitro and in vivo. After PEGylation, mPEG-rLDP still retained the binding capability to the enediyne AE and presented the physicochemical characteristics similar to that of native LDP. It is of interest that the PEGylation did not diminish the antitumor efficacy of LDM, implying the possibility that this derivative may function as a payload to deliver novel tumor-targeted drugs. PMID:26579455

  6. Optical painting and fluorescence activated sorting of single adherent cells labelled with photoswitchable Pdots

    PubMed Central

    Kuo, Chun-Ting; Thompson, Alison M.; Gallina, Maria Elena; Ye, Fangmao; Johnson, Eleanor S.; Sun, Wei; Zhao, Mengxia; Yu, Jiangbo; Wu, I-Che; Fujimoto, Bryant; DuFort, Christopher C.; Carlson, Markus A.; Hingorani, Sunil R.; Paguirigan, Amy L.; Radich, Jerald P.; Chiu, Daniel T.

    2016-01-01

    The efficient selection and isolation of individual cells of interest from a mixed population is desired in many biomedical and clinical applications. Here we show the concept of using photoswitchable semiconducting polymer dots (Pdots) as an optical ‘painting' tool, which enables the selection of certain adherent cells based on their fluorescence, and their spatial and morphological features, under a microscope. We first develop a Pdot that can switch between the bright (ON) and dark (OFF) states reversibly with a 150-fold contrast ratio on irradiation with ultraviolet or red light. With a focused 633-nm laser beam that acts as a ‘paintbrush' and the photoswitchable Pdots as the ‘paint', we select and ‘paint' individual Pdot-labelled adherent cells by turning on their fluorescence, then proceed to sort and recover the optically marked cells (with 90% recovery and near 100% purity), followed by genetic analysis. PMID:27118210

  7. Site-specifically labeled CA19.9-targeted immunoconjugates for the PET, NIRF, and multimodal PET/NIRF imaging of pancreatic cancer

    PubMed Central

    Houghton, Jacob L.; Zeglis, Brian M.; Abdel-Atti, Dalya; Aggeler, Robert; Sawada, Ritsuko; Agnew, Brian J.; Scholz, Wolfgang W.; Lewis, Jason S.

    2015-01-01

    Molecular imaging agents for preoperative positron emission tomography (PET) and near-infrared fluorescent (NIRF)-guided delineation of surgical margins could greatly enhance the diagnosis, staging, and resection of pancreatic cancer. PET and NIRF optical imaging offer complementary clinical applications, enabling the noninvasive whole-body imaging to localize disease and identification of tumor margins during surgery, respectively. We report the development of PET, NIRF, and dual-modal (PET/NIRF) imaging agents, using 5B1, a fully human monoclonal antibody that targets CA19.9, a well-established pancreatic cancer biomarker. Desferrioxamine (DFO) and/or a NIRF dye (FL) were conjugated to the heavy-chain glycans of 5B1, using a robust and reproducible site-specific (ss) labeling methodology to generate three constructs (ssDFO-5B1, ssFL-5B1, and ssdual-5B1) in which the immunoreactivity was not affected by the conjugation of either label. Each construct was evaluated in a s.c. xenograft model, using CA19.9-positive (BxPC3) and -negative (MIAPaCa-2) human pancreatic cancer cell lines. Each construct showed exceptional uptake and contrast in antigen-positive tumors with negligible nonspecific uptake in antigen-negative tumors. Additionally, the dual-modal construct was evaluated in an orthotopic murine pancreatic cancer model, using the human pancreatic cancer cell line, Suit-2. The ssdual-5B1 demonstrated a remarkable capacity to delineate metastases and to map the sentinel lymph nodes via tandem PET-computed tomography (PET/CT) and NIRF imaging. Fluorescence microscopy, histopathology, and autoradiography were performed on representative sections of excised tumors to visualize the distribution of the constructs within the tumors. These imaging tools have tremendous potential for further preclinical research and for clinical translation. PMID:26668398

  8. Hybrid [FeFe]-hydrogenases with modified active sites show remarkable residual enzymatic activity.

    PubMed

    Siebel, Judith F; Adamska-Venkatesh, Agnieszka; Weber, Katharina; Rumpel, Sigrun; Reijerse, Edward; Lubitz, Wolfgang

    2015-02-24

    [FeFe]-hydrogenases are to date the only enzymes for which it has been demonstrated that the native inorganic binuclear cofactor of the active site Fe2(adt)(CO)3(CN)2 (adt = azadithiolate = [S-CH2-NH-CH2-S](2-)) can be synthesized on the laboratory bench and subsequently inserted into the unmaturated enzyme to yield fully functional holo-enzyme (Berggren, G. et al. (2013) Nature 499, 66-70; Esselborn, J. et al. (2013) Nat. Chem. Biol. 9, 607-610). In the current study, we exploit this procedure to introduce non-native cofactors into the enzyme. Mimics of the binuclear subcluster with a modified bridging dithiolate ligand (thiodithiolate, N-methylazadithiolate, dimethyl-azadithiolate) and three variants containing only one CN(-) ligand were inserted into the active site of the enzyme. We investigated the activity of these variants for hydrogen oxidation as well as proton reduction and their structural accommodation within the active site was analyzed using Fourier transform infrared spectroscopy. Interestingly, the monocyanide variant with the azadithiolate bridge showed ∼50% of the native enzyme activity. This would suggest that the CN(-) ligands are not essential for catalytic activity, but rather serve to anchor the binuclear subsite inside the protein pocket through hydrogen bonding. The inserted artificial cofactors with a propanedithiolate and an N-methylazadithiolate bridge as well as their monocyanide variants also showed residual activity. However, these activities were less than 1% of the native enzyme. Our findings indicate that even small changes in the dithiolate bridge of the binuclear subsite lead to a rather strong decrease of the catalytic activity. We conclude that both the Brønsted base function and the conformational flexibility of the native azadithiolate amine moiety are essential for the high catalytic activity of the native enzyme. PMID:25633077

  9. Nucleotide-dependent displacement and dynamics of the α-1 helix in kinesin revealed by site-directed spin labeling EPR

    SciTech Connect

    Yasuda, Satoshi; Yanagi, Takanori; Yamada, Masafumi D.; Ueki, Shoji; Maruta, Shinsaku; Inoue, Akio; Arata, Toshiaki

    2014-01-17

    Highlights: •Dipolar EPR detects the distance between the spin-labeled kinesin α-1 and α-2 helices. •The distance has at least two populations: 1.5 nm (in crystal form: 20%) and >2.5 nm. •The short distance conformer was populated 40% in the apo state with microtubules. •ATP analog or ADP binding caused the 1.5 nm distance to be less populated (∼20%). •The α-1 helix moves closer to the neck-linker (away from α-2) to facilitate docking. -- Abstract: In kinesin X-ray crystal structures, the N-terminal region of the α-1 helix is adjacent to the adenine ring of the bound nucleotide, while the C-terminal region of the helix is near the neck-linker (NL). Here, we monitor the displacement of the α-1 helix within a kinesin monomer bound to microtubules (MTs) in the presence or absence of nucleotides using site-directed spin labeling EPR. Kinesin was doubly spin-labeled at the α-1 and α-2 helices, and the resulting EPR spectrum showed dipolar broadening. The inter-helix distance distribution showed that 20% of the spins have a peak characteristic of 1.4–1.7 nm separation, which is similar to what is predicted from the X-ray crystal structure, albeit 80% were beyond the sensitivity limit (>2.5 nm) of the method. Upon MT binding, the fraction of kinesin exhibiting an inter-helix distance of 1.4–1.7 nm in the presence of AMPPNP (a non-hydrolysable ATP analog) and ADP was 20% and 25%, respectively. In the absence of nucleotide, this fraction increased to 40–50%. These nucleotide-induced changes in the fraction of kinesin undergoing displacement of the α-1 helix were found to be related to the fraction in which the NL undocked from the motor core. It is therefore suggested that a shift in the α-1 helix conformational equilibrium occurs upon nucleotide binding and release, and this shift controls NL docking onto the motor core.

  10. Fluroine-18 labeled 28-carbomethoxy-3{beta}-(4-chlorophenyl)-8-[-3-fluoropropyl] nortropane(FPT): Synthesis and tissue distribution of a potential, radioligand for mapping cocaine receptor sites by PET

    SciTech Connect

    Keil, R.; Goodman, M.M.; Shoup, T.

    1995-05-01

    Highly potent and selective radioligands for the dopamine transporter labeled with fluorine-18 (t {1/2}=110 min) are attractive probes for longitudinal in vivo mapping of cocaine receptor sites in the caudate by PET. Recently, we reported an iodine-123 labeled 3{beta}-aryl analog of cocaine, 2{beta}-carbomethoxy-3{beta}-(4-chlorophenyl)-8-((E)-3-iodopropen-1-yl)nortropane, which was 125 times more potent than cocaine in inhibiting [I-125] RTI-55 binding to rat striatal homogenates and which showed high striatal (S) uptake (0.61% dose/g) and high S to cerebellum (C) ratio S/C=16.5 at 120 min in rats. These results demonstrated bulk tolerance at the 8-position of this I-123 analog. These findings prompted us to synthesize a new radioligand fluorine-18 labeled 2{beta}-carbomethoxy-3{beta}-(4-chlorophenyl)-8-3-fluoropropylnortropane (FPT) as a potential cocaine receptor PET imaging agent. Treatment of 2{beta}-carbomethoxy-3{beta}-(chlorophenyl) nortropane (1) with 1-bromo-3-fluoropropane (2) in CH3CN at 80{degrees}C afforded FPT (3). In Vitro binding studies in rat striatal homogenates using [I-125] RTI-55 resulted in a Ki (nM) of 8.2 for FPT. [F-18]FPT (3) was prepared by treating 1,3-diiodopropane (4) with NCA K[F-18]/K222 for 5 min in CH3CN at 85{degrees}C to give [F-18] 1-fluoro-3-iodopropane (5) in 50% E.O.B. yield. Coupling of [F-18] 5 with 1 in CH3CN at 60{degrees}C afforded [F-18]FPT in 5% yield (not optimized) E.O.B. following HPLC purification in a total synthesis time of 100 min.. [F-18]5 was >99% radiochemically pure with a specific activity of 8 Ci/{mu}mole. Following intravenous administration to rats [F-18]FPT showed high uptake in the striatum (S) with rapid washout from the cerebellum to afford a high S/C ratios=6.2 at 120 min. Primate imaging will also be presented. These results suggest that FPT is an excellent candidate for mapping cocaine receptor sites by PET.

  11. Activation of p70s6k is associated with phosphorylation of four clustered sites displaying Ser/Thr-Pro motifs.

    PubMed

    Ferrari, S; Bannwarth, W; Morley, S J; Totty, N F; Thomas, G

    1992-08-01

    Partial amino acid sequences were obtained from 22 internal tryptic peptides of rat liver p70s6k (M(r) 70,000 ribosomal protein S6 kinase), 3 of which were found to contain phosphorylated residues. To determine whether these sites were associated with p70s6k activation, the kinase was labeled to high specific activity with 32P(i) in Swiss mouse 3T3 cells. By sequential cleavage with CNBr and endoproteinase Lys-C followed by two-dimensional tryptic peptide analysis, it could be shown that all of the sites were located in a small endoproteinase Lys-C peptide of M(r) 2400. Analysis of the p70s6k protein sequence revealed a single candidate that could represent this peptide. Three tryptic peptides derived from the endoproteinase Lys-C fragment were chosen by a newly described computer program as the most likely candidates to contain the in vivo sites of phosphorylation. Synthetic peptides based on these sequences were phosphorylated either chemically or enzymatically and found to comigrate by two-dimensional thin-layer electrophoresis/chromatography with the four major in vivo labeled tryptic phosphopeptides. Three of the phosphorylation sites in these peptides were equivalent to those sequenced in the rat liver p70s6k. In addition, all four sites display the motif Ser/Thr-Pro, typical of cell cycle-regulated sites, and are clustered in a putative autoinhibitory domain of the enzyme.

  12. Investigating the Effect of Ligand Amount and Injected Therapeutic Activity: A Simulation Study for 177Lu-Labeled PSMA-Targeting Peptides.

    PubMed

    Kletting, Peter; Schuchardt, Christiane; Kulkarni, Harshad R; Shahinfar, Mostafa; Singh, Aviral; Glatting, Gerhard; Baum, Richard P; Beer, Ambros J

    2016-01-01

    In molecular radiotherapy with 177Lu-labeled prostate specific membrane antigen (PSMA) peptides, kidney and/or salivary glands doses limit the activity which can be administered. The aim of this work was to investigate the effect of the ligand amount and injected activity on the tumor-to-normal tissue biologically effective dose (BED) ratio for 177Lu-labeled PSMA peptides. For this retrospective study, a recently developed physiologically based pharmacokinetic model was adapted for PSMA targeting peptides. General physiological parameters were taken from the literature. Individual parameters were fitted to planar gamma camera measurements (177Lu-PSMA I&T) of five patients with metastasizing prostate cancer. Based on the estimated parameters, the pharmacokinetics of tumor, salivary glands, kidneys, total body and red marrow was simulated and time-integrated activity coefficients were calculated for different peptide amounts. Based on these simulations, the absorbed doses and BEDs for normal tissue and tumor were calculated for all activities leading to a maximal tolerable kidney BED of 10 Gy2.5/cycle, a maximal salivary gland absorbed dose of 7.5 Gy/cycle and a maximal red marrow BED of 0.25 Gy15/cycle. The fits yielded coefficients of determination > 0.85, acceptable relative standard errors and low parameter correlations. All estimated parameters were in a physiologically reasonable range. The amounts (for 25-29 nmol) and pertaining activities leading to a maximal tumor dose, considering the defined maximal tolerable doses to organs of risk, were calculated to be 272±253 nmol (452±420 μg) and 7.3±5.1 GBq. Using the actually injected amount (235±155 μg) and the same maximal tolerable doses, the potential improvement for the tumor BED was 1-3 fold. The results suggest that currently given amounts for therapy are in the appropriate order of magnitude for many lesions. However, for lesions with high binding site density or lower perfusion, optimizing the peptide

  13. Investigating the Effect of Ligand Amount and Injected Therapeutic Activity: A Simulation Study for 177Lu-Labeled PSMA-Targeting Peptides

    PubMed Central

    Schuchardt, Christiane; Kulkarni, Harshad R.; Shahinfar, Mostafa; Singh, Aviral; Glatting, Gerhard; Baum, Richard P.; Beer, Ambros J.

    2016-01-01

    In molecular radiotherapy with 177Lu-labeled prostate specific membrane antigen (PSMA) peptides, kidney and/or salivary glands doses limit the activity which can be administered. The aim of this work was to investigate the effect of the ligand amount and injected activity on the tumor-to-normal tissue biologically effective dose (BED) ratio for 177Lu-labeled PSMA peptides. For this retrospective study, a recently developed physiologically based pharmacokinetic model was adapted for PSMA targeting peptides. General physiological parameters were taken from the literature. Individual parameters were fitted to planar gamma camera measurements (177Lu-PSMA I&T) of five patients with metastasizing prostate cancer. Based on the estimated parameters, the pharmacokinetics of tumor, salivary glands, kidneys, total body and red marrow was simulated and time-integrated activity coefficients were calculated for different peptide amounts. Based on these simulations, the absorbed doses and BEDs for normal tissue and tumor were calculated for all activities leading to a maximal tolerable kidney BED of 10 Gy2.5/cycle, a maximal salivary gland absorbed dose of 7.5 Gy/cycle and a maximal red marrow BED of 0.25 Gy15/cycle. The fits yielded coefficients of determination > 0.85, acceptable relative standard errors and low parameter correlations. All estimated parameters were in a physiologically reasonable range. The amounts (for 25−29 nmol) and pertaining activities leading to a maximal tumor dose, considering the defined maximal tolerable doses to organs of risk, were calculated to be 272±253 nmol (452±420 μg) and 7.3±5.1 GBq. Using the actually injected amount (235±155 μg) and the same maximal tolerable doses, the potential improvement for the tumor BED was 1–3 fold. The results suggest that currently given amounts for therapy are in the appropriate order of magnitude for many lesions. However, for lesions with high binding site density or lower perfusion, optimizing the

  14. Investigating the Effect of Ligand Amount and Injected Therapeutic Activity: A Simulation Study for 177Lu-Labeled PSMA-Targeting Peptides.

    PubMed

    Kletting, Peter; Schuchardt, Christiane; Kulkarni, Harshad R; Shahinfar, Mostafa; Singh, Aviral; Glatting, Gerhard; Baum, Richard P; Beer, Ambros J

    2016-01-01

    In molecular radiotherapy with 177Lu-labeled prostate specific membrane antigen (PSMA) peptides, kidney and/or salivary glands doses limit the activity which can be administered. The aim of this work was to investigate the effect of the ligand amount and injected activity on the tumor-to-normal tissue biologically effective dose (BED) ratio for 177Lu-labeled PSMA peptides. For this retrospective study, a recently developed physiologically based pharmacokinetic model was adapted for PSMA targeting peptides. General physiological parameters were taken from the literature. Individual parameters were fitted to planar gamma camera measurements (177Lu-PSMA I&T) of five patients with metastasizing prostate cancer. Based on the estimated parameters, the pharmacokinetics of tumor, salivary glands, kidneys, total body and red marrow was simulated and time-integrated activity coefficients were calculated for different peptide amounts. Based on these simulations, the absorbed doses and BEDs for normal tissue and tumor were calculated for all activities leading to a maximal tolerable kidney BED of 10 Gy2.5/cycle, a maximal salivary gland absorbed dose of 7.5 Gy/cycle and a maximal red marrow BED of 0.25 Gy15/cycle. The fits yielded coefficients of determination > 0.85, acceptable relative standard errors and low parameter correlations. All estimated parameters were in a physiologically reasonable range. The amounts (for 25-29 nmol) and pertaining activities leading to a maximal tumor dose, considering the defined maximal tolerable doses to organs of risk, were calculated to be 272±253 nmol (452±420 μg) and 7.3±5.1 GBq. Using the actually injected amount (235±155 μg) and the same maximal tolerable doses, the potential improvement for the tumor BED was 1-3 fold. The results suggest that currently given amounts for therapy are in the appropriate order of magnitude for many lesions. However, for lesions with high binding site density or lower perfusion, optimizing the peptide

  15. Sulfonium Ion Derivatization, Isobaric Stable Isotope Labeling and Data Dependent CID- and ETD-MS/MS for Enhanced Phosphopeptide Quantitation, Identification and Phosphorylation Site Characterization

    PubMed Central

    Lu, Yali; Zhou, Xiao; Stemmer, Paul M.; Reid, Gavin E.

    2014-01-01

    An amine specific peptide derivatization strategy involving the use of novel isobaric stable isotope encoded ‘fixed charge’ sulfonium ion reagents, coupled with an analysis strategy employing capillary HPLC, ESI-MS, and automated data dependent ion trap CID-MS/MS, -MS3, and/or ETD-MS/MS, has been developed for the improved quantitative analysis of protein phosphorylation, and for identification and characterization of their site(s) of modification. Derivatization of 50 synthetic phosphopeptides with S,S′-dimethylthiobutanoylhydroxysuccinimide ester iodide (DMBNHS), followed by analysis using capillary HPLC-ESI-MS, yielded an average 2.5-fold increase in ionization efficiencies and a significant increase in the presence and/or abundance of higher charge state precursor ions compared to the non-derivatized phosphopeptides. Notably, 44% of the phosphopeptides (22 of 50) in their underivatized states yielded precursor ions whose maximum charge states corresponded to +2, while only 8% (4 of 50) remained at this maximum charge state following DMBNHS derivatization. Quantitative analysis was achieved by measuring the abundances of the diagnostic product ions corresponding to the neutral losses of ‘light’ (S(CH3)2) and ‘heavy’ (S(CD3)2) dimethylsulfide exclusively formed upon CID-MS/MS of isobaric stable isotope labeled forms of the DMBNHS derivatized phosphopeptides. Under these conditions, the phosphate group stayed intact. Access for a greater number of peptides to provide enhanced phosphopeptide sequence identification and phosphorylation site characterization was achieved via automated data-dependent CID-MS3 or ETD-MS/MS analysis due to the formation of the higher charge state precursor ions. Importantly, improved sequence coverage was observed using ETD-MS/MS following introduction of the sulfonium ion fixed charge, but with no detrimental effects on ETD fragmentation efficiency. PMID:21952753

  16. Label-Free Direct Visual Analysis of Hydrolytic Enzyme Activity Using Aqueous Two-Phase System Droplet Phase Transitions

    PubMed Central

    2015-01-01

    Dextran hydrolysis-mediated conversion of polyethylene glycol (PEG)-dextran (DEX) aqueous two-phase system droplets to a single phase was used to directly visualize Dextranase activity. DEX droplets were formed either by manual micropipetting or within a continuous PEG phase by computer controlled actuation of an orifice connecting rounded channels formed by backside diffused light lithography. The time required for the two-phase to one-phase transition was dependent on the Dextranase concentration, pH of the medium, and temperature. The apparent Michaelis constants for Dextranase were estimated based on previously reported catalytic constants, the binodal polymer concentration curves for PEG-DEX phase transition for each temperature, and pH condition. The combination of a microfluidic droplet system and phase transition observation provides a new method for label-free direct measurement of enzyme activity. PMID:24654925

  17. Label-free fluorescence turn-on detection of uracil DNA glycosylase activity based on G-quadruplex formation.

    PubMed

    Ma, Changbei; Wu, Kefeng; Liu, Haisheng; Xia, Kun; Wang, Kemin; Wang, Jun

    2016-11-01

    We have developed a new methodology for fluorescence turn-on detection of uracil DNA glycosylase (UDG) activity based on G-quadruplex formation using a thioflavin T probe. In the presence of UDG, it catalyzed the hydrolysis of the uracil bases in the duplex DNA, resulting in the dissociation of the duplex DNA owing to their low melting temperature. Then, the probe DNA can be recognized quickly by the ThT dye and resulting in an increase in fluorescence. This approach is highly selective and sensitive with a detection limit of 0.01U/mL. It is simple and cost effective without requirement of labeling with a fluorophore-quencher pair. This new method could be used to evaluate the inhibition effect of 5-fluorouracil on UDG activity, and become a useful tool in biomedical research. PMID:27591637

  18. High sensitive and label-free colorimetric DNA detection based on nicking endonuclease-assisted activation of DNAzymes.

    PubMed

    Li, Juan; Yao, Qiu-Hong; Fu, Hua-E; Zhang, Xiao-Long; Yang, Huang-Hao

    2011-07-15

    Horseradish peroxidase mimicking DNAzyme (HRP-DNAzyme) attracts growing interest as an amplifying label for biorecognition and biosensing events, especially for DNA detection. However, in the traditional designs, one target molecule can only generate one HRP-DNAzyme, which limits the signal enhancement and thus its sensitivity. In this article, we propose an amplified and label-free colorimetric DNA detection strategy based on nicking endonuclease (NEase)-assisted activation of HRP-DNAzymes (NEAA-DNAzymes). This new strategy relies on the hairpin-DNAzyme probe and NEase-assisted target recycling. In the hairpin-DNAzyme probe, the HRP-DNAzyme sequence is protected in a "caged" inactive structure, whereas the loop region includes the target complementary sequence. Upon hybridization with target, the beacon is opened, resulting in the activation of the HRP-DNAzyme. Meanwhile, upon formation of the duplex, the NEase recognizes a specific nucleotide sequence and cleaves the hairpin-DNAzyme probe into two fragments. After nicking, the fragments of the hairpin-DNAzyme probe spontaneously dissociate from the target DNA. Amplification is accomplished by another hairpin-DNAzyme probe hybridizing to the released intact target to continue the strand-scission cycle, which results in activation of numerous DNAzymes. The activated HRP-DNAzymes generate colorimetric or chemiluminescence readout signals, thus providing the amplified detection of DNA. The detection limit of the colorimetric method is 10 pmol/L, which are three orders of magnitude lower than that without NEase. In addition, the detection limit of the chemiluminescence method is 0.2 pmol/L. Meanwhile, this strategy also exhibits high discrimination ability even against single-base mismatch.

  19. Probing Structural Dynamics and Topology of the KCNE1 Membrane Protein in Lipid Bilayers via Site-Directed Spin Labeling and Electron Paramagnetic Resonance Spectroscopy.

    PubMed

    Sahu, Indra D; Craig, Andrew F; Dunagan, Megan M; Troxel, Kaylee R; Zhang, Rongfu; Meiberg, Andrew G; Harmon, Corrinne N; McCarrick, Robert M; Kroncke, Brett M; Sanders, Charles R; Lorigan, Gary A

    2015-10-20

    KCNE1 is a single transmembrane protein that modulates the function of voltage-gated potassium channels, including KCNQ1. Hereditary mutations in the genes encoding either protein can result in diseases such as congenital deafness, long QT syndrome, ventricular tachyarrhythmia, syncope, and sudden cardiac death. Despite the biological significance of KCNE1, the structure and dynamic properties of its physiologically relevant native membrane-bound state are not fully understood. In this study, the structural dynamics and topology of KCNE1 in bilayered lipid vesicles was investigated using site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy. A 53-residue nitroxide EPR scan of the KCNE1 protein sequence including all 27 residues of the transmembrane domain (45-71) and 26 residues of the N- and C-termini of KCNE1 in lipid bilayered vesicles was analyzed in terms of nitroxide side-chain motion. Continuous wave-EPR spectral line shape analysis indicated the nitroxide spin label side-chains located in the KCNE1 TMD are less mobile when compared to the extracellular region of KCNE1. The EPR data also revealed that the C-terminus of KCNE1 is more mobile when compared to the N-terminus. EPR power saturation experiments were performed on 41 sites including 18 residues previously proposed to reside in the transmembrane domain (TMD) and 23 residues of the N- and C-termini to determine the topology of KCNE1 with respect to the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) lipid bilayers. The results indicated that the transmembrane domain is indeed buried within the membrane, spanning the width of the lipid bilayer. Power saturation data also revealed that the extracellular region of KCNE1 is solvent-exposed with some of the portions partially or weakly interacting with the membrane surface. These results are consistent with the previously published solution NMR

  20. 21 CFR 201.64 - Sodium labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... contains sodium bicarbonate, sodium phosphate, or sodium biphosphate as an active ingredient for oral... 21 Food and Drugs 4 2011-04-01 2011-04-01 false Sodium labeling. 201.64 Section 201.64 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.64 Sodium labeling. (a) The labeling...

  1. 21 CFR 201.64 - Sodium labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... contains sodium bicarbonate, sodium phosphate, or sodium biphosphate as an active ingredient for oral... 21 Food and Drugs 4 2012-04-01 2012-04-01 false Sodium labeling. 201.64 Section 201.64 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.64 Sodium labeling. (a) The labeling...

  2. 21 CFR 201.64 - Sodium labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... contains sodium bicarbonate, sodium phosphate, or sodium biphosphate as an active ingredient for oral... 21 Food and Drugs 4 2013-04-01 2013-04-01 false Sodium labeling. 201.64 Section 201.64 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.64 Sodium labeling. (a) The labeling...

  3. 21 CFR 201.64 - Sodium labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... contains sodium bicarbonate, sodium phosphate, or sodium biphosphate as an active ingredient for oral... 21 Food and Drugs 4 2014-04-01 2014-04-01 false Sodium labeling. 201.64 Section 201.64 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.64 Sodium labeling. (a) The labeling...

  4. 21 CFR 201.64 - Sodium labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... contains sodium bicarbonate, sodium phosphate, or sodium biphosphate as an active ingredient for oral... 21 Food and Drugs 4 2010-04-01 2010-04-01 false Sodium labeling. 201.64 Section 201.64 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.64 Sodium labeling. (a) The labeling...

  5. Use of stable isotope-labelled cells to identify active grazers of picocyanobacteria in ocean surface waters

    PubMed Central

    Frias-Lopez, Jorge; Thompson, Anne; Waldbauer, Jacob; Chisholm, Sallie W

    2009-01-01

    Prochlorococcus and Synechococcus are the two most abundant marine cyanobacteria. They represent a significant fraction of the total primary production of the world oceans and comprise a major fraction of the prey biomass available to phagotrophic protists. Despite relatively rapid growth rates, picocyanobacterial cell densities in open-ocean surface waters remain fairly constant, implying steady mortality due to viral infection and consumption by predators. There have been several studies on grazing by specific protists on Prochlorococcus and Synechococcus in culture, and of cell loss rates due to overall grazing in the field. However, the specific sources of mortality of these primary producers in the wild remain unknown. Here, we use a modification of the RNA stable isotope probing technique (RNA-SIP), which involves adding labelled cells to natural seawater, to identify active predators that are specifically consuming Prochlorococcus and Synechococcus in the surface waters of the Pacific Ocean. Four major groups were identified as having their 18S rRNA highly labelled: Prymnesiophyceae (Haptophyta), Dictyochophyceae (Stramenopiles), Bolidomonas (Stramenopiles) and Dinoflagellata (Alveolata). For the first three of these, the closest relative of the sequences identified was a photosynthetic organism, indicating the presence of mixotrophs among picocyanobacterial predators. We conclude that the use of RNA-SIP is a useful method to identity specific predators for picocyanobacteria in situ, and that the method could possibly be used to identify other bacterial predators important in the microbial food-web. PMID:19196281

  6. Use of stable isotope-labelled cells to identify active grazers of picocyanobacteria in ocean surface waters.

    PubMed

    Frias-Lopez, Jorge; Thompson, Anne; Waldbauer, Jacob; Chisholm, Sallie W

    2009-02-01

    Prochlorococcus and Synechococcus are the two most abundant marine cyanobacteria. They represent a significant fraction of the total primary production of the world oceans and comprise a major fraction of the prey biomass available to phagotrophic protists. Despite relatively rapid growth rates, picocyanobacterial cell densities in open-ocean surface waters remain fairly constant, implying steady mortality due to viral infection and consumption by predators. There have been several studies on grazing by specific protists on Prochlorococcus and Synechococcus in culture, and of cell loss rates due to overall grazing in the field. However, the specific sources of mortality of these primary producers in the wild remain unknown. Here, we use a modification of the RNA stable isotope probing technique (RNA-SIP), which involves adding labelled cells to natural seawater, to identify active predators that are specifically consuming Prochlorococcus and Synechococcus in the surface waters of the Pacific Ocean. Four major groups were identified as having their 18S rRNA highly labelled: Prymnesiophyceae (Haptophyta), Dictyochophyceae (Stramenopiles), Bolidomonas (Stramenopiles) and Dinoflagellata (Alveolata). For the first three of these, the closest relative of the sequences identified was a photosynthetic organism, indicating the presence of mixotrophs among picocyanobacterial predators. We conclude that the use of RNA-SIP is a useful method to identity specific predators for picocyanobacteria in situ, and that the method could possibly be used to identify other bacterial predators important in the microbial food-web.

  7. Characterization of the active site of chloroperoxidase using physical techniques

    SciTech Connect

    Hall, K.S.

    1986-01-01

    Chloroperoxidase (CPO) and Cytochrome P-450, two very different hemeproteins, have been shown to have similar active sites by several techniques. Recent work has demonstrated thiolate ligation from a cysteine residue to the iron in P-450. A major portion of this research has been devoted to obtaining direct evidence that CPO also has a thiolate 5th ligand from a cysteine residue. This information will provide the framework for a detailed analysis of the structure-function relationships between peroxidases, catalase and cytochrome P-450 hemeproteins. To determine whether the 5th ligand is a cysteine, methionine or a unique amino acid, specific isotope enrichment experiments were used. Preliminary /sup 1/H-NMR studies show that the carbon monoxide-CPO complex has a peak in the upfield region corresponding to alpha-protons of a thiolate amino acid. C. fumago was grown on 95% D/sub 2/O media with a small amount of /sup 1/H-cysteine added. Under these conditions C. fumago slows down the biosynthesis of cysteine by at least 50% and utilizes the exogenous cysteine in the media. GC-MS was able to show that the methylene protons next to the sulfur atom in cysteine are 80-90% protonated while these positions in methionine are approximately 73% deuterated. Comparison of the /sup 1/H-NMR spectra of CO-CPO and CO-CPO indicate the presence of a cysteine ligand in chloroperoxidase.

  8. N6-Methyldeoxyadenosine Marks Active Transcription Start Sites in Chlamydomonas

    PubMed Central

    Chen, Kai; Deng, Xin; Yu, Miao; Han, Dali; Hao, Ziyang; Liu, Jianzhao; Lu, Xingyu; Dore, Louis C; Weng, Xiaocheng; Ji, Quanjiang; Mets, Laurens; He, Chuan

    2015-01-01

    SUMMARY N6-methyldeoxyadenosine (6mA or m6A) is a DNA modification preserved in prokaryotes to eukaryotes. It is widespread in bacteria, and functions in DNA mismatch repair, chromosome segregation, and virulence regulation. In contrast, the distribution and function of 6mA in eukaryotes have been unclear. Here we present a comprehensive analysis of the 6mA landscape in the genome of Chlamydomonas using new sequencing approaches. We identified the 6mA modification in 84% of genes in Chlamydomonas. We found that 6mA mainly locates at ApT dinucleotides around transcription start sites (TSS) with a bimodal distribution, and appears to mark active genes. A periodic pattern of 6mA deposition was also observed at base resolution, which is associated with nucleosome distribution near the TSS, suggesting a possible role in nucleosome positioning. The new genome-wide mapping of 6mA and its unique distribution in the Chlamydomonas genome suggest potential regulatory roles of 6mA in gene expression in eukaryotic organisms. PMID:25936837

  9. Detection limit for activation measurements in ultralow background sites

    NASA Astrophysics Data System (ADS)

    Trache, Livius; Chesneanu, D.; Margineanu, R.; Pantelica, A.; Ghita, D. G.; Burducea, I.; Straticiuc, M.; Tang, X. D.

    2014-09-01

    We used 12C +13C fusion at the beam energies E = 6, 7 and 8 MeV to determine the sensitivity and the limits of activation method measurements in ultralow background sites. A 13C beam of 0.5 μA from the 3 MV Tandem accelerator of the Horia Hulubei National Institute of Physics and Nuclear Engineering - IFIN HH impinged on thick graphite targets. After about 24 hrs of irradiation targets were measured in two different laboratories: one with a heavy shielded Ge detector in the institute (at the surface) and one located underground in the microBequerel laboratory, in the salt mine of Slanic-Prahova, Romania. The 1369- and 2754 keV peaks from 24Na deactivation were clearly observed in the γ-ray spectra obtained for acquisitions lasting a few hours, or a few days. Determination of the detection limit in evaluating the cross sections for the target irradiated at Ec . m = 3 MeV indicates the fact that it is possible to measure gamma spectrum in underground laboratory down to Ec . m = 2 . 6 MeV. Cleaning the spectra with beta-gamma coincidences and increasing beam intensity 20 times will take as further down. The measurements are motivated by the study of the 12 C +12 C reaction at astrophysical energies.

  10. Disturbance opens recruitment sites for bacterial colonization in activated sludge.

    PubMed

    Vuono, David C; Munakata-Marr, Junko; Spear, John R; Drewes, Jörg E

    2016-01-01

    Little is known about the role of immigration in shaping bacterial communities or the factors that may dictate success or failure of colonization by bacteria from regional species pools. To address these knowledge gaps, the influence of bacterial colonization into an ecosystem (activated sludge bioreactor) was measured through a disturbance gradient (successive decreases in the parameter solids retention time) relative to stable operational conditions. Through a DNA sequencing approach, we show that the most abundant bacteria within the immigrant community have a greater probability of colonizing the receiving ecosystem, but mostly as low abundance community members. Only during the disturbance do some of these bacterial populations significantly increase in abundance beyond background levels and in few cases become dominant community members post-disturbance. Two mechanisms facilitate the enhanced enrichment of immigrant populations during disturbance: (i) the availability of resources left unconsumed by established species and (ii) the increased availability of niche space for colonizers to establish and displace resident populations. Thus, as a disturbance decreases local diversity, recruitment sites become available to promote colonization. This work advances our understanding of microbial resource management and diversity maintenance in complex ecosystems. PMID:25727891

  11. Active Site Characterization of Proteases Sequences from Different Species of Aspergillus.

    PubMed

    Morya, V K; Yadav, Virendra K; Yadav, Sangeeta; Yadav, Dinesh

    2016-09-01

    A total of 129 proteases sequences comprising 43 serine proteases, 36 aspartic proteases, 24 cysteine protease, 21 metalloproteases, and 05 neutral proteases from different Aspergillus species were analyzed for the catalytically active site residues using MEROPS database and various bioinformatics tools. Different proteases have predominance of variable active site residues. In case of 24 cysteine proteases of Aspergilli, the predominant active site residues observed were Gln193, Cys199, His364, Asn384 while for 43 serine proteases, the active site residues namely Asp164, His193, Asn284, Ser349 and Asp325, His357, Asn454, Ser519 were frequently observed. The analysis of 21 metalloproteases of Aspergilli revealed Glu298 and Glu388, Tyr476 as predominant active site residues. In general, Aspergilli species-specific active site residues were observed for different types of protease sequences analyzed. The phylogenetic analysis of these 129 proteases sequences revealed 14 different clans representing different types of proteases with diverse active site residues.

  12. A proposed definition of the 'activity' of surface sites on lactose carriers for dry powder inhalation.

    PubMed

    Grasmeijer, Floris; Frijlink, Henderik W; de Boer, Anne H

    2014-06-01

    A new definition of the activity of surface sites on lactose carriers for dry powder inhalation is proposed which relates to drug detachment during dispersion. The new definition is expected to improve the understanding of 'carrier surface site activity', which stimulates the unambiguous communication about this subject and may aid in the rational design and interpretation of future formulation studies. In contrast to the currently prevailing view on carrier surface site activity, it follows from the newly proposed definition that carrier surface site activity depends on more variables than just the physicochemical properties of the carrier surface. Because the term 'active sites' is ambiguous, it is recommended to use the term 'highly active sites' instead to denote carrier surface sites with a relatively high activity. PMID:24613490

  13. Thioredoxin binding site of phosphoribulokinase overlaps the catalytic site. [R

    SciTech Connect

    Porter, M.A.; Hartman, F.C.

    1986-01-01

    The ATP-regulatory binding site of phosphoribulokinase was studied using bromoacetylethanolamine phosphate (BrAcNHEtOP). BrAcNHEtOP binds to the active-regulatory binding site of the protein. Following trypsin degradation of the labeled protein, fragments were separated by HPLC and sequenced. (DT)

  14. Total-hip arthroplasty: Periprosthetic indium-111-labeled leukocyte activity and complementary technetium-99m-sulfur colloid imaging in suspected infection

    SciTech Connect

    Palestro, C.J.; Kim, C.K.; Swyer, A.J.; Capozzi, J.D.; Solomon, R.W.; Goldsmith, S.J. )

    1990-12-01

    Indium-111-labeled leukocyte images of 92 cemented total-hip arthroplasties were correlated with final diagnoses. Prostheses were divided into four zones: head (including acetabulum), trochanter, shaft, and tip. The presence (or absence) and intensity of activity in each zone was noted, and compared to the corresponding contralateral zone. Though present in all 23 infected arthroplasties, periprosthetic activity was also present in 77% of uninfected arthroplasties, and was greater than the contralateral zone 51% of the time. When analyzed by zone, head zone activity was the best criterion for infection (87% sensitivity, 94% specificity, 92% accuracy). Fifty of the arthroplasties were studied with combined labeled leukocyte/sulfur colloid imaging. Using incongruence of images as the criterion for infection, the sensitivity, specificity, and accuracy of the study were 100%, 97%, and 98%, respectively. While variable periprosthetic activity makes labeled leukocyte imaging alone unreliable for diagnosing hip arthroplasty infection, the addition of sulfur colloid imaging results in a highly accurate diagnostic procedure.

  15. Small molecule binding sites on the Ras:SOS complex can be exploited for inhibition of Ras activation.

    PubMed

    Winter, Jon J G; Anderson, Malcolm; Blades, Kevin; Brassington, Claire; Breeze, Alexander L; Chresta, Christine; Embrey, Kevin; Fairley, Gary; Faulder, Paul; Finlay, M Raymond V; Kettle, Jason G; Nowak, Thorsten; Overman, Ross; Patel, S Joe; Perkins, Paula; Spadola, Loredana; Tart, Jonathan; Tucker, Julie A; Wrigley, Gail

    2015-03-12

    Constitutively active mutant KRas displays a reduced rate of GTP hydrolysis via both intrinsic and GTPase-activating protein-catalyzed mechanisms, resulting in the perpetual activation of Ras pathways. We describe a fragment screening campaign using X-ray crystallography that led to the discovery of three fragment binding sites on the Ras:SOS complex. The identification of tool compounds binding at each of these sites allowed exploration of two new approaches to Ras pathway inhibition by stabilizing or covalently modifying the Ras:SOS complex to prevent the reloading of Ras with GTP. Initially, we identified ligands that bound reversibly to the Ras:SOS complex in two distinct sites, but these compounds were not sufficiently potent inhibitors to validate our stabilization hypothesis. We conclude by demonstrating that covalent modification of Cys118 on Ras leads to a novel mechanism of inhibition of the SOS-mediated interaction between Ras and Raf and is effective at inhibiting the exchange of labeled GDP in both mutant (G12C and G12V) and wild type Ras.

  16. Discovering the enzyme mimetic activity of metal-organic framework (MOF) for label-free and colorimetric sensing of biomolecules.

    PubMed

    Wang, Ying; Zhu, Yingjing; Binyam, Atsebeha; Liu, Misha; Wu, Yinan; Li, Fengting

    2016-12-15

    A label-free sensing strategy based on the enzyme-mimicking activity of MOF was demonstrated for colorimetric detection of biomolecules. Firstly obvious blue color was observed due to the high efficiency of peroxidase-like catalytic activity of Fe-MIL-88A (an ion-based MOF material) toward 3,3',5,5'-tetramethylbenzidine (TMB). Then in the presence of target biomolecule and corresponding aptamer, the mimetic activity of Fe-MIL-88A can be strongly inhibited and used directly to realize the colorimetric detection. On the basis of the interesting findings, we designed a straightforward, label-free and sensitive colorimetric method for biomolecule detection by using the enzyme mimetic property of MOF coupling with molecular recognition element. Compared with the existed publications, our work breaks the routine way by setting up an inorganic-organic MOF-aptamer hybrid platform for colorimetric determination of biomolecules, expanding the targets scope from H2O2 or glucose to biomolecules. As a proof of concept, thrombin and thrombin aptamer was used as a model analyte. The limit of detection of 10nM can be achieved with naked eyes and ultrahigh selectivity of thrombin toward numerous interfering substances with 10-fold concentration was demonstrated significantly. Of note, the method was further applied for the detection of thrombin in human serum samples, showing the results in agreement with those values obtained in an immobilization buffer by the colorimetric method. This inorganic-organic MOF-aptamer sensing strategy may in principle be universally applicable for the detection of a range of environmental or biomedical molecules of interests. PMID:27419909

  17. Discovering the enzyme mimetic activity of metal-organic framework (MOF) for label-free and colorimetric sensing of biomolecules.

    PubMed

    Wang, Ying; Zhu, Yingjing; Binyam, Atsebeha; Liu, Misha; Wu, Yinan; Li, Fengting

    2016-12-15

    A label-free sensing strategy based on the enzyme-mimicking activity of MOF was demonstrated for colorimetric detection of biomolecules. Firstly obvious blue color was observed due to the high efficiency of peroxidase-like catalytic activity of Fe-MIL-88A (an ion-based MOF material) toward 3,3',5,5'-tetramethylbenzidine (TMB). Then in the presence of target biomolecule and corresponding aptamer, the mimetic activity of Fe-MIL-88A can be strongly inhibited and used directly to realize the colorimetric detection. On the basis of the interesting findings, we designed a straightforward, label-free and sensitive colorimetric method for biomolecule detection by using the enzyme mimetic property of MOF coupling with molecular recognition element. Compared with the existed publications, our work breaks the routine way by setting up an inorganic-organic MOF-aptamer hybrid platform for colorimetric determination of biomolecules, expanding the targets scope from H2O2 or glucose to biomolecules. As a proof of concept, thrombin and thrombin aptamer was used as a model analyte. The limit of detection of 10nM can be achieved with naked eyes and ultrahigh selectivity of thrombin toward numerous interfering substances with 10-fold concentration was demonstrated significantly. Of note, the method was further applied for the detection of thrombin in human serum samples, showing the results in agreement with those values obtained in an immobilization buffer by the colorimetric method. This inorganic-organic MOF-aptamer sensing strategy may in principle be universally applicable for the detection of a range of environmental or biomedical molecules of interests.

  18. Labeled nucleotide phosphate (NP) probes

    SciTech Connect

    Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

    2009-02-03

    The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

  19. Site-specific protein backbone and side-chain NMR chemical shift and relaxation analysis of human vinexin SH3 domain using a genetically encoded {sup 15}N/{sup 19}F-labeled unnatural amino acid

    SciTech Connect

    Shi, Pan; Xi, Zhaoyong; Wang, Hu; Shi, Chaowei; Xiong, Ying; Tian, Changlin

    2010-11-19

    Research highlights: {yields} Chemical synthesis of {sup 15}N/{sup 19}F-trifluomethyl phenylalanine. {yields} Site-specific incorporation of {sup 15}N/{sup 19}F-trifluomethyl phenylalanine to SH3. {yields} Site-specific backbone and side chain chemical shift and relaxation analysis. {yields} Different internal motions at different sites of SH3 domain upon ligand binding. -- Abstract: SH3 is a ubiquitous domain mediating protein-protein interactions. Recent solution NMR structural studies have shown that a proline-rich peptide is capable of binding to the human vinexin SH3 domain. Here, an orthogonal amber tRNA/tRNA synthetase pair for {sup 15}N/{sup 19}F-trifluoromethyl-phenylalanine ({sup 15}N/{sup 19}F-tfmF) has been applied to achieve site-specific labeling of SH3 at three different sites. One-dimensional solution NMR spectra of backbone amide ({sup 15}N){sup 1}H and side-chain {sup 19}F were obtained for SH3 with three different site-specific labels. Site-specific backbone amide ({sup 15}N){sup 1}H and side-chain {sup 19}F chemical shift and relaxation analysis of SH3 in the absence or presence of a peptide ligand demonstrated different internal motions upon ligand binding at the three different sites. This site-specific NMR analysis might be very useful for studying large-sized proteins or protein complexes.

  20. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of... conduct of site characterization activities at the Yucca Mountain site, DOE shall report the nature and... activities at the Yucca Mountain site, NRC staff shall be permitted to visit and inspect the locations...

  1. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of... conduct of site characterization activities at the Yucca Mountain site, DOE shall report the nature and... activities at the Yucca Mountain site, NRC staff shall be permitted to visit and inspect the locations...

  2. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of... conduct of site characterization activities at the Yucca Mountain site, DOE shall report the nature and... activities at the Yucca Mountain site, NRC staff shall be permitted to visit and inspect the locations...

  3. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of... conduct of site characterization activities at the Yucca Mountain site, DOE shall report the nature and... activities at the Yucca Mountain site, NRC staff shall be permitted to visit and inspect the locations...

  4. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of... conduct of site characterization activities at the Yucca Mountain site, DOE shall report the nature and... activities at the Yucca Mountain site, NRC staff shall be permitted to visit and inspect the locations...

  5. Site-specific scFv labelling with invertase via Sortase A mechanism as a platform for antibody-antigen detection using the personal glucose meter

    PubMed Central

    Ismail, Nur Faezee; Lim, Theam Soon

    2016-01-01

    Antibody labelling to reporter molecules is gaining popularity due to its many potential applications for diagnostics and therapeutics. However, non-directional bioconjugation methods which are commonly used often results in the loss of target binding capabilities. Therefore, a site-specific enzymatic based bioconjugation such as sortase-mediated transpeptidation allows for a more rapid and efficient method of antibody conjugation for diagnostic applications. Here we describe the utilization of sortase A bioconjugation to conjugate a single chain fragment variable (scFv) to the extracellular invertase (invB) from Zymomonas mobilis with the aim of developing an invertase based immunoassay. In addition, conjugation to enhanced green fluorescent protein (eGFP) was also validated to show the flexibility of the method. The invertase conjugated complex was successfully applied for the detection of antibody-antigen interaction using a personal glucose meter (PGM) for assay readout. The setup was used in both a direct and competitive assay highlighting the robustness of the conjugate for assay development. The method provides an alternative conjugation process to allow easy exchange of antibodies to facilitate rapid development of diagnostic assays for various diseases on the PGM platform. PMID:26782912

  6. Site-specific scFv labelling with invertase via Sortase A mechanism as a platform for antibody-antigen detection using the personal glucose meter.

    PubMed

    Ismail, Nur Faezee; Lim, Theam Soon

    2016-01-01

    Antibody labelling to reporter molecules is gaining popularity due to its many potential applications for diagnostics and therapeutics. However, non-directional bioconjugation methods which are commonly used often results in the loss of target binding capabilities. Therefore, a site-specific enzymatic based bioconjugation such as sortase-mediated transpeptidation allows for a more rapid and efficient method of antibody conjugation for diagnostic applications. Here we describe the utilization of sortase A bioconjugation to conjugate a single chain fragment variable (scFv) to the extracellular invertase (invB) from Zymomonas mobilis with the aim of developing an invertase based immunoassay. In addition, conjugation to enhanced green fluorescent protein (eGFP) was also validated to show the flexibility of the method. The invertase conjugated complex was successfully applied for the detection of antibody-antigen interaction using a personal glucose meter (PGM) for assay readout. The setup was used in both a direct and competitive assay highlighting the robustness of the conjugate for assay development. The method provides an alternative conjugation process to allow easy exchange of antibodies to facilitate rapid development of diagnostic assays for various diseases on the PGM platform. PMID:26782912

  7. Enzyme immunoassay of benzyl penicilloyl (BPO) groups using acetylcholinesterase as label. Application to the study of the BPO-binding sites on albumin.

    PubMed

    Wal, J M; Yvon, M; Pradelles, P; Grassi, J

    1991-01-01

    Benzyl penicilloyl groups (BPO) derive from penicillin G by cleavage of the beta lactam ring; they covalently bind to proteins to give conjugates which have lost all antibiotic properties but are considered as the major allergenic determinants in penicillin allergy. A solid-phase Enzyme Immuno Assay (EIA) of BPO groups in different biological fluids is described. It is a competitive immunoassay using acetylcholinesterase as label. In all biological fluids, very low non-specific binding values are observed. The sensitivity and the precision of the assay are good since ca. 0.5 ng/ml can be measured with a coefficient of variation less than 10%. Cross reactions between BPO and penicillin or penicillin derivatives are nil or very low. This assay is more sensitive, much more rapid and easier to handle than the other methods available and is thus suitable for routine determinations. In association with reversed-phase high performance liquid chromatography this EIA has allowed an initial investigation of the location of BPO-binding sites on micro quantities of serum albumin (ca. 1 mg) from penicillin treated patients.

  8. Development of an efficient signal amplification strategy for label-free enzyme immunoassay using two site-specific biotinylated recombinant proteins.

    PubMed

    Tang, Jin-Bao; Tang, Ying; Yang, Hong-Ming

    2015-02-15

    Constructing a recombinant protein between a reporter enzyme and a detector protein to produce a homogeneous immunological reagent is advantageous over random chemical conjugation. However, the approach hardly recombines multiple enzymes in a difunctional fusion protein, which results in insufficient amplification of the enzymatic signal, thereby limiting its application in further enhancement of analytical signal. In this study, two site-specific biotinylated recombinant proteins, namely, divalent biotinylated alkaline phosphatase (AP) and monovalent biotinylated ZZ domain, were produced by employing the Avitag-BirA system. Through the high streptavidin (SA)-biotin interaction, the divalent biotinylated APs were clustered in the SA-biotin complex and then incorporated with the biotinylated ZZ. This incorporation results in the formation of a functional macromolecule that involves numerous APs, thereby enhancing the enzymatic signal, and in the production of several ZZ molecules for the interaction with immunoglobulin G (IgG) antibody. The advantage of this signal amplification strategy is demonstrated through ELISA, in which the analytical signal was substantially enhanced, with a 32-fold increase in the detection sensitivity compared with the ZZ-AP fusion protein approach. The proposed immunoassay without chemical modification can be an alternative strategy to enhance the analytical signals in various applications involving immunosensors and diagnostic chips, given that the label-free IgG antibody is suitable for the ZZ protein.

  9. Site-directed fluorescence labeling reveals a revised N-terminal membrane topology and functional periplasmic residues in the Escherichia coli cell division protein FtsK.

    PubMed

    Berezuk, Alison M; Goodyear, Mara; Khursigara, Cezar M

    2014-08-22

    In Escherichia coli, FtsK is a large integral membrane protein that coordinates chromosome segregation and cell division. The N-terminal domain of FtsK (FtsKN) is essential for division, and the C terminus (FtsKC) is a well characterized DNA translocase. Although the function of FtsKN is unknown, it is suggested that FtsK acts as a checkpoint to ensure DNA is properly segregated before septation. This may occur through modulation of protein interactions between FtsKN and other division proteins in both the periplasm and cytoplasm; thus, a clear understanding of how FtsKN is positioned in the membrane is required to characterize these interactions. The membrane topology of FtsKN was initially determined using site-directed reporter fusions; however, questions regarding this topology persist. Here, we report a revised membrane topology generated by site-directed fluorescence labeling. The revised topology confirms the presence of four transmembrane segments and reveals a newly identified periplasmic loop between the third and fourth transmembrane domains. Within this loop, four residues were identified that, when mutated, resulted in the appearance of cellular voids. High resolution transmission electron microscopy of these voids showed asymmetric division of the cytoplasm in the absence of outer membrane invagination or visible cell wall ingrowth. This uncoupling reveals a novel role for FtsK in linking cell envelope septation events and yields further evidence for FtsK as a critical checkpoint of cell division. The revised topology of FtsKN also provides an important platform for future studies on essential interactions required for this process.

  10. Site-directed Fluorescence Labeling Reveals a Revised N-terminal Membrane Topology and Functional Periplasmic Residues in the Escherichia coli Cell Division Protein FtsK*

    PubMed Central

    Berezuk, Alison M.; Goodyear, Mara; Khursigara, Cezar M.

    2014-01-01

    In Escherichia coli, FtsK is a large integral membrane protein that coordinates chromosome segregation and cell division. The N-terminal domain of FtsK (FtsKN) is essential for division, and the C terminus (FtsKC) is a well characterized DNA translocase. Although the function of FtsKN is unknown, it is suggested that FtsK acts as a checkpoint to ensure DNA is properly segregated before septation. This may occur through modulation of protein interactions between FtsKN and other division proteins in both the periplasm and cytoplasm; thus, a clear understanding of how FtsKN is positioned in the membrane is required to characterize these interactions. The membrane topology of FtsKN was initially determined using site-directed reporter fusions; however, questions regarding this topology persist. Here, we report a revised membrane topology generated by site-directed fluorescence labeling. The revised topology confirms the presence of four transmembrane segments and reveals a newly identified periplasmic loop between the third and fourth transmembrane domains. Within this loop, four residues were identified that, when mutated, resulted in the appearance of cellular voids. High resolution transmission electron microscopy of these voids showed asymmetric division of the cytoplasm in the absence of outer membrane invagination or visible cell wall ingrowth. This uncoupling reveals a novel role for FtsK in linking cell envelope septation events and yields further evidence for FtsK as a critical checkpoint of cell division. The revised topology of FtsKN also provides an important platform for future studies on essential interactions required for this process. PMID:25002583

  11. Proliferative activity of adrenal glands with adrenocortical cytomegaly measured by MIB-1 labeling index.

    PubMed

    Fasano, M; Greco, M A

    1996-01-01

    To investigate the proliferative activity of cytomegalic cells in the fetal adrenal cortex, we studied adrenal glands with cytomegaly by immunohistochemistry using the nuclear proliferation maker MIB-1. The percentage of positively stained nuclei was quantified using the SAMBA 4000 image analysis system. Only one case showed occasional positively stained cytomegalic cell nuclei. The permanent cortices showed proliferative activity that decreased with increasing gestational age. No proliferative activity was seen in normal fetal cortices except in one case that received corticosteroid therapy and had a maternal history of diabetes. The near absence of proliferative activity of the cytomegalic cells supports the previously proposed theory of cellular exhaustion following hyperactivity. The high proliferative activity in the fetal cortex of the infant receiving corticosteroid therapy may provide insight into the stimulus causing the hypermetabolic state. PMID:9025875

  12. A single-label phenylpyrrolocytidine provides a molecular beacon-like response reporting HIV-1 RT RNase H activity.

    PubMed

    Wahba, Alexander S; Esmaeili, Abbasali; Damha, Masad J; Hudson, Robert H E

    2010-01-01

    6-Phenylpyrrolocytidine (PhpC), a structurally conservative and highly fluorescent cytidine analog, was incorporated into oligoribonucleotides. The PhpC-containing RNA formed native-like duplex structures with complementary DNA or RNA. The PhpC-modification was found to act as a sensitive reporter group being non-disruptive to structure and the enzymatic activity of RNase H. A RNA/DNA hybrid possessing a single PhpC insert was an excellent substrate for HIV-1 RT Ribonuclease H and rapidly reported cleavage of the RNA strand with a 14-fold increase in fluorescence intensity. The PhpC-based assay for RNase H was superior to the traditional molecular beacon approach in terms of responsiveness, rapidity and ease (single label versus dual). Furthermore, the PhpC-based assay is amenable to high-throughput microplate assay format and may form the basis for a new screen for inhibitors of HIV-RT RNase H.

  13. GAS HYDRATES AT TWO SITES OF AN ACTIVE CONTINENTAL MARGIN.

    USGS Publications Warehouse

    Kvenvolden, K.A.

    1985-01-01

    Sediment containing gas hydrates from two distant Deep Sea Drilling Project sites (565 and 568), located about 670 km apart on the landward flank of the Middle America Trench, was studied to determine the geochemical conditions that characterize the occurrence of gas hydrates. Site 565 was located in the Pacific Ocean offshore the Nicoya Peninsula of Costa Rica in 3,111 m of water. The depth of the hole at this site was 328 m, and gas hydrates were recovered from 285 and 319 m. Site 568 was located about 670 km to the northwest offshore Guatemala in 2,031 m of water. At this site the hole penetrated to 418 m, and gas hydrates were encountered at 404 m.

  14. Control of active sites in selective flocculation: III -- Mechanism of site blocking

    SciTech Connect

    Behl, S.; Moudgil, B.M. . Dept. of Materials Science and Engineering)

    1993-12-01

    It has been shown in Parts I and II of this paper that heteroflocculation can be controlled by poisoning the sites for flocculant adsorption using a site blocking agent (SBA). An efficient SBA was determined to be the lower molecular weight fraction of the flocculant. In this paper, the underlying mechanism of SBA action is described. Also, the mathematical model detailed in Part I is used to determine the effect of different SBAs on apatite-dolomite separation efficiency. It has been demonstrated that the depression in flocculation is directly related to the site blocking parameter ([bar [Phi

  15. Dynamically achieved active site precision in enzyme catalysis.

    PubMed

    Klinman, Judith P

    2015-02-17

    CONSPECTUS: The grand challenge in enzymology is to define and understand all of the parameters that contribute to enzymes' enormous rate accelerations. The property of hydrogen tunneling in enzyme reactions has moved the focus of research away from an exclusive focus on transition state stabilization toward the importance of the motions of the heavy atoms of the protein, a role for reduced barrier width in catalysis, and the sampling of a protein conformational landscape to achieve a family of protein substates that optimize enzyme-substrate interactions and beyond. This Account focuses on a thermophilic alcohol dehydrogenase for which the chemical step of hydride transfer is rate determining across a wide range of experimental conditions. The properties of the chemical coordinate have been probed using kinetic isotope effects, indicating a transition in behavior below 30 °C that distinguishes nonoptimal from optimal C-H activation. Further, the introduction of single site mutants has the impact of either enhancing or eliminating the temperature dependent transition in catalysis. Biophysical probes, which include time dependent hydrogen/deuterium exchange and fluorescent lifetimes and Stokes shifts, have also been pursued. These studies allow the correlation of spatially resolved transitions in protein motions with catalysis. It is now possible to define a long-range network of protein motions in ht-ADH that extends from a dimer interface to the substrate binding domain across to the cofactor binding domain, over a distance of ca. 30 Å. The ongoing challenge to obtaining spatial and temporal resolution of catalysis-linked protein motions is discussed.

  16. Labeled content of two furanocoumarins in dietary supplements correlates with neither actual content nor CYP3A inhibitory activity.

    PubMed

    VanderMolen, Karen M; Ainslie, Garrett R; Paine, Mary F; Oberlies, Nicholas H

    2014-09-01

    Dietary supplements are a multi-billion dollar business, with yearly profit increases. Allegedly safe, these supplements are marketed to a variety of niches, encompassing claims from immune support to weight loss. Six sports nutrition supplements were acquired that were labeled to contain the furanocoumarin(s) bergamottin and/or 6',7'-dihydroxybergamottin (DHB), both of which are potent irreversible inhibitors of the prominent drug metabolizing enzyme cytochrome P450 3A (CYP3A). Both furanocoumarins are typically present in grapefruit juice, which has been shown to inhibit intestinal CYP3A, perpetrating an increase in the systemic exposure of certain concomitant 'victim' drugs. The acquired supplements were analyzed using ultra-performance liquid chromatography coupled to both a photodiode array (PDA) detector and a triple quadrupole mass spectrometer (MS). Contrary to the product labeling, four of the supplements contained no detectable quantities of either furanocoumarin (LOD 0.060μg/capsule), while two of the supplements contained minimal amounts (one contained 12.13 (±0.23) μg bergamottin and 65.51 (±0.64) μg DHB per capsule; the other contained 2.705 (±0.069) μg bergamottin per capsule and no detectable quantities of DHB). A CYP3A inhibition bioassay was used to assess whether the actual content of the furanocoumarins correlated with CYP3A inhibitory activity. Despite the low amounts of bergamottin and DHB, CYP3A inhibition by the supplements was greater than could be accounted for by the two furanocoumarins. The additional activity suggests the presence of other potent or highly abundant CYP3A inhibitors.

  17. Labeled Content of Two Furanocoumarins in Dietary Supplements Correlates with neither Actual Content nor CYP3A Inhibitory Activity

    PubMed Central

    VanderMolen, Karen M.; Ainslie, Garrett R.; Paine, Mary F.; Oberlies, Nicholas H.

    2014-01-01

    Dietary supplements are a multi-billion dollar business, with yearly profit increases. Allegedly safe, these supplements are marketed to a variety of niches, encompassing claims from immune support to weight loss. Six sports nutrition supplements were acquired that were labeled to contain the furanocoumarin(s) bergamottin and/or 6′,7′-dihydroxybergamottin (DHB), both of which are potent irreversible inhibitors of the prominent drug metabolizing enzyme cytochrome P450 3A (CYP3A). Both furanocoumarins are typically present in grapefruit juice, which has been shown to inhibit intestinal CYP3A, perpetrating an increase in the systemic exposure of certain concomitant ‘victim’ drugs. The acquired supplements were analyzed using ultra-performance liquid chromatography coupled to both a photodiode array (PDA) detector and a triple quadrupole mass spectrometer (MS). Contrary to the product labeling, four of the supplements contained no detectable quantities of either furanocoumarin (LOD 0.060 μg/capsule), while two of the supplements contained minimal amounts (one contained 12.13 (± 0.23) μg bergamottin and 65.51 (± 0.64) μg DHB per capsule; the other contained 2.705 (± 0.069) μg bergamottin per capsule and no detectable quantities of DHB). A CYP3A inhibition bioassay was used to assess whether the actual content of the furanocoumarins correlated with CYP3A inhibitory activity. Despite the low amounts of bergamottin and DHB, CYP3A inhibition by the supplements was greater than could be accounted for by the two furanocoumarins. The additional activity suggests the presence of other potent or highly abundant CYP3A inhibitors. PMID:24951959

  18. Lethal Factor Active-Site Mutations Affect Catalytic Activity In Vitro

    PubMed Central

    Hammond, S. E.; Hanna, P. C.

    1998-01-01

    The lethal factor (LF) protein of Bacillus anthracis lethal toxin contains the thermolysin-like active-site and zinc-binding consensus motif HEXXH (K. R. Klimpel, N. Arora, and S. H. Leppla, Mol. Microbiol. 13:1093–1100, 1994). LF is hypothesized to act as a Zn2+ metalloprotease in the cytoplasm of macrophages, but no proteolytic activities have been previously shown on any target substrate. Here, synthetic peptides are hydrolyzed by LF in vitro. Mass spectroscopy and peptide sequencing of isolated cleavage products separated by reverse-phase high-pressure liquid chromatography indicate that LF seems to prefer proline-containing substrates. Substitution mutations within the consensus active-site residues completely abolish all in vitro catalytic functions, as does addition of 1,10-phenanthroline, EDTA, and certain amino acid hydroxamates, including the novel zinc metalloprotease inhibitor ZINCOV. In contrast, the protease inhibitors bestatin and lysine CMK, previously shown to block LF activity on macrophages, did not block LF activity in vitro. These data provide the first direct evidence that LF may act as an endopeptidase. PMID:9573135

  19. Monoclonal antibody against the active site of caeruloplasmin and the ELISA system detecting active caeruloplasmin.

    PubMed

    Hiyamuta, S; Ito, K

    1994-04-01

    Serum caeruloplasmin deficiency is a characteristic biochemical abnormality found in patients with Wilson's disease, but the mechanism of this disease is unknown. Although the phenylenediamine oxidase activity of serum caeruloplasmin is markedly low in patients with Wilson's disease, mRNA of caeruloplasmin exists to some extent. To investigate the deficiency of caeruloplasmin oxidase activity in Wilson's disease, we generated 14 monoclonal antibodies (MAbs) and selected ID1, which had the strongest reactivity, and ID2, which had neutralizing ability. We also established a system to measure active caeruloplasmin specifically using these MAbs. These MAbs and the system will be useful tools in analyzing the active site of caeruloplasmin in patients with Wilson's disease.

  20. A label-free and sensitive fluorescent assay for one step detection of protein kinase activity and inhibition.

    PubMed

    Wang, Lei; Yan, Xu; Su, Xingguang

    2016-09-01

    In this paper, a label-free, highly sensitive and simple assay for one step detection of protein kinase (PKA) activity and inhibition that avoids the fluorescent dye process has been established. The detection was based on the fluorescence (FL) quenching of peptide-Ag nanoclusters (Ag NCs) caused by antibody modified Au nanoparticles (anti-Au NPs) via fluorescence resonance energy transfer (FRET). With PKA and adenosine 5'-triphosphate (ATP) introduced, the substrate peptide of Ag NCs could react with PKA via targeted phosphorylation, and followed by the linking interactions between peptide-Ag NCs and anti-Au NPs. According to the fluorescence quenching of Ag NCs, the activity of protein kinase can be facilely monitored in the range of 0.1-2000 mU/μL with high sensitivity. The detection limit for PKA is 0.039 mU/μL. We further explored the inhibitory effect of H-89 for protein kinase activity. The developed method was also applied to the investigation of drug-induced PKA activation in HeLa cells, which provides a promising means for screening of kinase-related drugs and the clinical diagnosis of disease. PMID:27543031

  1. Scintigraphic assessment of indium-111-labeled granulocyte splenic pooling: A new approach to inflammatory bowel disease activity

    SciTech Connect

    Loreal, O.; Moisan, A.; Bretagne, J.F.; LeCloirec, J.; Raoul, J.L.; Gastard, J.; Herry, J.Y. )

    1990-09-01

    We have conducted a prospective study into the sensitivity and the specificity of the fall in splenic activity (FSA) as an index of activity in inflammatory bowel disease (IBD). FSA was measured on scintiscans obtained at 3 and 24 hr postinjection of indium-111-labeled granulocytes. One hundred and twenty-two scans were acquired in 96 patients who were divided into six groups: Gr. I = normal volunteers (n = 10); Gr. II = inflammatory rheumatism (n = 10); Gr. III = abscesses (n = 17); Gr. IV = ulcerative colitis (UC: n = 23); Gr. V = colonic Crohn's disease (CCD: n = 22); and Gr. VI = ileal Crohn's disease (ICD: n = 14). FSA for Groups I and II was constantly below 10%, but it was increased in the other four groups (abscesses: 39% +/- 12%; UC: 35% +/- 13.5%; CCD: 23.7% +/- 14.7%; ICD: 21.5% +/- 11.7%). There was a significant correlation between fecal excretion of 111In (FEI) and FSA in patients with IBD (UC: r = 0.71, p less than 0.001; CCD: r = 0.74, p less than 0.001; ICD: r = 0.43, p less than 0.001). FSA was followed in 16 patients with IBD after medical treatment and there was a significant correlation between variations in FSA and in FEI (r = 0.879, p less than 0.001). FSA is a very sensitive although nonspecific index of disease activity in IBD and may replace FEI in the assessment of IBD activity.

  2. Robotics and Automation Activities at the Savannah River Site: A Site Report for SUBWOG 39F

    SciTech Connect

    Teese, G.D.

    1995-09-28

    The Savannah River Site has successfully used robots, teleoperators, and remote video to reduce exposure to ionizing radiation, improve worker safety, and improve the quality of operations. Previous reports have described the use of mobile teleoperators in coping with a high level liquid waste spill, the removal of highly contaminated equipment, and the inspection of nuclear reactor vessels. This report will cover recent applications at the Savannah River, as well as systems which SRS has delivered to other DOE site customers.

  3. Control of active sites in selective flocculation: II -- Role of site blocking agents

    SciTech Connect

    Behl, S.; Moudgil, B.M. . Dept. of Materials Science and Engineering)

    1993-12-01

    Control of heteroflocculation using a lower molecular weight fraction of the flocculant as a site blocking agent is demonstrated in the apatite-dolomite-polyethylene oxide system. The most effective SBA (site blocking agent) was determined to be the highest molecular weight fraction of the flocculant itself which was not capable of flocculating any of the components of the mixture. In the presence of the SBA, flocculant adsorption decreased significantly on apatite particles, thereby inhibiting coflocculation.

  4. Active-site mutations of diphtheria toxin: effects of replacing glutamic acid-148 with aspartic acid, glutamine, or serine.

    PubMed

    Wilson, B A; Reich, K A; Weinstein, B R; Collier, R J

    1990-09-18

    Glutamic acid-148, an active-site residue of diphtheria toxin identified by photoaffinity labeling with NAD, was replaced with aspartic acid, glutamine, or serine by directed mutagenesis of the F2 fragment of the toxin gene. Wild-type and mutant F2 proteins were synthesized in Escherichia coli, and the corresponding enzymic fragment A moieties (DTA) were derived, purified, and characterized. The Glu----Asp (E148D), Glu----Gln (E148Q), and Glu----Ser (E148S) mutations caused reductions in NAD:EF-2 ADP-ribosyltransferase activity of ca. 100-, 250-, and 300-fold, respectively, while causing only minimal changes in substrate affinity. The effects of the mutations on NAD-glycohydrolase activity were considerably different; only a 10-fold reduction in activity was observed for E148S, and the E148D and E148Q mutants actually exhibited a small but reproducible increase in NAD-glycohydrolytic activity. Photolabeling by nicotinamide-radiolabeled NAD was diminished ca. 8-fold in the E148D mutant and was undetectable in the other mutants. The results confirm that Glu-148 plays a crucial role in the ADP-ribosylation of EF-2 and imply an important function for the side-chain carboxyl group in catalysis. The carboxyl group is also important for photochemical labeling by NAD but not for NAD-glycohydrolase activity. The pH dependence of the catalytic parameters for the ADP-ribosyltransferase reaction revealed a group in DTA-wt that titrates with an apparent pKa of 6.2-6.3 and is in the protonated state in the rate-determining step.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. Nutrition Labeling

    NASA Astrophysics Data System (ADS)

    Metzger, Lloyd E.

    Nutrition labeling regulations differ in countries around the world. The focus of this chapter is on nutrition labeling regulations in the USA, as specified by the Food and Drug Administration (FDA) and the Food Safety and Inspection Service (FSIS) of the United States Department of Agriculture (USDA). A major reason for analyzing the chemical components of foods in the USA is nutrition labeling regulations. Nutrition label information is not only legally required in many countries, but also is of increasing importance to consumers as they focus more on health and wellness.

  6. Specific affinity-labeling of the nociceptin ORL1 receptor using a thiol-activated Cys(Npys)-containing peptide ligand.

    PubMed

    Matsushima, Ayami; Nishimura, Hirokazu; Matsuyama, Yutaka; Liu, Xiaohui; Costa, Tommaso; Shimohigashi, Yasuyuki

    2016-11-01

    We previously showed that an antagonist-based peptide ligand, H-Cys(Npys)-Arg-Tyr-Tyr-Arg- Ile-Lys-NH2 , captures the free thiol groups in the ligand-binding site of the nociceptin receptor ORL1. However, the exact receptor sites of this thiol-disulfide exchange reaction have not been uncovered, although such identification would help to clarify the ligand recognition site. Since the Cys→Ala substitution prevents the reaction, we performed the so-called Ala scanning for all the Cys residues in the transmembrane (TM) domains of the ORL1 receptor. Seven different mutant receptors were soundly expressed in the COS-7 cells and examined for their specific affinity labeling by a competitive binding assay using nociceptin and [(3) H]nociceptin. The results of in vitro Ala scanning analyses revealed that the labeled residues were Cys59 in TM1, Cys215 and Cys231 in TM5, and Cys310 in TM7. The present study has provided a novel method of Cys(Npys)-affinity labeling for identification of the ligand-binding sites in the ORL1 receptor. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 460-469, 2016.

  7. Activity-based labeling of matrix metalloproteinases in living vertebrate embryos.

    PubMed

    Keow, Jonathan Y; Pond, Eric D; Cisar, Justin S; Cravatt, Benjamin F; Crawford, Bryan D

    2012-01-01

    Extracellular matrix (ECM) remodeling is a physiologically and developmentally essential process mediated by a family of zinc-dependent extracellular proteases called matrix metalloproteinases (MMPs). In addition to complex transcriptional control, MMPs are subject to extensive post-translational regulation. Because of this, classical biochemical, molecular and histological techniques that detect the expression of specific gene products provide useful but limited data regarding the biologically relevant activity of MMPs. Using benzophenone-bearing hydroxamate-based probes that interact with the catalytic zinc ion in MMPs, active proteases can be covalently 'tagged' by UV cross-linking. This approach has been successfully used to tag MMP-2 in vitro in tissue culture supernatants, and we show here that this probe tags proteins with mobilities consistent with known MMPs and detectable gelatinolytic activity in homogenates of zebrafish embryos. Furthermore, because of the transparency of the zebrafish embryo, UV-photocroslinking can be accomplished in vivo, and rhodamated benzophenone probe is detected in striking spatial patterns consistent with known distributions of active matrix remodeling in embryos. Finally, in metamorphosing Xenopus tadpoles, this probe can be used to biotinylate active MMP-2 by injecting it and cross-linking it in vivo, allowing the protein to be subsequently extracted and biochemically identified. PMID:22952682

  8. Spin-labeled polyribonucleotides.

    PubMed Central

    Petrov, A I; Sukhorukov, B I

    1980-01-01

    Poly (U), poly (C) and poly (A) were spin labeled with N-(2,2,5,5-tetramethyl-3-carbonylpyrroline-1-oxyl)-imidazole. This spin label interacts selectively with 2' OH ribose groups of polynucleotides and does not modify the nucleic acid bases. The extent of spin labeling is not dependent upon the nature of the base and is entirely determined by rigidity of the secondary structure of the polynucleotide. The extent of modification for poly (U), poly (C) and poly (A) was 4.2, 1.7 and 1.5 per cent, respectively, the secondary structure of the polynucleotides being practically unchanged. Some physico-chemical properties of the spin-labeled polynucleotides were investigated by ESR spectroscopy. Rotational correlation times of the spin label and activation energy of its motion were calculated. PMID:6253911

  9. Diurnal feeding rhythms in north sea copepods measured by Gut fluorescence, digestive enzyme activity and grazing on labelled food

    NASA Astrophysics Data System (ADS)

    Baars, M. A.; Oosterhuis, S. S.

    Results obtained with three methods for measuring feeding rhythms of copepods were different. Gut fluorescence showed clear day-night variation during 2 out of 3 cruises at the Oyster Ground in the North Sea. The species studied ( Pseudocalanus, Temora, Centropages, Calanus) had highest gut fluorescence during the night in May and September, the larger species demonstrating the largest difference. Gut fluorescence was positively correlated with ambient chlorophyll concentrations. Gut clearance rate was not dependent on temperature but on gut fullness. Gut passage times at high gut fluorescence levels were ˜25 minutes, at low levels 2 hours. In grazing experiments with 14C labelled food, filtering rates declined after 5 to 15 minutes, presumably before the first defecation of radioactive material. Filtering rates in Temora were higher at night than by day during May and July, but not in Pseudocalanus and Calanus during September. No diurnal pattern of amylase and tryptic activity was found, except in July for amylase but then probably due to vertical migration. The activity of these digestive enzymes appeared to be least and gut fluorescence most suitable for the detection of grazing rhythms. The occurrence of high fluorescence levels at night in all species studied suggests that intermittent feeding by copepods on phytoplankton is a general phenomenon from spring to autumn. The increase in foraging activity appeared to start well before complete darkness, during May and July even one hour or more before sunset.

  10. 21 CFR 347.60 - Labeling of permitted combinations of active ingredients.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...) Combinations of skin protectant and sunscreen active ingredients in § 347.20(d). In addition to any or all of... sunscreen drug products should be used and any or all of the additional indications for sunscreen drug... a skin protectant and a sunscreen identified in §§ 347.20(d) and 352.20(b). The warnings...

  11. 21 CFR 347.60 - Labeling of permitted combinations of active ingredients.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...) Combinations of skin protectant and sunscreen active ingredients in § 347.20(d). In addition to any or all of... sunscreen drug products should be used and any or all of the additional indications for sunscreen drug... a skin protectant and a sunscreen identified in §§ 347.20(d) and 352.20(b). The warnings...

  12. 21 CFR 347.60 - Labeling of permitted combinations of active ingredients.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...) Combinations of skin protectant and sunscreen active ingredients in § 347.20(d). In addition to any or all of... sunscreen drug products should be used and any or all of the additional indications for sunscreen drug... a skin protectant and a sunscreen identified in §§ 347.20(d) and 352.20(b). The warnings...

  13. 21 CFR 347.60 - Labeling of permitted combinations of active ingredients.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...) Combinations of skin protectant and sunscreen active ingredients in § 347.20(d). In addition to any or all of... sunscreen drug products should be used and any or all of the additional indications for sunscreen drug... a skin protectant and a sunscreen identified in §§ 347.20(d) and 352.20(b). The warnings...

  14. 21 CFR 347.60 - Labeling of permitted combinations of active ingredients.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ...) Combinations of skin protectant and sunscreen active ingredients in § 347.20(d). In addition to any or all of... sunscreen drug products should be used and any or all of the additional indications for sunscreen drug... a skin protectant and a sunscreen identified in §§ 347.20(d) and 352.20(b). The warnings...

  15. A label-free electrochemical strategy for highly sensitive methyltransferase activity assays.

    PubMed

    Zhou, Jiawan; Zhang, Xiaohua; Xiong, Erhu; Yu, Peng; Chen, Jinhua

    2015-03-25

    A new electrochemical strategy for the simple and ultrasensitive evaluation of DNA methyltransferase (MTase) activity was developed based on electrocatalytic oxidation of ascorbic acid by graphene. In addition, the suitability of this sensing platform for MTase inhibitor screening was also demonstrated.

  16. Metabolism and excretion kinetics of {sup 14}C-labeled and non-labeled difloxacin in pigs after oral administration, and antimicrobial activity of manure containing difloxacin and its metabolites

    SciTech Connect

    Sukul, Premasis; Lamshoeft, Marc; Kusari, Souvik; Zuehlke, Sebastian; Spiteller, Michael

    2009-04-15

    Fluoroquinolones are amongst the most important antibiotics used in veterinary medicine. On this account the behavior of difloxacin (DIF) and its metabolites was investigated by administering the {sup 14}C-labeled and non-labeled veterinary drug to fattening pigs. The excretion kinetics were determined after daily collection of manure. Sarafloxacin (SAR) was found to be the major metabolite, three further trace metabolites were also recovered, applying high-resolution (HR) mass spectrometric technique. The identification of DIF and SAR was confirmed by comparison with the spectroscopic and chromatographic data of the authentic references. The identification of the three trace metabolites was performed by HR-MS/MS. Only 8.1% of the administered radioactivity remained in the pig after 10 days and DIF accounted for 95.9% of the radioactivity excreted. More than 99% of the labeled compounds were detected and identified in the manure. The mean recoveries for all single electrolytes were {>=}94%. Linearity was established over concentration range 10-10,000 {mu}g/kg manure with a correlation coefficient {>=}0.99. By using in vitro antimicrobial activity tests against a group of standard pathogenic control strains, the results showed that the residual antibiotic concentrations in the manure of pigs are high enough to exhibit antibacterial activity.

  17. Mutation at a Strictly Conserved, Active Site Tyrosine in the Copper Amine Oxidase Leads to Uncontrolled Oxygenase Activity

    SciTech Connect

    Chen, Zhi-wei; Datta, Saumen; DuBois, Jennifer L.; Klinman, Judith P.; Mathews, F. Scott

    2010-09-07

    The copper amine oxidases carry out two copper-dependent processes: production of their own redox-active cofactor (2,4,5-trihydroxyphenylalanine quinone, TPQ) and the subsequent oxidative deamination of substrate amines. Because the same active site pocket must facilitate both reactions, individual active site residues may serve multiple roles. We have examined the roles of a strictly conserved active site tyrosine Y305 in the copper amine oxidase from Hansenula polymorpha kinetically, spetroscopically (Dubois and Klinman (2006) Biochemistry 45, 3178), and, in the present work, structurally. While the Y305A enzyme is almost identical to the wild type, a novel, highly oxygenated species replaces TPQ in the Y305F active sites. This new structure not only provides the first direct detection of peroxy intermediates in cofactor biogenesis but also indicates the critical control of oxidation chemistry that can be conferred by a single active site residue.

  18. A [Cu2O]2+ core in Cu-ZSM-5, the active site in the oxidation of methane to methanol

    PubMed Central

    Woertink, Julia S.; Smeets, Pieter J.; Groothaert, Marijke H.; Vance, Michael A.; Sels, Bert F.; Schoonheydt, Robert A.; Solomon, Edward I.

    2009-01-01

    Driven by the depletion of crude oil, the direct oxidation of methane to methanol has been of considerable interest. Promising low-temperature activity of an oxygen-activated zeolite, Cu-ZSM-5, has recently been reported in this selective oxidation and the active site in this reaction correlates with an absorption feature at 22,700 cm−1. In the present study, this absorption band is used to selectively resonance enhance Raman vibrations of this active site. 18O2 labeling experiments allow definitive assignment of the observed vibrations and exclude all previously characterized copper-oxygen species for the active site. In combination with DFT and normal coordinate analysis calculations, the oxygen activated Cu core is uniquely defined as a bent mono-(μ-oxo)dicupric site. Spectroscopically validated electronic structure calculations show polarization of the low-lying singly-occupied molecular orbital of the [Cu2O]2+ core, which is directed into the zeolite channel, upon approach of CH4. This induces significant oxyl character into the bridging O atom leading to a low transition state energy consistent with experiment and explains why the bent mono-(μ-oxo)dicupric core is highly activated for H atom abstraction from CH4. The oxygen intermediate of Cu-ZSM-5 is now the most well defined species active in the methane monooxygenase reaction. PMID:19864626

  19. A [Cu2O]2+ core in Cu-ZSM-5, the active site in the oxidation of methane to methanol.

    PubMed

    Woertink, Julia S; Smeets, Pieter J; Groothaert, Marijke H; Vance, Michael A; Sels, Bert F; Schoonheydt, Robert A; Solomon, Edward I

    2009-11-10

    Driven by the depletion of crude oil, the direct oxidation of methane to methanol has been of considerable interest. Promising low-temperature activity of an oxygen-activated zeolite, Cu-ZSM-5, has recently been reported in this selective oxidation and the active site in this reaction correlates with an absorption feature at 22,700 cm(-1). In the present study, this absorption band is used to selectively resonance enhance Raman vibrations of this active site. (18)O(2) labeling experiments allow definitive assignment of the observed vibrations and exclude all previously characterized copper-oxygen species for the active site. In combination with DFT and normal coordinate analysis calculations, the oxygen activated Cu core is uniquely defined as a bent mono-(mu-oxo)dicupric site. Spectroscopically validated electronic structure calculations show polarization of the low-lying singly-occupied molecular orbital of the [Cu(2)O](2+) core, which is directed into the zeolite channel, upon approach of CH(4). This induces significant oxyl character into the bridging O atom leading to a low transition state energy consistent with experiment and explains why the bent mono-(mu-oxo)dicupric core is highly activated for H atom abstraction from CH(4). The oxygen intermediate of Cu-ZSM-5 is now the most well defined species active in the methane monooxygenase reaction.

  20. Gastric ulcer localization: Potential use of in vivo labeling

    SciTech Connect

    Pera, A.; Rose, H.; Seavers, R.; Bekerman, C.; Pinsky, S.

    1984-01-01

    A previous work suggests that sucralfate labeled by binding to Tc-99m HSA permits the visualization of gastric ulcers. Potential problems with this technique are: 1) decreased binding of sucralfate to ulcer sites due to the labeling method of binding to exogenous protein (HSA); 2) overlying activity that may obscure identification of the ulcer. Because of these problems we have examined the possibility of direct in vivo Tc-99m labeling of sucralfate after it has already bound to the ulcer. In vitro studies were done to determine the binding of Tc-99m pertechnetate to sucralfate in the presence of tin in HCl solution at pHs comparable to those found in the stomach. Rapid and efficient labeling was achieved with 75-95% of the label bound to sucralfate at 30 minutes. In vivo studies were performed in rabbits with aspirin induced ulcers and in ulcer free human volunteers. The animal studies confirm that orally administered Tc-99m pertechnetate will bind to previously ingested sucralfate and that the labeled material will bind to the ulcers. Tc-99m pertechnetate was also shown to bind well to previously ingested sucralfate in humans. The results suggest that it is possible to label sucralfate in vivo. This method would offer the following advantages: 1) a simpler labeling procedure; 2) the potential of increased sensitivity by delaying the labeling until much of the sucralfate not bound to ulcer has passed, and thus decreasing the activity that remains in the stomach; and also by leaving the protein binding sites of the sucralfate free to interact with the ulcer since no exogenous protein is involved in labeling.

  1. Identification of promiscuous ene-reductase activity by mining structural databases using active site constellations

    PubMed Central

    Steinkellner, Georg; Gruber, Christian C.; Pavkov-Keller, Tea; Binter, Alexandra; Steiner, Kerstin; Winkler, Christoph; Łyskowski, Andrzej; Schwamberger, Orsolya; Oberer, Monika; Schwab, Helmut; Faber, Kurt; Macheroux, Peter; Gruber, Karl

    2014-01-01

    The exploitation of catalytic promiscuity and the application of de novo design have recently opened the access to novel, non-natural enzymatic activities. Here we describe a structural bioinformatic method for predicting catalytic activities of enzymes based on three-dimensional constellations of functional groups in active sites (‘catalophores’). As a proof-of-concept we identify two enzymes with predicted promiscuous ene-reductase activity (reduction of activated C–C double bonds) and compare them with known ene-reductases, that is, members of the Old Yellow Enzyme family. Despite completely different amino acid sequences, overall structures and protein folds, high-resolution crystal structures reveal equivalent binding modes of typical Old Yellow Enzyme substrates and ligands. Biochemical and biocatalytic data show that the two enzymes indeed possess ene-reductase activity and reveal an inverted stereopreference compared with Old Yellow Enzymes for some substrates. This method could thus be a tool for the identification of viable starting points for the development and engineering of novel biocatalysts. PMID:24954722

  2. iLoc-Virus: a multi-label learning classifier for identifying the subcellular localization of virus proteins with both single and multiple sites.

    PubMed

    Xiao, Xuan; Wu, Zhi-Cheng; Chou, Kuo-Chen

    2011-09-01

    In the last two decades or so, although many computational methods were developed for predicting the subcellular locations of proteins according to their sequence information, it is still remains as a challenging problem, particularly when the system concerned contains both single- and multiple-location proteins. Also, among the existing methods, very few were developed specialized for dealing with viral proteins, those generated by viruses. Actually, knowledge of the subcellular localization of viral proteins in a host cell or virus-infected cell is very important because it is closely related to their destructive tendencies and consequences. In this paper, by introducing the "multi-label scale" and by hybridizing the gene ontology information with the sequential evolution information, a predictor called iLoc-Virus is developed. It can be utilized to identify viral proteins among the following six locations: (1) viral capsid, (2) host cell membrane, (3) host endoplasmic reticulum, (4) host cytoplasm, (5) host nucleus, and (6) secreted. The iLoc-Virus predictor not only can more accurately predict the location sites of viral proteins in a host cell, but also have the capacity to deal with virus proteins having more than one location. As a user-friendly web-server, iLoc-Virus is freely accessible to the public at http://icpr.jci.edu.cn/bioinfo/iLoc-Virus. Meanwhile, a step-by-step guide is provided on how to use the web-server to get the desired results. Furthermore, for the user's convenience, the iLoc-Virus web-server also has the function to accept the batch job submission. It is anticipated that iLoc-Virus may become a useful high throughput tool for both basic research and drug development.

  3. An ionizable active-site tryptophan imparts catalase activity to a peroxidase core.

    PubMed

    Loewen, Peter C; Carpena, Xavi; Vidossich, Pietro; Fita, Ignacio; Rovira, Carme

    2014-05-21

    Catalase peroxidases (KatG's) are bifunctional heme proteins that can disproportionate hydrogen peroxide (catalatic reaction) despite their structural dissimilarity with monofunctional catalases. Using X-ray crystallography and QM/MM calculations, we demonstrate that the catalatic reaction of KatG's involves deprotonation of the active-site Trp, which plays a role similar to that of the distal His in monofunctional catalases. The interaction of a nearby mobile arginine with the distal Met-Tyr-Trp essential adduct (in/out) acts as an electronic switch, triggering deprotonation of the adduct Trp.

  4. Label-free electrochemical detection of botulinum neurotoxin type E based on its enzymatic activity using interdigitated electrodes

    NASA Astrophysics Data System (ADS)

    Hyun, Sang Hwa; Park, Dae Keun; Kang, Aeyeon; Kim, Soohyun; Kim, Daehee; Shin, Yu Mi; Song, Ji-Joon; Yun, Wan Soo

    2016-02-01

    We report a simple label-free electrochemical method of detecting low concentrations of botulinum neurotoxin type E light chain (BoNT/E LC) based on its peptide cleavage activity. Dual-mode cyclic voltammetry was employed to observe changes in the redox signal of ferri-/ferro-cyanide on interdigitated microelectrodes, whose surfaces were covered by peptides designed from synaptosomal-associated protein 25 to be cleaved by BoNT/E LC. With the introduction of BoNT/E LC, the redox signal showed a time-dependent increase due to cleavage of the immobilized peptide molecules. In addition to the increased redox signal intensity, its time-dependence can be considered as a strong evidence of BoNT/E sensing, since the time-dependent increase can only result from the enzymatic activity of BoNT/E LC. Using this method, BoNT/E LC, at concentrations as low as 5 pg/ml, was readily measurable with only an hour of incubation.

  5. Nuclear Site Security in the Event of Terrorist Activity

    SciTech Connect

    Thomson, M.L.; Sims, J.

    2008-07-01

    This paper, presented as a poster, identifies why ballistic protection should now be considered at nuclear sites to counter terrorist threats. A proven and flexible form of multi purpose protection is described in detail with identification of trial results that show its suitability for this role. (authors)

  6. Preliminary siting activities for new waste handling facilities at the Idaho National Engineering Laboratory

    SciTech Connect

    Taylor, D.D.; Hoskinson, R.L.; Kingsford, C.O.; Ball, L.W.

    1994-09-01

    The Idaho Waste Processing Facility, the Mixed and Low-Level Waste Treatment Facility, and the Mixed and Low-Level Waste Disposal Facility are new waste treatment, storage, and disposal facilities that have been proposed at the Idaho National Engineering Laboratory (INEL). A prime consideration in planning for such facilities is the selection of a site. Since spring of 1992, waste management personnel at the INEL have been involved in activities directed to this end. These activities have resulted in the (a) identification of generic siting criteria, considered applicable to either treatment or disposal facilities for the purpose of preliminary site evaluations and comparisons, (b) selection of six candidate locations for siting,and (c) site-specific characterization of candidate sites relative to selected siting criteria. This report describes the information gathered in the above three categories for the six candidate sites. However, a single, preferred site has not yet been identified. Such a determination requires an overall, composite ranking of the candidate sites, which accounts for the fact that the sites under consideration have different advantages and disadvantages, that no single site is superior to all the others in all the siting criteria, and that the criteria should be assigned different weighing factors depending on whether a site is to host a treatment or a disposal facility. Stakeholder input should now be solicited to help guide the final selection. This input will include (a) siting issues not already identified in the siting, work to date, and (b) relative importances of the individual siting criteria. Final site selection will not be completed until stakeholder input (from the State of Idaho, regulatory agencies, the public, etc.) in the above areas has been obtained and a strategy has been developed to make a composite ranking of all candidate sites that accounts for all the siting criteria.

  7. Active Layer and Moisture Measurements for Intensive Site 0 and 1, Barrow, Alaska

    DOE Data Explorer

    John Peterson

    2015-04-17

    These are measurements of Active Layer Thickness collected along several lines beginning in September, 2011 to the present. The data were collected at several time periods along the Site0 L2 Line, the Site1 AB Line, and an ERT Monitoring Line near Area A in Site1.

  8. A label-free gold nanocluster fluorescent probe for protease activity monitoring.

    PubMed

    Zhang, Jiajing; Zhang, Zhen; Nie, Xin; Zhang, Zhen; Wu, Xiaochun; Chen, Chunying; Fang, Xiaohong

    2014-06-01

    Water soluble BSA-stabilized gold nanoclusters (Au NCs) were synthesized with a simple one-pot procedure. The as-prepared Au NCs were able to emit intensive red fluorescence under the excitation of ultraviolet light, and the fluorescence could be quenched by enzymatic hydrolysis. In this contribution, BSA-stabilized Au NCs as novel fluorescent probes were successfully utilized for the detection and real-time monitoring of proteolytic activity of trypsin and chymotrypsin. High performance liquid chromatography-inductively coupled plasma mass spectrometry, X-ray photoelectron spectroscopy, and X-ray absorption fine structure were performed to investigate the quenching mechanism, and the results indicated that BSA scaffold degradation caused by enzymatic proteolysis led to the decrease in fluorescence intensity. Furthermore, this method would be potentially extended to the detection of other enzymes with Au NCs stabilized by different biomolecules.

  9. Structural mechanism of RuBisCO activation by carbamylation of the active site lysine

    PubMed Central

    Stec, Boguslaw

    2012-01-01

    Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is a crucial enzyme in carbon fixation and the most abundant protein on earth. It has been studied extensively by biochemical and structural methods; however, the most essential activation step has not yet been described. Here, we describe the mechanistic details of Lys carbamylation that leads to RuBisCO activation by atmospheric CO2. We report two crystal structures of nitrosylated RuBisCO from the red algae Galdieria sulphuraria with O2 and CO2 bound at the active site. G. sulphuraria RuBisCO is inhibited by cysteine nitrosylation that results in trapping of these gaseous ligands. The structure with CO2 defines an elusive, preactivation complex that contains a metal cation Mg2+ surrounded by three H2O/OH molecules. Both structures suggest the mechanism for discriminating gaseous ligands by their quadrupole electric moments. We describe conformational changes that allow for intermittent binding of the metal ion required for activation. On the basis of these structures we propose the individual steps of the activation mechanism. Knowledge of all these elements is indispensable for engineering RuBisCO into a more efficient enzyme for crop enhancement or as a remedy to global warming. PMID:23112176

  10. Label-free ultrasensitive detection of telomerase activity via multiple telomeric hemin/G-quadruplex triggered polyaniline deposition and a DNA tetrahedron-structure regulated signal.

    PubMed

    Liu, Yuanjian; Wei, Min; Liu, Xu; Wei, Wei; Zhao, Hongyu; Zhang, Yuanjian; Liu, Songqin

    2016-01-31

    Label-free detection of telomerase activity was done by using telomeric hemin/G-quadruplex triggered polyaniline deposition, not only on themselves but also on the DNA tetrahedron-structure (DTS). DTS size has a great impact on telomerase accessibility, reactivity and detection sensitivity. The method has been used to evaluate bladder cancer development.

  11. Using catalytic atom maps to predict the catalytic functions present in enzyme active sites.

    PubMed

    Nosrati, Geoffrey R; Houk, K N

    2012-09-18

    Catalytic atom maps (CAMs) are minimal models of enzyme active sites. The structures in the Protein Data Bank (PDB) were examined to determine if proteins with CAM-like geometries in their active sites all share the same catalytic function. We combined the CAM-based search protocol with a filter based on the weighted contact number (WCN) of the catalytic residues, a measure of the "crowdedness" of the microenvironment around a protein residue. Using this technique, a CAM based on the Ser-His-Asp catalytic triad of trypsin was able to correctly identify catalytic triads in other enzymes within 0.5 Å rmsd of the CAM with 96% accuracy. A CAM based on the Cys-Arg-(Asp/Glu) active site residues from the tyrosine phosphatase active site achieved 89% accuracy in identifying this type of catalytic functionality. Both of these CAMs were able to identify active sites across different fold types. Finally, the PDB was searched to locate proteins with catalytic functionality similar to that present in the active site of orotidine 5'-monophosphate decarboxylase (ODCase), whose mechanism is not known with certainty. A CAM, based on the conserved Lys-Asp-Lys-Asp tetrad in the ODCase active site, was used to search the PDB for enzymes with similar active sites. The ODCase active site has a geometry similar to that of Schiff base-forming Class I aldolases, with lowest aldolase rmsd to the ODCase CAM at 0.48 Å. The similarity between this CAM and the aldolase active site suggests that ODCase has the correct catalytic functionality present in its active site for the generation of a nucleophilic lysine. PMID:22909276

  12. Using Catalytic Atom Maps to Predict the Catalytic Functions Present in Enzyme Active Sites

    PubMed Central

    Nosrati, Geoffrey R.; Houk, K. N.

    2012-01-01

    Catalytic Atom Maps (CAMs) are minimal models of enzyme active sites. The structures in the Protein Data Bank (PDB) were examined to determine if proteins with CAM-like geometries in their active sites all share the same catalytic function. We combined the CAM-based search protocol with a filter based on the weighted contact number (WCN) of the catalytic residues, a measure of the “crowdedness” of the microenvironment around a protein residue. Using this technique, a CAM based on the Ser-His-Asp catalytic triad of trypsin was able to correctly identify catalytic triads in other enzymes within 0.5 Å RMSD of the Catalytic Atom Map with 96% accuracy. A CAM based on the Cys-Arg-(Asp/Glu) active site residues from the tyrosine phosphatase active site achieved 89% accuracy in identifying this type of catalytic functionality. Both of these Catalytic Atom Maps were able to identify active sites across different fold types. Finally, the PDB was searched to locate proteins with catalytic functionality similar to that present in the active site of orotidine 5′-monophosphate decarboxylase (ODCase), whose mechanism is not known with certainty. A CAM, based on the conserved Lys-Asp-Lys-Asp tetrad in the ODCase active site, was used to search the PDB for enzymes with similar active sites. The ODCase active site has a geometry similar to that of Schiff base-forming Class I aldolases, with lowest aldolase RMSD to the ODCase CAM at 0.48 Å. The similarity between this CAM and the aldolase active site suggests that ODCase has the correct catalytic functionality present in its active site for the generation of a nucleophilic lysine. PMID:22909276

  13. Active-Site Hydration and Water Diffusion in Cytochrome P450cam: A Highly Dynamic Process

    SciTech Connect

    Miao, Yinglong; Baudry, Jerome Y

    2011-01-01

    Long-timescale molecular dynamics simulations (300 ns) are performed on both the apo- (i.e., camphor-free) and camphor-bound cytochrome P450cam (CYP101). Water diffusion into and out of the protein active site is observed without biased sampling methods. During the course of the molecular dynamics simulation, an average of 6.4 water molecules is observed in the camphor-binding site of the apo form, compared to zero water molecules in the binding site of the substrate-bound form, in agreement with the number of water molecules observed in crystal structures of the same species. However, as many as 12 water molecules can be present at a given time in the camphor-binding region of the active site in the case of apo-P450cam, revealing a highly dynamic process for hydration of the protein active site, with water molecules exchanging rapidly with the bulk solvent. Water molecules are also found to exchange locations frequently inside the active site, preferentially clustering in regions surrounding the water molecules observed in the crystal structure. Potential-of-mean-force calculations identify thermodynamically favored trans-protein pathways for the diffusion of water molecules between the protein active site and the bulk solvent. Binding of camphor in the active site modifies the free-energy landscape of P450cam channels toward favoring the diffusion of water molecules out of the protein active site.

  14. Parameterization of an Active Thermal Erosion Site, Caribou Creek, Alaska

    NASA Astrophysics Data System (ADS)

    Busey, R.; Bolton, W. R.; Cherry, J. E.; Hinzman, L. D.

    2012-12-01

    Thermokarst features are thought to be an important mechanism for landscape change in permafrost-dominated cold regions, but few such features have been incorporated into full featured landscape models. The root of this shortcoming is that historic observations are not detailed enough to parameterize a model, and the models typically do not include the relevant processes for thermal erosion. A new, dynamic thermokarst feature has been identified at the Caribou-Poker Creek Research Watershed (CPCRW) in the boreal forest of Interior Alaska. Located adjacent to a traditional use trail, this feature terminates directly in Caribou Creek. Erosion within the feature is driven predominantly by fluvial interflow. CPCRW is a Long-Term Ecological Research site underlain by varying degrees of relatively warm, discontinuous permafrost. This poster will describe the suite of measurements that have been undertaken to parameterize the ERODE model for this site, including thorough surveys, time lapse- and aerial photography, and 3-D structure from motion algorithms.

  15. Blogs and Social Network Sites as Activity Systems: Exploring Adult Informal Learning Process through Activity Theory Framework

    ERIC Educational Resources Information Center

    Heo, Gyeong Mi; Lee, Romee

    2013-01-01

    This paper uses an Activity Theory framework to explore adult user activities and informal learning processes as reflected in their blogs and social network sites (SNS). Using the assumption that a web-based space is an activity system in which learning occurs, typical features of the components were investigated and each activity system then…

  16. Origins of intersubject variability of blood oxygenation level dependent and arterial spin labeling fMRI: implications for quantification of brain activity.

    PubMed

    Gaxiola-Valdez, Ismael; Goodyear, Bradley G

    2012-12-01

    Accurate localization of brain activity using blood oxygenation level-dependent (BOLD) functional magnetic resonance imaging (fMRI) has been challenged because of the large BOLD signal within distal veins. Arterial spin labeling (ASL) techniques offer greater sensitivity to the microvasculature but possess low temporal resolution and limited brain coverage. In this study, we show that the physiological origins of BOLD and ASL depend on whether percent change or statistical significance is being considered. For BOLD and ASL fMRI data collected during a simple unilateral hand movement task, we found that in the area of the contralateral motor cortex the centre of gravity (CoG) of the intersubject coefficient of variation (CV) of BOLD fMRI was near the brain surface for percent change in signal, whereas the CoG of the intersubject CV for Z-score was in close proximity of sites of brain activity for both BOLD and ASL. These findings suggest that intersubject variability of BOLD percent change is vascular in origin, whereas the origin of inter-subject variability of Z-score is neuronal for both BOLD and ASL. For longer duration tasks (12 s or greater), however, there was a significant correlation between BOLD and ASL percent change, which was not evident for short duration tasks (6 s). These findings suggest that analyses directly comparing percent change in BOLD signal between pre-defined regions of interest using short duration stimuli, as for example in event-related designs, may be heavily weighted by large-vessel responses rather than neuronal responses.

  17. Novel Chemical Strategies for Labeling Small Molecule Ligands for Androgen, Progestin, and Peroxisome Proliferator-Activated Receptors for Imaging Prostate and Breast Cancer and the Heart

    SciTech Connect

    Katzenellenbogen, John, A.

    2007-04-19

    Summary of Progress The specific aims of this project can be summarized as follows: • Aim 1: Prepare and evaluate radiolabeled ligands for the peroxisome proliferator-activated receptor (PPAR), a new nuclear hormone receptor target for tumor imaging and hormone therapy. • Aim 2: Prepare steroids labeled with a cyclopentadienyl tricarbonyl technetium or rhenium unit. • Aim 3: Prepare and evaluate other organometallic systems of novel design as ligand mimics and halogenated ligands for nuclear hormone receptor-based tumor imaging. As is described in detail below, we made excellent progress on all three of these aims; the highlights of our progress are the following: • we have prepared the first fluorine-18 labeled analogs of ligands for the PPAR receptor and used these in tissue distribution studies in rats • we have developed three new methods for the synthesis of cyclopentadienyltricarbonyl rhenium and technetium (CpRe(CO)3 and CpTc(CO)3) systems and we have adapted these to the synthesis of steroids labeled with these metals, as well as ligands for other receptor systems • we have prepared a number of fluorine-18 labeled steroidal and non-steroidal androgens and measured their tissue distribution in rats • we have prepared iodine and bromine-labeled progestins with high progesterone receptor binding affinity • we have prepared inorganic metal tricarbonyl complexes and steroid receptor ligands in which the metal tricarbonyl unit is an integral part off the ligand core.

  18. Reduced mitochondrial malate dehydrogenase activity has a strong effect on photorespiratory metabolism as revealed by 13C labelling

    PubMed Central

    Lindén, Pernilla; Keech, Olivier; Stenlund, Hans; Gardeström, Per; Moritz, Thomas

    2016-01-01

    Mitochondrial malate dehydrogenase (mMDH) catalyses the interconversion of malate and oxaloacetate (OAA) in the tricarboxylic acid (TCA) cycle. Its activity is important for redox control of the mitochondrial matrix, through which it may participate in regulation of TCA cycle turnover. In Arabidopsis, there are two isoforms of mMDH. Here, we investigated to which extent the lack of the major isoform, mMDH1 accounting for about 60% of the activity, affected leaf metabolism. In air, rosettes of mmdh1 plants were only slightly smaller than wild type plants although the fresh weight was decreased by about 50%. In low CO2 the difference was much bigger, with mutant plants accumulating only 14% of fresh weight as compared to wild type. To investigate the metabolic background to the differences in growth, we developed a 13CO2 labelling method, using a custom-built chamber that enabled simultaneous treatment of sets of plants under controlled conditions. The metabolic profiles were analysed by gas- and liquid- chromatography coupled to mass spectrometry to investigate the metabolic adjustments between wild type and mmdh1. The genotypes responded similarly to high CO2 treatment both with respect to metabolite pools and 13C incorporation during a 2-h treatment. However, under low CO2 several metabolites differed between the two genotypes and, interestingly most of these were closely associated with photorespiration. We found that while the glycine/serine ratio increased, a concomitant altered glutamine/glutamate/α-ketoglutarate relation occurred. Taken together, our results indicate that adequate mMDH activity is essential to shuttle reductants out from the mitochondria to support the photorespiratory flux, and strengthen the idea that photorespiration is tightly intertwined with peripheral metabolic reactions. PMID:26889011

  19. Fluorogen-activating proteins provide tunable labeling densities for tracking FcεRI independent of IgE.

    PubMed

    Schwartz, Samantha L; Yan, Qi; Telmer, Cheryl A; Lidke, Keith A; Bruchez, Marcel P; Lidke, Diane S

    2015-02-20

    Crosslinking of IgE bound FcεRI on mast cells and basophils by multivalent antigen leads to degranulation and the release of key inflammatory mediators that stimulate the allergic response. Here, we present and characterize the use of fluorogen-activating proteins (FAPs) for single particle tracking of FcεRI to investigate how receptor mobility is influenced after IgE-induced changes in mast cell behavior. FAPs are genetically encoded tags that bind a fluorogen dye and increase its brightness upon binding up to 20,000-fold. We demonstrate that, by titrating fluorogen concentration, labeling densities from ensemble to single particle can be achieved, independent of expression level and without the need for wash steps or photobleaching. The FcεRI γ-subunit fused to a FAP (FAP-γ) provides, for the first time, an IgE-independent probe for tracking this signaling subunit of FcεRI at the single molecule level. We show that the FcεRI γ-subunit dynamics are controlled by the IgE-binding α-subunit and that the cytokinergic IgE, SPE-7, induces mast cell activation without altering FcεRI mobility or promoting internalization. We take advantage of the far-red emission of the malachite green (MG) fluorogen to track FcεRI relative to dynamin-GFP and find that immobilized receptors readily correlate with locations of dynamin recruitment only under conditions that promote rapid endocytosis. These studies demonstrate the usefulness of the FAP system for single molecule studies and have provided new insights into the relationship among FcεRI structure, activity, and mobility. PMID:25343439

  20. Early Site Permit Demonstration Program: Recommendations for communication activities and public participation in the Early Site Permit Demonstration Program

    SciTech Connect

    Not Available

    1993-01-27

    On October 24, 1992, President Bush signed into law the National Energy Policy Act of 1992. The bill is a sweeping, comprehensive overhaul of the Nation`s energy laws, the first in more than a decade. Among other provisions, the National Energy Policy Act reforms the licensing process for new nuclear power plants by adopting a new approach developed by the US Nuclear Regulatory Commission (NRC) in 1989, and upheld in court in 1992. The NRC 10 CFR Part 52 rule is a three-step process that guarantees public participation at each step. The steps are: early site permit approval; standard design certifications; and, combined construction/operating licenses for nuclear power reactors. Licensing reform increases an organization`s ability to respond to future baseload electricity generation needs with less financial risk for ratepayers and the organization. Costly delays can be avoided because design, safety and siting issues will be resolved before a company starts to build a plant. Specifically, early site permit approval allows for site suitability and acceptability issues to be addressed prior to an organization`s commitment to build a plant. Responsibility for site-specific activities, including communications and public participation, rests with those organizations selected to try out early site approval. This plan has been prepared to assist those companies (referred to as sponsoring organizations) in planning their communications and public involvement programs. It provides research findings, information and recommendations to be used by organizations as a resource and starting point in developing their own plans.

  1. Active Site Structure and Peroxidase Activity of Oxidatively Modified Cytochrome c Species in Complexes with Cardiolipin.

    PubMed

    Capdevila, Daiana A; Oviedo Rouco, Santiago; Tomasina, Florencia; Tortora, Verónica; Demicheli, Verónica; Radi, Rafael; Murgida, Daniel H

    2015-12-29

    We report a resonance Raman and UV-vis characterization of the active site structure of oxidatively modified forms of cytochrome c (Cyt-c) free in solution and in complexes with cardiolipin (CL). The studied post-translational modifications of Cyt-c include methionine sulfoxidation and tyrosine nitration, which lead to altered heme axial ligation and increased peroxidase activity with respect to those of the wild-type protein. In spite of the structural and activity differences between the protein variants free in solution, binding to CL liposomes induces in all cases the formation of a spectroscopically identical bis-His axial coordination conformer that more efficiently promotes lipid peroxidation. The spectroscopic results indicate that the bis-His form is in equilibrium with small amounts of high-spin species, thus suggesting a labile distal His ligand as the basis for the CL-induced increase in enzymatic activity observed for all protein variants. For Cyt-c nitrated at Tyr74 and sulfoxidized at Met80, the measured apparent binding affinities for CL are ∼4 times larger than for wild-type Cyt-c. On the basis of these results, we propose that these post-translational modifications may amplify the pro-apoptotic signal of Cyt-c under oxidative stress conditions at CL concentrations lower than for the unmodified protein.

  2. Identification of ice nucleation active sites on feldspar dust particles.

    PubMed

    Zolles, Tobias; Burkart, Julia; Häusler, Thomas; Pummer, Bernhard; Hitzenberger, Regina; Grothe, Hinrich

    2015-03-19

    Mineral dusts originating from Earth's crust are known to be important atmospheric ice nuclei. In agreement with earlier studies, feldspar was found as the most active of the tested natural mineral dusts. Here we investigated in closer detail the reasons for its activity and the difference in the activity of the different feldspars. Conclusions are drawn from scanning electron microscopy, X-ray powder diffraction, infrared spectroscopy, and oil-immersion freezing experiments. K-feldspar showed by far the highest ice nucleation activity. Finally, we give a potential explanation of this effect, finding alkali-metal ions having different hydration shells and thus an influence on the ice nucleation activity of feldspar surfaces. PMID:25584435

  3. Identification of Ice Nucleation Active Sites on Feldspar Dust Particles

    PubMed Central

    2015-01-01

    Mineral dusts originating from Earth’s crust are known to be important atmospheric ice nuclei. In agreement with earlier studies, feldspar was found as the most active of the tested natural mineral dusts. Here we investigated in closer detail the reasons for its activity and the difference in the activity of the different feldspars. Conclusions are drawn from scanning electron microscopy, X-ray powder diffraction, infrared spectroscopy, and oil-immersion freezing experiments. K-feldspar showed by far the highest ice nucleation activity. Finally, we give a potential explanation of this effect, finding alkali-metal ions having different hydration shells and thus an influence on the ice nucleation activity of feldspar surfaces. PMID:25584435

  4. Structure of the high-affinity binding site for noncompetitive blockers of the acetylcholine receptor: (/sup 3/H)chlorpromazine labels homologous residues in the. beta. and delta chains

    SciTech Connect

    Giraudat, J.; Dennis, M.; Heidmann, T.; Haumont, P.Y.; Lederer, F.; Changeux, J.P.

    1987-05-05

    The membrane-bound acetylcholine receptor from Torpedo marmorata was photolabeled by the noncompetitive channel blocker (/sup 3/H)chlorpromazine under equilibrium conditions in the presence of the agonist carbamoylcholine. The amount of radioactivity incorporated into all subunits was reduced by addition of phencyclidine, a specific ligand for the high-affinity site for noncompetitive blockers. The labeled ..beta.. chain was purified and digested with trypsin or CNBr, and the resulting fragments were fractionated by high-performance liquid chromatography. Sequence analysis resulted in the identification of Ser-254 and Leu-257 as residues labeled by (/sup 3/H)chlorpromazine in a phencyclidine-sensitive manner. These residues are located in the hydrophobic and potentially transmembrane segment M II of the ..beta.. chain, a region homologous to that containing the chlorpromazine-labeled Ser-262 in the delta chain. These results show that homologous regions of different receptor subunits contribute to the unique high-affinity site for noncompetitive blockers, a finding consistent with the location of this site on the axis of symmetry of the receptor molecule.

  5. Stereospecific suppression of active site mutants by methylphosphonate substituted substrates reveals the stereochemical course of site-specific DNA recombination.

    PubMed

    Rowley, Paul A; Kachroo, Aashiq H; Ma, Chien-Hui; Maciaszek, Anna D; Guga, Piotr; Jayaram, Makkuni

    2015-07-13

    Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5' to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions. PMID:25999343

  6. Stereospecific suppression of active site mutants by methylphosphonate substituted substrates reveals the stereochemical course of site-specific DNA recombination.

    PubMed

    Rowley, Paul A; Kachroo, Aashiq H; Ma, Chien-Hui; Maciaszek, Anna D; Guga, Piotr; Jayaram, Makkuni

    2015-07-13

    Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5' to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions.

  7. Active site densities, oxygen activation and adsorbed reactive oxygen in alcohol activation on npAu catalysts.

    PubMed

    Wang, Lu-Cun; Friend, C M; Fushimi, Rebecca; Madix, Robert J

    2016-07-01

    The activation of molecular O2 as well as the reactivity of adsorbed oxygen species is of central importance in aerobic selective oxidation chemistry on Au-based catalysts. Herein, we address the issue of O2 activation on unsupported nanoporous gold (npAu) catalysts by applying a transient pressure technique, a temporal analysis of products (TAP) reactor, to measure the saturation coverage of atomic oxygen, its collisional dissociation probability, the activation barrier for O2 dissociation, and the facility with which adsorbed O species activate methanol, the initial step in the catalytic cycle of esterification. The results from these experiments indicate that molecular O2 dissociation is associated with surface silver, that the density of reactive sites is quite low, that adsorbed oxygen atoms do not spill over from the sites of activation onto the surrounding surface, and that methanol reacts quite facilely with the adsorbed oxygen atoms. In addition, the O species from O2 dissociation exhibits reactivity for the selective oxidation of methanol but not for CO. The TAP experiments also revealed that the surface of the npAu catalyst is saturated with adsorbed O under steady state reaction conditions, at least for the pulse reaction. PMID:27376884

  8. Site-directed immobilization of a genetically engineered anti-methotrexate antibody via an enzymatically introduced biotin label significantly increases the binding capacity of immunoaffinity columns.

    PubMed

    Davenport, Kaitlynn R; Smith, Christopher A; Hofstetter, Heike; Horn, James R; Hofstetter, Oliver

    2016-05-15

    In this study, the effect of random vs. site-directed immobilization techniques on the performance of antibody-based HPLC columns was investigated using a single-domain camelid antibody (VHH) directed against methotrexate (MTX) as a model system. First, the high flow-through support material POROS-OH was activated with disuccinimidyl carbonate (DSC), and the VHH was bound in a random manner via amines located on the protein's surface. The resulting column was characterized by Frontal Affinity Chromatography (FAC). Then, two site-directed techniques were explored to increase column efficiency by immobilizing the antibody via its C-terminus, i.e., away from the antigen-binding site. In one approach, a tetra-lysine tail was added, and the antibody was immobilized onto DSC-activated POROS. In the second site-directed approach, the VHH was modified with the AviTag peptide, and a biotin-residue was enzymatically incorporated at the C-terminus using the biotin ligase BirA. The biotinylated antibody was subsequently immobilized onto NeutrAvidin-derivatized POROS. A comparison of the FAC analyses, which for all three columns showed excellent linearity (R(2)>0.999), revealed that both site-directed approaches yield better results than the random immobilization; the by far highest efficiency, however, was determined for the immunoaffinity column based on AviTag-biotinylated antibody. As proof of concept, all three columns were evaluated for quantification of MTX dissolved in phosphate buffered saline (PBS). Validation using UV-detection showed excellent linearity in the range of 0.04-12μM (R(2)>0.993). The lower limit of detection (LOD) and lower limit of quantification (LLOQ) were found to be independent of the immobilization strategy and were 40nM and 132nM, respectively. The intra- and inter-day precision was below 11.6%, and accuracy was between 90.7% and 112%. To the best of our knowledge, this is the first report of the AviTag-system in chromatography, and the first

  9. Site-directed immobilization of a genetically engineered anti-methotrexate antibody via an enzymatically introduced biotin label significantly increases the binding capacity of immunoaffinity columns.

    PubMed

    Davenport, Kaitlynn R; Smith, Christopher A; Hofstetter, Heike; Horn, James R; Hofstetter, Oliver

    2016-05-15

    In this study, the effect of random vs. site-directed immobilization techniques on the performance of antibody-based HPLC columns was investigated using a single-domain camelid antibody (VHH) directed against methotrexate (MTX) as a model system. First, the high flow-through support material POROS-OH was activated with disuccinimidyl carbonate (DSC), and the VHH was bound in a random manner via amines located on the protein's surface. The resulting column was characterized by Frontal Affinity Chromatography (FAC). Then, two site-directed techniques were explored to increase column efficiency by immobilizing the antibody via its C-terminus, i.e., away from the antigen-binding site. In one approach, a tetra-lysine tail was added, and the antibody was immobilized onto DSC-activated POROS. In the second site-directed approach, the VHH was modified with the AviTag peptide, and a biotin-residue was enzymatically incorporated at the C-terminus using the biotin ligase BirA. The biotinylated antibody was subsequently immobilized onto NeutrAvidin-derivatized POROS. A comparison of the FAC analyses, which for all three columns showed excellent linearity (R(2)>0.999), revealed that both site-directed approaches yield better results than the random immobilization; the by far highest efficiency, however, was determined for the immunoaffinity column based on AviTag-biotinylated antibody. As proof of concept, all three columns were evaluated for quantification of MTX dissolved in phosphate buffered saline (PBS). Validation using UV-detection showed excellent linearity in the range of 0.04-12μM (R(2)>0.993). The lower limit of detection (LOD) and lower limit of quantification (LLOQ) were found to be independent of the immobilization strategy and were 40nM and 132nM, respectively. The intra- and inter-day precision was below 11.6%, and accuracy was between 90.7% and 112%. To the best of our knowledge, this is the first report of the AviTag-system in chromatography, and the first

  10. Whole abdominal wall segmentation using augmented active shape models (AASM) with multi-atlas label fusion and level set

    NASA Astrophysics Data System (ADS)

    Xu, Zhoubing; Baucom, Rebeccah B.; Abramson, Richard G.; Poulose, Benjamin K.; Landman, Bennett A.

    2016-03-01

    The abdominal wall is an important structure differentiating subcutaneous and visceral compartments and intimately involved with maintaining abdominal structure. Segmentation of the whole abdominal wall on routinely acquired computed tomography (CT) scans remains challenging due to variations and complexities of the wall and surrounding tissues. In this study, we propose a slice-wise augmented active shape model (AASM) approach to robustly segment both the outer and inner surfaces of the abdominal wall. Multi-atlas label fusion (MALF) and level set (LS) techniques are integrated into the traditional ASM framework. The AASM approach globally optimizes the landmark updates in the presence of complicated underlying local anatomical contexts. The proposed approach was validated on 184 axial slices of 20 CT scans. The Hausdorff distance against the manual segmentation was significantly reduced using proposed approach compared to that using ASM, MALF, and LS individually. Our segmentation of the whole abdominal wall enables the subcutaneous and visceral fat measurement, with high correlation to the measurement derived from manual segmentation. This study presents the first generic algorithm that combines ASM, MALF, and LS, and demonstrates practical application for automatically capturing visceral and subcutaneous fat volumes.

  11. Chip electrophoresis of active banana ingredients with label-free detection utilizing deep UV native fluorescence and mass spectrometry.

    PubMed

    Ohla, Stefan; Schulze, Philipp; Fritzsche, Stefanie; Belder, Detlev

    2011-02-01

    In the present work, we report on a rapid and straightforward approach for the determination of biologically active compounds in bananas applying microchip electrophoresis (MCE). For this purpose, we applied label-free detection utilizing deep UV fluorescence detection with excitation at 266 nm. Using this approach, we could identify dopamine and serotonin, their precursors tryptophan and tyrosine and also the isoquinoline alkaloid salsolinol in less than 1 min. In bananas, after 10 days of ripening, we additionally found the compound levodopa which is a metabolite of the tyrosine pathway. Quantitative analysis of extracts by external calibration revealed concentrations of serotonin, tryptophan, and tyrosine from 2.7 to 7.6 μg/mL with relative standard deviations of less than 3.5%. The corresponding calibration plots showed good linearity with correlation coefficients higher than 0.985. For reliable peak assignment, the compounds were also analyzed by coupling chip electrophoresis with mass spectrometry. This paper demonstrates exemplarily the applicability of MCE with native fluorescence detection for rapid analysis of natural compounds in fruits and reveals the potential of chip-based separation systems for the analysis of complex mixtures. PMID:21181134

  12. Whole Abdominal Wall Segmentation using Augmented Active Shape Models (AASM) with Multi-Atlas Label Fusion and Level Set

    PubMed Central

    Xu, Zhoubing; Baucom, Rebeccah B.; Abramson, Richard G.; Poulose, Benjamin K.; Landman, Bennett A.

    2016-01-01

    The abdominal wall is an important structure differentiating subcutaneous and visceral compartments and intimately involved with maintaining abdominal structure. Segmentation of the whole abdominal wall on routinely acquired computed tomography (CT) scans remains challenging due to variations and complexities of the wall and surrounding tissues. In this study, we propose a slice-wise augmented active shape model (AASM) approach to robustly segment both the outer and inner surfaces of the abdominal wall. Multi-atlas label fusion (MALF) and level set (LS) techniques are integrated into the traditional ASM framework. The AASM approach globally optimizes the landmark updates in the presence of complicated underlying local anatomical contexts. The proposed approach was validated on 184 axial slices of 20 CT scans. The Hausdorff distance against the manual segmentation was significantly reduced using proposed approach compared to that using ASM, MALF, and LS individually. Our segmentation of the whole abdominal wall enables the subcutaneous and visceral fat measurement, with high correlation to the measurement derived from manual segmentation. This study presents the first generic algorithm that combines ASM, MALF, and LS, and demonstrates practical application for automatically capturing visceral and subcutaneous fat volumes. PMID:27127333

  13. Multiple Transport-Active Binding Sites Are Available for a Single Substrate on Human P-Glycoprotein (ABCB1)

    PubMed Central

    Chufan, Eduardo E.; Kapoor, Khyati; Sim, Hong-May; Singh, Satyakam; Talele, Tanaji T.; Durell, Stewart R.; Ambudkar, Suresh V.

    2013-01-01

    P-glycoprotein (Pgp, ABCB1) is an ATP-Binding Cassette (ABC) transporter that is associated with the development of multidrug resistance in cancer cells. Pgp transports a variety of chemically dissimilar amphipathic compounds using the energy from ATP hydrolysis. In the present study, to elucidate the binding sites on Pgp for substrates and modulators, we employed site-directed mutagenesis, cell- and membrane-based assays, molecular modeling and docking. We generated single, double and triple mutants with substitutions of the Y307, F343, Q725, F728, F978 and V982 residues at the proposed drug-binding site with cys in a cysless Pgp, and expressed them in insect and mammalian cells using a baculovirus expression system. All the mutant proteins were expressed at the cell surface to the same extent as the cysless wild-type Pgp. With substitution of three residues of the pocket (Y307, Q725 and V982) with cysteine in a cysless Pgp, QZ59S-SSS, cyclosporine A, tariquidar, valinomycin and FSBA lose the ability to inhibit the labeling of Pgp with a transport substrate, [125I]-Iodoarylazidoprazosin, indicating these drugs cannot bind at their primary binding sites. However, the drugs can modulate the ATP hydrolysis of the mutant Pgps, demonstrating that they bind at secondary sites. In addition, the transport of six fluorescent substrates in HeLa cells expressing triple mutant (Y307C/Q725C/V982C) Pgp is also not significantly altered, showing that substrates bound at secondary sites are still transported. The homology modeling of human Pgp and substrate and modulator docking studies support the biochemical and transport data. In aggregate, our results demonstrate that a large flexible pocket in the Pgp transmembrane domains is able to bind chemically diverse compounds. When residues of the primary drug-binding site are mutated, substrates and modulators bind to secondary sites on the transporter and more than one transport-active binding site is available for each substrate

  14. Possible active site of the sweet-tasting protein thaumatin.

    PubMed

    Slootstra, J W; De Geus, P; Haas, H; Verrips, C T; Meloen, R H

    1995-10-01

    Epitopes on thaumatin and monellin were studied using the PEPSCAN-technology. The antibodies used were raised against thaumatin. Only antibodies that, in an ELISA, both recognized thaumatin and monellin were used in the PEPSCAN-analyses. On thaumatin two major overlapping epitopes were identified. On monellin no epitopes could be identified. The identified epitope region on thaumatin shares structural features with various peptide and protein sweeteners. It contains an aspartame-like site which is formed by Asp21 and Phe80, tips of the two extruding loops KGDAALDAGGR19-29 and CKRFGRPP77-84, which are spatially positioned next to each other. Furthermore, sub-sequences of the KGDAALDAGGR19-29 loop are similar to peptide-sweeteners such as L-Asp-D-Ala-L-Ala-methyl ester and L-Asp-D-Ala-Gly-methyl ester. Since the aspartame-like Asp21-Phe80 site and the peptide-sweetener-like sequences are also not present in non-sweet thaumatin-like proteins it is postulated that the KGDAALDAGGR19-29- and CKRFGRPP77-84 loop contain important sweet-taste determinants. This region has previously not been implicated as a sweet-taste determinant of thaumatin.

  15. A multi-label classifier for predicting the subcellular localization of gram-negative bacterial proteins with both single and multiple sites.

    PubMed

    Xiao, Xuan; Wu, Zhi-Cheng; Chou, Kuo-Chen

    2011-01-01

    Prediction of protein subcellular localization is a challenging problem, particularly when the system concerned contains both singleplex and multiplex proteins. In this paper, by introducing the "multi-label scale" and hybridizing the information of gene ontology with the sequential evolution information, a novel predictor called iLoc-Gneg is developed for predicting the subcellular localization of gram-positive bacterial proteins with both single-location and multiple-location sites. For facilitating comparison, the same stringent benchmark dataset used to estimate the accuracy of Gneg-mPLoc was adopted to demonstrate the power of iLoc-Gneg. The dataset contains 1,392 gram-negative bacterial proteins classified into the following eight locations: (1) cytoplasm, (2) extracellular, (3) fimbrium, (4) flagellum, (5) inner membrane, (6) nucleoid, (7) outer membrane, and (8) periplasm. Of the 1,392 proteins, 1,328 are each with only one subcellular location and the other 64 are each with two subcellular locations, but none of the proteins included has pairwise sequence identity to any other in a same subset (subcellular location). It was observed that the overall success rate by jackknife test on such a stringent benchmark dataset by iLoc-Gneg was over 91%, which is about 6% higher than that by Gneg-mPLoc. As a user-friendly web-server, iLoc-Gneg is freely accessible to the public at http://icpr.jci.edu.cn/bioinfo/iLoc-Gneg. Meanwhile, a step-by-step guide is provided on how to use the web-server to get the desired results. Furthermore, for the user's convenience, the iLoc-Gneg web-server also has the function to accept the batch job submission, which is not available in the existing version of Gneg-mPLoc web-server. It is anticipated that iLoc-Gneg may become a useful high throughput tool for Molecular Cell Biology, Proteomics, System Biology, and Drug Development.

  16. 21 CFR 358.760 - Labeling of permitted combinations of active ingredients for the control of dandruff.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    .../anti-itch shampoo” or “antidandruff/anti-itch shampoo”. (2) (b) Indications. The labeling of the... hair apply shampoo and work into a lather rinse thoroughly for best results, use at least twice a...

  17. Assessment of activation products in the Savannah River Site environment

    SciTech Connect

    Carlton, W.H.; Denham, M.

    1996-07-01

    This document assesses the impact of radioactive activation products released from SRS facilities since the first reactor became operational late in 1953. The isotopes reported here are those whose release resulted in the highest dose to people living near SRS: {sup 32}P, {sup 51}Cr, {sup 60}C, and {sup 65}Zn. Release pathways, emission control features, and annual releases to the aqueous and atmospheric environments are discussed. No single incident has resulted in a major acute release of activation products to the environment. The releases were the result of normal operations of the reactors and separations facilities. Releases declined over the years as better controls were established and production was reduced. The overall radiological impact of SRS activation product atmospheric releases from 1954 through 1994 on the offsite maximally exposed individual can be characterized by a total dose of 0.76 mrem. During the same period, such an individual received a total dose of 14,400 mrem from non-SRS sources of ionizing radiation present in the environment. SRS activation product aqueous releases between 1954 and 1994 resulted in a total dose of 54 mrem to the offsite maximally exposed individual. The impact of SRS activation product releases on offsite populations also has been evaluated.

  18. Quantifying the density and utilization of active sites in non-precious metal oxygen electroreduction catalysts

    PubMed Central

    Sahraie, Nastaran Ranjbar; Kramm, Ulrike I.; Steinberg, Julian; Zhang, Yuanjian; Thomas, Arne; Reier, Tobias; Paraknowitsch, Jens-Peter; Strasser, Peter

    2015-01-01

    Carbon materials doped with transition metal and nitrogen are highly active, non-precious metal catalysts for the electrochemical conversion of molecular oxygen in fuel cells, metal air batteries, and electrolytic processes. However, accurate measurement of their intrinsic turn-over frequency and active-site density based on metal centres in bulk and surface has remained difficult to date, which has hampered a more rational catalyst design. Here we report a successful quantification of bulk and surface-based active-site density and associated turn-over frequency values of mono- and bimetallic Fe/N-doped carbons using a combination of chemisorption, desorption and 57Fe Mössbauer spectroscopy techniques. Our general approach yields an experimental descriptor for the intrinsic activity and the active-site utilization, aiding in the catalyst development process and enabling a previously unachieved level of understanding of reactivity trends owing to a deconvolution of site density and intrinsic activity. PMID:26486465

  19. Characterization of an Active Thermal Erosion Site, Caribou Creek, Alaska

    NASA Astrophysics Data System (ADS)

    Busey, R.; Bolton, W. R.; Cherry, J. E.; Hinzman, L. D.

    2013-12-01

    The goal of this project is to estimate volume loss of soil over time from this site, provide parameterizations on erodibility of ice rich permafrost and serve as a baseline for future landscape evolution simulations. Located in the zone of discontinuous permafrost, the interior region of Alaska (USA) is home to a large quantity of warm, unstable permafrost that is both high in ice content and has soil temperatures near the freezing point. Much of this permafrost maintains a frozen state despite the general warming air temperature trend in the region due to the presence of a thick insulating organic mat and a dense root network in the upper sub-surface of the soil column. At a rapidly evolving thermo-erosion site, located within the Caribou-Poker Creeks Research Watershed (part of the Bonanza Creek LTER) near Chatanika, Alaska (N65.140, W147.570), the protective organic layer and associated plants were disturbed by an adjacent traditional use trail and the shifting of a groundwater spring. These triggers have led to rapid geomorphological change on the landscape as the soil thaws and sediment is transported into the creek at the valley bottom. Since 2006 (approximately the time of initiation), the thermal erosion has grown to 170 meters length, 3 meters max depth, and 15 meters maximum width. This research combines several data sets: DGPS survey, imagery from an extremely low altitude pole-based remote sensing (3 to 5 meters above ground level), and imagery from an Unmanned Aerial System (UAS) at about 60m altitude.

  20. Molecular Basis for Enzymatic Sulfite Oxidation -- HOW THREE CONSERVED ACTIVE SITE RESIDUES SHAPE ENZYME ACTIVITY

    SciTech Connect

    Bailey, Susan; Rapson, Trevor; Johnson-Winters, Kayunta; Astashkin, Andrei; Enemark, John; Kappler, Ulrike

    2008-11-10

    Sulfite dehydrogenases (SDHs) catalyze the oxidation and detoxification of sulfite to sulfate, a reaction critical to all forms of life. Sulfite-oxidizing enzymes contain three conserved active site amino acids (Arg-55, His-57, and Tyr-236) that are crucial for catalytic competency. Here we have studied the kinetic and structural effects of two novel and one previously reported substitution (R55M, H57A, Y236F) in these residues on SDH catalysis. Both Arg-55 and His-57 were found to have key roles in substrate binding. An R55M substitution increased Km(sulfite)(app) by 2-3 orders of magnitude, whereas His-57 was required for maintaining a high substrate affinity at low pH when the imidazole ring is fully protonated. This effect may be mediated by interactions of His-57 with Arg-55 that stabilize the position of the Arg-55 side chain or, alternatively, may reflect changes in the protonation state of sulfite. Unlike what is seen for SDHWT and SDHY236F, the catalytic turnover rates of SDHR55M and SDHH57A are relatively insensitive to pH (~;;60 and 200 s-1, respectively). On the structural level, striking kinetic effects appeared to correlate with disorder (in SDHH57A and SDHY236F) or absence of Arg-55 (SDHR55M), suggesting that Arg-55 and the hydrogen bonding interactions it engages in are crucial for substrate binding and catalysis. The structure of SDHR55M has sulfate bound at the active site, a fact that coincides with a significant increase in the inhibitory effect of sulfate in SDHR55M. Thus, Arg-55 also appears to be involved in enabling discrimination between the substrate and product in SDH.

  1. Active Site Metal Occupancy and Cyclic Di-GMP Phosphodiesterase Activity of Thermotoga maritima HD-GYP.

    PubMed

    Miner, Kyle D; Kurtz, Donald M

    2016-02-16

    HD-GYPs make up a subclass of the metal-dependent HD phosphohydrolase superfamily and catalyze conversion of cyclic di(3',5')-guanosine monophosphate (c-di-GMP) to 5'-phosphoguanylyl-(3'→5')-guanosine (pGpG) and GMP. Until now, the only reported crystal structure of an HD-GYP that also exhibits c-di-GMP phosphodiesterase activity contains a His/carboxylate ligated triiron active site. However, other structural and phylogenetic correlations indicate that some HD-GYPs contain dimetal active sites. Here we provide evidence that an HD-GYP c-di-GMP phosphodiesterase, TM0186, from Thermotoga maritima can accommodate both di- and trimetal active sites. We show that an as-isolated iron-containing TM0186 has an oxo/carboxylato-bridged diferric site, and that the reduced (diferrous) form is necessary and sufficient to catalyze conversion of c-di-GMP to pGpG, but that conversion of pGpG to GMP requires more than two metals per active site. Similar c-di-GMP phosphodiesterase activities were obtained with divalent iron or manganese. On the basis of activity correlations with several putative metal ligand residue variants and molecular dynamics simulations, we propose that TM0186 can accommodate both di- and trimetal active sites. Our results also suggest that a Glu residue conserved in a subset of HD-GYPs is required for formation of the trimetal site and can also serve as a labile ligand to the dimetal site. Given the anaerobic growth requirement of T. maritima, we suggest that this HD-GYP can function in vivo with either divalent iron or manganese occupying di- and trimetal sites.

  2. Investigations of enzymatic alterations of 2,4-dichlorophenol using {sup 13}C-nuclear magnetic resonance in combination with site-specific {sup 13}C-labeling: Understanding the environmental fate of this pollutant

    SciTech Connect

    Nanny, M.A.; Bortiatynski, J.M.; Tien, M.; Hatcher, P.G.

    1996-11-01

    The biodegradation of {sup 13}C-labeled 2,4-dichlorophenol (DCP labeled at the C-2 and C-6 positions), in the presence and absence of natural organic matter (NOM), by the white-rot fungus Phanerochaete chrysosporium, was examined using {sup 13}C-nuclear magnetic resonance (NMR). Using this method permitted the chemistry occurring at or near the labeled site to be followed. The formation of alkyl ethers and alkene ethers was observed. No aromatic by-products were detected, indicating that aromatic compounds are quickly degraded. Examining the reaction with time shows the exponential removal of 2,4-DCP and the consequential formation of labeled by-products, whose concentration reaches a maximum just before all 2,4-DCP is consumed. After this, the by-products degrade exponentially. The presence of NOM causes 2,4-DCP to be removed from the aqueous phase more quickly than in its absence and also causes the by-products to reach their maximum concentration much earlier. Degradation of the by-products occurs at a much greater rate in the presence of NOM. One hypothesis for this behavior is that the NOM interacts with 2,4-DCP and its by-products, allowing them to be incorporated into the fungal biomass. {sup 13}C-nuclear magnetic resonance spectra of the fungal biomass after NaOH extraction show the presence of alkanes and a small amount of 2,4-DCP.

  3. A rapid and direct method for the determination of active site accessibility in proteins based on ESI-MS and active site titrations.

    PubMed

    O'Farrell, Norah; Kreiner, Michaela; Moore, Barry D; Parker, Marie-Claire

    2006-11-01

    We have developed an electrospray ionisation mass spectrometry (ESI-MS) technique that can be applied to rapidly determine the number of intact active sites in proteins. The methodology relies on inhibiting the protein with an active-site irreversible inhibitor and then using ESI-MS to determine the extent of inhibition. We have applied this methodology to a test system: a serine protease, subtilisin Carlsberg, and monitored the extent of inhibition by phenylmethylsulfonyl fluoride (PMSF), an irreversible serine hydrolase inhibitor as a function of the changes in immobilisation and hydration conditions. Two types of enzyme preparation were investigated, lyophilised enzymes and protein-coated microcrystals (PCMC).

  4. Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand.

    PubMed

    Parashar, Abhinav; Venkatachalam, Avanthika; Gideon, Daniel Andrew; Manoj, Kelath Murali

    2014-12-12

    The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins' active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations in heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes.

  5. Marine Biology Field Trip Sites. Ocean Related Curriculum Activities.

    ERIC Educational Resources Information Center

    Pauls, John

    The ocean affects all of our lives. Therefore, awareness of and information about the interconnections between humans and oceans are prerequisites to making sound decisions for the future. Project ORCA (Ocean Related Curriculum Activities) has developed interdisciplinary curriculum materials designed to meet the needs of students and teachers…

  6. Endolysosomes Are the Principal Intracellular Sites of Acid Hydrolase Activity.

    PubMed

    Bright, Nicholas A; Davis, Luther J; Luzio, J Paul

    2016-09-12

    The endocytic delivery of macromolecules from the mammalian cell surface for degradation by lysosomal acid hydrolases requires traffic through early endosomes to late endosomes followed by transient (kissing) or complete fusions between late endosomes and lysosomes. Transient or complete fusion results in the formation of endolysosomes, which are hybrid organelles from which lysosomes are re-formed. We have used synthetic membrane-permeable cathepsin substrates, which liberate fluorescent reporters upon proteolytic cleavage, as well as acid phosphatase cytochemistry to identify which endocytic compartments are acid hydrolase active. We found that endolysosomes are the principal organelles in which acid hydrolase substrates are cleaved. Endolysosomes also accumulated acidotropic probes and could be distinguished from terminal storage lysosomes, which were acid hydrolase inactive and did not accumulate acidotropic probes. Using live-cell microscopy, we have demonstrated that fusion events, which form endolysosomes, precede the onset of acid hydrolase activity. By means of sucrose and invertase uptake experiments, we have also shown that acid-hydrolase-active endolysosomes and acid-hydrolase-inactive, terminal storage lysosomes exist in dynamic equilibrium. We conclude that the terminal endocytic compartment is composed of acid-hydrolase-active, acidic endolysosomes and acid hydrolase-inactive, non-acidic, terminal storage lysosomes, which are linked and function in a lysosome regeneration cycle. PMID:27498570

  7. Outside-binding site mutations modify the active site's shapes in neuraminidase from influenza A H1N1.

    PubMed

    Tolentino-Lopez, Luis; Segura-Cabrera, Aldo; Reyes-Loyola, Paola; Zimic, Mirko; Quiliano, Miguel; Briz, Veronica; Muñoz-Fernández, Angeles; Rodríguez-Pérez, Mario; Ilizaliturri-Flores, Ian; Correa-Basurto, Jose

    2013-01-01

    The recent occurrence of 2009 influenza A (H1N1) pandemic as well as others has raised concern of a far more dangerous outcome should this virus becomes resistant to current drug therapies. The number of clinical cases that are resistant to oseltamivir (Tamiflu®) is larger than the limited number of neuraminidase (NA) mutations (H275Y, N295S, and I223R) that have been identified at the active site and that are associated to oseltamivir resistance. In this study, we have performed a comparative analysis between a set of NAs that have the most representative mutations located outside the active site. The recently crystallized NA-oseltamivir complex (PDB ID: 3NSS) was used as a wild-type structure. After selecting the target NA sequences, their three-dimensional (3D) structure was built using 3NSS as a template by homology modeling. The 3D NA models were refined by molecular dynamics (MD) simulations. The refined models were used to perform a docking study, using oseltamivir as a ligand. Furthermore, the docking results were refined by free-energy analysis using the MM-PBSA method. The analysis of the MD simulation results showed that the NA models reached convergence during the first 10 ns. Visual inspection and structural measures showed that the mutated NA active sites show structural variations. The docking and MM-PBSA results from the complexes showed different binding modes and free energy values. These results suggest that distant mutations located outside the active site of NA affect its structure and could be considered to be a new source of resistance to oseltamivir, which agrees with reports in the clinical literature.

  8. Active site proton delivery and the lyase activity of human CYP17A1

    SciTech Connect

    Khatri, Yogan; Gregory, Michael C.; Grinkova, Yelena V.; Denisov, Ilia G.; Sligar, Stephen G.

    2014-01-03

    equivalents and protons are funneled into non-productive pathways. This is similar to previous work with other P450 catalyzed hydroxylation. However, catalysis of carbon–carbon bond scission by the T306A mutant was largely unimpeded by disruption of the CYP17A1 acid-alcohol pair. The unique response of CYP17A1 lyase activity to mutation of Thr306 is consistent with a reactive intermediate formed independently of proton delivery in the active site, and supports involvement of a nucleophilic peroxo-anion rather than the traditional Compound I in catalysis.

  9. Identification of inhibitors against the potential ligandable sites in the active cholera toxin.

    PubMed

    Gangopadhyay, Aditi; Datta, Abhijit

    2015-04-01

    The active cholera toxin responsible for the massive loss of water and ions in cholera patients via its ADP ribosylation activity is a heterodimer of the A1 subunit of the bacterial holotoxin and the human cytosolic ARF6 (ADP Ribosylation Factor 6). The active toxin is a potential target for the design of inhibitors against cholera. In this study we identified the potential ligandable sites of the active cholera toxin which can serve as binding sites for drug-like molecules. By employing an energy-based approach to identify ligand binding sites, and comparison with the results of computational solvent mapping, we identified two potential ligandable sites in the active toxin which can be targeted during structure-based drug design against cholera. Based on the probe affinities of the identified ligandable regions, docking-based virtual screening was employed to identify probable inhibitors against these sites. Several indole-based alkaloids and phosphates showed strong interactions to the important residues of the ligandable region at the A1 active site. On the other hand, 26 top scoring hits were identified against the ligandable region at the A1 ARF6 interface which showed strong hydrogen bonding interactions, including guanidines, phosphates, Leucopterin and Aristolochic acid VIa. This study has important implications in the application of hybrid structure-based and ligand-based methods against the identified ligandable sites using the identified inhibitors as reference ligands, for drug design against the active cholera toxin.

  10. Encroachment of Human Activity on Sea Turtle Nesting Sites

    NASA Astrophysics Data System (ADS)

    Ziskin, D.; Aubrecht, C.; Elvidge, C.; Tuttle, B.; Baugh, K.; Ghosh, T.

    2008-12-01

    The encroachment of anthropogenic lighting on sea turtle nesting sites poses a serious threat to the survival of these animals [Nicholas, 2001]. This danger is quantified by combining two established data sets. The first is the Nighttime Lights data produced by the NOAA National Geophysical Data Center [Elvidge et al., 1997]. The second is the Marine Turtle Database produced by the World Conservation Monitoring Centre (WCMC). The technique used to quantify the threat of encroachment is an adaptation of the method described in Aubrecht et al. [2008], which analyzes the stress on coral reef systems by proximity to nighttime lights near the shore. Nighttime lights near beaches have both a direct impact on turtle reproductive success since they disorient hatchlings when they mistake land-based lights for the sky-lit surf [Lorne and Salmon, 2007] and the lights are also a proxy for other anthropogenic threats. The identification of turtle nesting sites with high rates of encroachment will hopefully steer conservation efforts to mitigate their effects [Witherington, 1999]. Aubrecht, C, CD Elvidge, T Longcore, C Rich, J Safran, A Strong, M Eakin, KE Baugh, BT Tuttle, AT Howard, EH Erwin, 2008, A global inventory of coral reef stressors based on satellite observed nighttime lights, Geocarto International, London, England: Taylor and Francis. In press. Elvidge, CD, KE Baugh, EA Kihn, HW Kroehl, ER Davis, 1997, Mapping City Lights with Nighttime Data from the DMSP Operational Linescan System, Photogrammatic Engineering and Remote Sensing, 63:6, pp. 727-734. Lorne, JK, M Salmon, 2007, Effects of exposure to artificial lighting on orientation of hatchling sea turtles on the beach and in the ocean, Endangered Species Research, Vol. 3: 23-30. Nicholas, M, 2001, Light Pollution and Marine Turtle Hatchlings: The Straw that Breaks the Camel's Back?, George Wright Forum, 18:4, p77-82. Witherington, BE, 1999, Reducing Threats To Nesting Habitat, Research and Management Techniques for

  11. Reduction of Urease Activity by Interaction with the Flap Covering the Active Site

    PubMed Central

    Macomber, Lee; Minkara, Mona S.; Hausinger, Robert P.; Merz, Kenneth M.

    2015-01-01

    With the increasing appreciation for the human microbiome coupled with the global rise of antibiotic resistant organisms, it is imperative that new methods be developed to specifically target pathogens. To that end, a novel computational approach was devised to identify compounds that reduce the activity of urease, a medically important enzyme of Helicobacter pylori, Proteus mirabilis, and many other microorganisms. Urease contains a flexible loop that covers its active site; Glide was used to identify small molecules predicted to lock this loop in an open conformation. These compounds were screened against the model urease from Klebsiella aerogenes and the natural products epigallocatechin and quercetin were shown to inhibit at low and high micromolar concentrations, respectively. These molecules exhibit a strong time-dependent inactivation of urease that was not due to their oxygen sensitivity. Rather, these compounds appear to inactivate urease by reacting with a specific Cys residue located on the flexible loop. Substitution of this cysteine by alanine in the C319A variant increased the urease resistance to both epigallocatechin and quercetin, as predicted by the computational studies. Protein dynamics are integral to the function of many enzymes; thus, identification of compounds that lock an enzyme into a single conformation presents a useful approach to define potential inhibitors. PMID:25594724

  12. The Sedentary Time and Activity Reporting Questionnaire (STAR-Q): reliability and validity against doubly labeled water and 7-day activity diaries.

    PubMed

    Csizmadi, Ilona; Neilson, Heather K; Kopciuk, Karen A; Khandwala, Farah; Liu, Andrew; Friedenreich, Christine M; Yasui, Yutaka; Rabasa-Lhoret, Rémi; Bryant, Heather E; Lau, David C W; Robson, Paula J

    2014-08-15

    We determined measurement properties of the Sedentary Time and Activity Reporting Questionnaire (STAR-Q), which was designed to estimate past-month activity energy expenditure (AEE). STAR-Q validity and reliability were assessed in 102 adults in Alberta, Canada (2009-2011), who completed 14-day doubly labeled water (DLW) protocols, 7-day activity diaries on day 15, and the STAR-Q on day 14 and again at 3 and 6 months. Three-month reliability was substantial for total energy expenditure (TEE) and AEE (intraclass correlation coefficients of 0.84 and 0.73, respectively), while 6-month reliability was moderate. STAR-Q-derived TEE and AEE were moderately correlated with DLW estimates (Spearman's ρs of 0.53 and 0.40, respectively; P < 0.001), and on average, the STAR-Q overestimated TEE and AEE (median differences were 367 kcal/day and 293 kcal/day, respectively). Body mass index-, age-, sex-, and season-adjusted concordance correlation coefficients (CCCs) were 0.24 (95% confidence interval (CI): 0.07, 0.36) and 0.21 (95% CI: 0.11, 0.32) for STAR-Q-derived versus DLW-derived TEE and AEE, respectively. Agreement between the diaries and STAR-Q (metabolic equivalent-hours/day) was strongest for occupational sedentary time (adjusted CCC = 0.76, 95% CI: 0.64, 0.85) and overall strenuous activity (adjusted CCC = 0.64, 95% CI: 0.49, 0.76). The STAR-Q demonstrated substantial validity for estimating occupational sedentary time and strenuous activity and fair validity for ranking individuals by AEE.

  13. 3,4-Methylenedioxymethamphetamine and 3,4-methylenedioxyamphetamine destroy serotonin terminals in rat brain: quantification of neurodegeneration by measurement of (/sup 3/H)paroxetine-labeled serotonin uptake sites

    SciTech Connect

    Battaglia, G.; Yeh, S.Y.; O'Hearn, E.; Molliver, M.E.; Kuhar, M.J.; De Souza, E.B.

    1987-09-01

    This study examines the effects of repeated systemic administration (20 mg/kg s.c., twice daily for 4 days) of 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA) on levels of brain monoamines, their metabolites and on the density of monoamine uptake sites in various regions of rat brain. Marked reductions (30-60%) in the concentration of 5-hydroxyindoleacetic acid were observed in cerebral cortex, hippocampus, striatum, hypothalamus and midbrain at 2 weeks after a 4-day treatment regimen of MDMA or MDA; less consistent reductions in serotonin (5-HT) content were observed in these brain regions. In addition, both MDMA and MDA caused comparable and substantial reductions (50-75%) in the density of (/sup 3/H)paroxetine-labeled 5-HT uptake sites in all brain regions examined. In contrast, neither MDMA nor MDA caused any widespread or long-term changes in the content of the catecholaminergic markers (i.e., norepinephrine, dopamine, 3,4 dihydroxyphenylacetic acid and homovanillic acid) or in the number of (/sup 3/H)mazindol-labeled norepinephrine or dopamine uptake sites in the brain regions examined. These data demonstrate that MDMA and MDA cause long-lasting neurotoxic effects with respect to both the functional and structural integrity of serotonergic neurons in brain. Furthermore, our measurement of reductions in the density of 5-HT uptake sites provides a means for quantification of the neurodegenerative effects of MDMA and MDA on presynaptic 5-HT terminals.

  14. Indium-111-labeled platelets: effect of heparin on uptake by venous thrombi and relationship to the activated partial thromboplastin time

    SciTech Connect

    Fedullo, P.F.; Moser, K.M.; Moser, K.S.; Konopka, R.; Hartman, M.T.

    1982-09-01

    The goal of heparin thepapy in deep vein thrombosis is to prevent thrombus extension. The relationship between thrombus extension and the results of coagulation tests used to monitor heparin thepapy is unclear. To expose this relationship, we studied the effect of several heparin regimens on the accretion of indium-111-labeled platelets on fresh venous thrombi, as detected by gamma imaging, and monitored the activated partial thromboplastin time (APTT). Six dogs were treated with a 300-U/kg bolus of heparin followed by a 90-U/kg/hour heparin infusion, a dose of heparin sufficient to increase the APTT to levels greater than eight times baseline (APTT ratio); platelet accretion (thrombus imaging) occurred only after the heparin effect was reversed with protamine sulfate. Nineteen dogs were treated with a 150-U/kg bolus of heparin followed by a 4-hour, 45-U/kg/hour heparin infusion; a thrombus was demonstrated only after protamine injection in 12 (mean APTT ratio 1.3 +/- 0.19) and before protamine injection in seven. In thirteen of these 19 dogs, 30 minutes separated the platelet injection from heparin therapy, while in six this duration was less than 30 minutes. In four of these six dogs, thrombi were demonstrated before protamine therapy and at APTT ratios greater than 3.0. Finally, 10 dogs were treated with a 100-U/kg bolus followed by a 3-hour, 50-U/kg/hour heparin infusion, after which the APTT was allowed to return to baseline values spontaneously. In all 10 dogs, a thrombus was demonstrated only after cessation of the heparin infusion, and at a mean APTT ratio of 1.4 +/- 0.15 times baseline. These results suggest that, except with very early platelet injection, platelet accretion by thrombi is consistently inhibited by heparin at APTT ratios greater than 2.5.

  15. Spectroscopic definition of the copper active sites in mordenite: selective methane oxidation.

    PubMed

    Vanelderen, Pieter; Snyder, Benjamin E R; Tsai, Ming-Li; Hadt, Ryan G; Vancauwenbergh, Julie; Coussens, Olivier; Schoonheydt, Robert A; Sels, Bert F; Solomon, Edward I

    2015-05-20

    Two distinct [Cu-O-Cu](2+) sites with methane monooxygenase activity are identified in the zeolite Cu-MOR, emphasizing that this Cu-O-Cu active site geometry, having a ∠Cu-O-Cu ∼140°, is particularly formed and stabilized in zeolite topologies. Whereas in ZSM-5 a similar [Cu-O-Cu](2+) active site is located in the intersection of the two 10 membered rings, Cu-MOR provides two distinct local structures, situated in the 8 membered ring windows of the side pockets. Despite their structural similarity, as ascertained by electronic absorption and resonance Raman spectroscopy, the two Cu-O-Cu active sites in Cu-MOR clearly show different kinetic behaviors in selective methane oxidation. This difference in reactivity is too large to be ascribed to subtle differences in the ground states of the Cu-O-Cu sites, indicating the zeolite lattice tunes their reactivity through second-sphere effects. The MOR lattice is therefore functionally analogous to the active site pocket of a metalloenzyme, demonstrating that both the active site and its framework environment contribute to and direct reactivity in transition metal ion-zeolites.

  16. School Pharmacist/School Environmental Hygienic Activities at School Site.

    PubMed

    Muramatsu, Akiyoshi

    2016-01-01

    The "School Health and Safety Act" was enforced in April 2009 in Japan, and "school environmental health standards" were established by the Minister of Education, Culture, Sports, Science and Technology. In Article 24 of the Enforcement Regulations, the duties of the school pharmacist have been clarified; school pharmacists have charged with promoting health activities in schools and carrying out complete and regular checks based on the "school environmental health standards" in order to protect the health of students and staff. In supported of this, the school pharmacist group of Japan Pharmaceutical Association has created and distributed digital video discs (DVDs) on "check methods of school environmental health standards" as support material. We use the DVD to ensure the basic issues that scho