michaelis constant values: Topics by Science.gov

Sample records for michaelis constant values

Catalytic oxidation of o-aminophenols and aromatic amines by mushroom tyrosinase.

PubMed

Muñoz-Muñoz, Jose Luis; Garcia-Molina, Francisco; Garcia-Ruiz, Pedro Antonio; Varon, Ramon; Tudela, Jose; Rodriguez-Lopez, Jose N; Garcia-Canovas, Francisco

2011-12-01

The kinetics of tyrosinase acting on o-aminophenols and aromatic amines as substrates was studied. The catalytic constants of aromatic monoamines and o-diamines were both low, these results are consistent with our previous mechanism in which the slow step is the transfer of a proton by a hydroxyl to the peroxide in oxy-tyrosinase (Fenoll et al., Biochem. J. 380 (2004) 643-650). In the case of o-aminophenols, the hydroxyl group indirectly cooperates in the transfer of the proton and consequently the catalytic constants in the action of tyrosinase on these compounds are higher. In the case of aromatic monoamines, the Michaelis constants are of the same order of magnitude than for monophenols, which suggests that the monophenols bind better (higher binding constant) to the enzyme to facilitate the π-π interactions between the aromatic ring and a possible histidine of the active site. In the case of aromatic o-diamines, both the catalytic and Michaelis constants are low, the values of the catalytic constants being lower than those of the corresponding o-diphenols. The values of the Michaelis constants of the aromatic o-diamines are slightly lower than those of their corresponding o-diphenols, confirming that the aromatic o-diamines bind less well (lower binding constant) to the enzyme. Copyright © 2011 Elsevier B.V. All rights reserved.
Comparison of in situ and in vitro techniques for measuring ruminal degradation of animal by-product proteins.

PubMed

England, M L; Broderick, G A; Shaver, R D; Combs, D K

1997-11-01

Ruminally undegraded protein (RUP) values of blood meal (n = 2), hydrolyzed feather meal (n = 2), fish meal (n = 2), meat and bone meal, and soybean meal were estimated using an in situ method, an inhibitor in vitro method, and an inhibitor in vitro technique applying Michaelis-Menten saturation kinetics. Degradation rates for in situ and inhibitor in vitro methods were calculated by regression of the natural log of the proportion of crude protein (CP) remaining undegraded versus time. Nonlinear regression analysis of the integrated Michaelis-Menten equation was used to determine maximum velocity, the Michaelis constant, and degradation rate (the ratio of maximum velocity to the Michaelis constant). A ruminal passage rate of 0.06/h was assumed in the calculation of RUP. The in situ and inhibitor in vitro techniques yielded similar estimates of ruminal degradation. Mean RUP estimated for soybean meal, blood meal, hydrolyzed feather meal, fish meal, and meat and bone meal were, respectively, 28.6, 86.0, 77.4, 52.9, and 52.6% of CP by the in situ method and 26.4, 86.1, 76.0, 59.6, and 49.5% of CP by the inhibitor in vitro technique. The Michaelis-Menten inhibitor in vitro technique yielded more rapid CP degradation rates and decreased estimates of RUP. The inhibitor in vitro method required less time and labor than did the other two techniques to estimate the RUP values of animal by-product proteins. Results from in vitro incubations with pepsin.HCl suggested that low postruminal digestibility of hydrolyzed feather meal may impair its value as a source of RUP.
The Original Michaelis Constant: Translation of the 1913 Michaelis-Menten Paper

PubMed Central

Johnson, Kenneth A.; Goody, Roger S.

2011-01-01

Nearly 100 years ago Michaelis and Menten published their now classic paper (Michaelis, L., and Menten, M. L. (1913) Die Kinetik der Invertinwirkung, Biochemische Zeitschrift 49, 333–369), in which they show that the rate of an enzyme-catalyzed reaction is proportional to the concentration of enzyme-substrate complex predicted by the Michaelis-Menten equation. Because the original text was written in German, yet is often quoted by English speaking authors, we undertook a complete translation of the 1913 publication, which we provide as an online supplement (http://pubs.acs.org). Here we introduce the translation, describe the historical context of the work, and show a new analysis of the original data. In doing so, we uncovered several surprises that reveal an interesting glimpse into the early history of enzymology. In particular, our re-analysis of Michaelis and Menten’s data using modern computational methods revealed an unanticipated rigor and precision in the original publication and uncovered a sophisticated, comprehensive analysis that has been overlooked in the century since their work was published. Michaelis and Menten not only analyzed initial velocity measurements, but they also fit their full time course data to the integrated form of the rate equations, including product inhibition, and derived a single global constant to represent all of their data. That constant was not the Michaelis constant, but rather, Vmax/Km, the specificity constant times the enzyme concentration (kcat/Km*E0). PMID:21888353
Co-solvent effects on reaction rate and reaction equilibrium of an enzymatic peptide hydrolysis.

PubMed

Wangler, A; Canales, R; Held, C; Luong, T Q; Winter, R; Zaitsau, D H; Verevkin, S P; Sadowski, G

2018-04-25

This work presents an approach that expresses the Michaelis constant KaM and the equilibrium constant Kth of an enzymatic peptide hydrolysis based on thermodynamic activities instead of concentrations. This provides KaM and Kth values that are independent of any co-solvent. To this end, the hydrolysis reaction of N-succinyl-l-phenylalanine-p-nitroanilide catalysed by the enzyme α-chymotrypsin was studied in pure buffer and in the presence of the co-solvents dimethyl sulfoxide, trimethylamine-N-oxide, urea, and two salts. A strong influence of the co-solvents on the measured Michaelis constant (KM) and equilibrium constant (Kx) was observed, which was found to be caused by molecular interactions expressed as activity coefficients. Substrate and product activity coefficients were used to calculate the activity-based values KaM and Kth for the co-solvent free reaction. Based on these constants, the co-solvent effect on KM and Kx was predicted in almost quantitative agreement with the experimental data. The approach presented here does not only reveal the importance of understanding the thermodynamic non-ideality of reactions taking place in biological solutions and in many technological applications, it also provides a framework for interpreting and quantifying the multifaceted co-solvent effects on enzyme-catalysed reactions that are known and have been observed experimentally for a long time.
Exact and Approximate Solutions for the Decades-Old Michaelis-Menten Equation: Progress-Curve Analysis through Integrated Rate Equations

ERIC Educational Resources Information Center

Golicnik, Marko

2011-01-01

The Michaelis-Menten rate equation can be found in most general biochemistry textbooks, where the time derivative of the substrate is a hyperbolic function of two kinetic parameters (the limiting rate "V", and the Michaelis constant "K"[subscript M]) and the amount of substrate. However, fundamental concepts of enzyme kinetics can be difficult to…
Bringing metabolic networks to life: convenience rate law and thermodynamic constraints

PubMed Central

Liebermeister, Wolfram; Klipp, Edda

2006-01-01

Background Translating a known metabolic network into a dynamic model requires rate laws for all chemical reactions. The mathematical expressions depend on the underlying enzymatic mechanism; they can become quite involved and may contain a large number of parameters. Rate laws and enzyme parameters are still unknown for most enzymes. Results We introduce a simple and general rate law called "convenience kinetics". It can be derived from a simple random-order enzyme mechanism. Thermodynamic laws can impose dependencies on the kinetic parameters. Hence, to facilitate model fitting and parameter optimisation for large networks, we introduce thermodynamically independent system parameters: their values can be varied independently, without violating thermodynamical constraints. We achieve this by expressing the equilibrium constants either by Gibbs free energies of formation or by a set of independent equilibrium constants. The remaining system parameters are mean turnover rates, generalised Michaelis-Menten constants, and constants for inhibition and activation. All parameters correspond to molecular energies, for instance, binding energies between reactants and enzyme. Conclusion Convenience kinetics can be used to translate a biochemical network – manually or automatically - into a dynamical model with plausible biological properties. It implements enzyme saturation and regulation by activators and inhibitors, covers all possible reaction stoichiometries, and can be specified by a small number of parameters. Its mathematical form makes it especially suitable for parameter estimation and optimisation. Parameter estimates can be easily computed from a least-squares fit to Michaelis-Menten values, turnover rates, equilibrium constants, and other quantities that are routinely measured in enzyme assays and stored in kinetic databases. PMID:17173669
Exact and approximate solutions for the decades-old Michaelis-Menten equation: Progress-curve analysis through integrated rate equations.

PubMed

Goličnik, Marko

2011-01-01

The Michaelis-Menten rate equation can be found in most general biochemistry textbooks, where the time derivative of the substrate is a hyperbolic function of two kinetic parameters (the limiting rate V, and the Michaelis constant K(M) ) and the amount of substrate. However, fundamental concepts of enzyme kinetics can be difficult to understand fully, or can even be misunderstood, by students when based only on the differential form of the Michaelis-Menten equation, and the variety of methods available to calculate the kinetic constants from rate versus substrate concentration "textbook data." Consequently, enzyme kinetics can be confusing if an analytical solution of the Michaelis-Menten equation is not available. Therefore, the still rarely known exact solution to the Michaelis-Menten equation is presented here through the explicit closed-form equation in terms of the Lambert W(x) function. Unfortunately, as the W(x) is not available in standard curve-fitting computer programs, the practical use of this direct solution is limited for most life-science students. Thus, the purpose of this article is to provide analytical approximations to the equation for modeling Michaelis-Menten kinetics. The elementary and explicit nature of these approximations can provide students with direct and simple estimations of kinetic parameters from raw experimental time-course data. The Michaelis-Menten kinetics studied in the latter context can provide an ideal alternative to the 100-year-old problems of data transformation, graphical visualization, and data analysis of enzyme-catalyzed reactions. Hence, the content of the course presented here could gradually become an important component of the modern biochemistry curriculum in the 21st century. Copyright © 2011 Wiley Periodicals, Inc.
Diffusional falsification of kinetic constants on Lineweaver-Burk plots.

PubMed

Ghim, Y S; Chang, H N

1983-11-07

The effect of mass transfer resistances on the Lineweaver-Burk plots in immobilized enzyme systems has been investigated numerically and with analytical approximate solutions. While Hamilton, Gardner & Colton (1974) studied the effect of internal diffusion resistances in planar geometry, our study was extended to the combined effect of internal and external diffusion in cylindrical and spherical geometries as well. The variation of Lineweaver-Burk plots with respect to the geometries was minimized by modifying the Thiele modulus and the Biot number with the shape factor. Especially for a small Biot number all the three Lineweaver-Burk plots fell on a single line. As was discussed by Hamilton et al. (1974), the curvature of the line for large external diffusion resistances was small enough to be assumed linear, which was confirmed from the two approximate solutions for large and small substrate concentrations. Two methods for obtaining intrinsic kinetic constants were proposed: First, we obtained both maximum reaction rate and Michaelis constant by fitting experimental data to a straight line where external diffusion resistance was relatively large, and second, we obtained Michaelis constant from apparent Michaelis constant from the figure in case we knew maximum reaction rate a priori.
Beyond Popper and Polanyi: Leonor Michaelis, a critical and passionate pioneer of research at the interface of medicine, enzymology, and physical chemistry.

PubMed

Deichmann, Ute

2012-01-01

The biochemist and biophysicist Leonor Michaelis (1875-1949) was a renowned pioneer who worked at the interface of physical chemistry and biochemistry. He is best known for his work on the physical chemistry of proteins and enzymes and for the mathematical derivation, together with Maud Menten, of the affinity constant of the enzyme substrate bond, now known as the Michaelis-Menten constant. His thorough experimentation and careful theorizing made him critical of his contemporaries in medical biochemistry, whose work did not withstand scrutiny. His strong influence resulted from combining new concepts and approaches with traditional ones, thus bridging conceptual gaps. Most importantly, his success was brought about because he combined a critical and sharp mind with competence, passion, and determination. A review of Michaelis's approach and achievements shows that critical theory testing, as suggested by Popper, cannot explain scientific advance if taken alone; the existence of a passionate commitment to scientific beliefs, as emphasized by Polanyi, is another necessary prerequisite for the development of science.
Reformulation of the Michaelis-Menten Equation: How Enzyme-Catalyzed Reactions Depend on Gibbs Energy

ERIC Educational Resources Information Center

Bozlee, Brian J.

2007-01-01

The impact of raising Gibbs energy of the enzyme-substrate complex (G[subscript 3]) and the reformulation of the Michaelis-Menten equation are discussed. The maximum velocity of the reaction (v[subscript m]) and characteristic constant for the enzyme (K[subscript M]) will increase with increase in Gibbs energy, indicating that the rate of reaction…
Perturbation theory in the catalytic rate constant of the Henri-Michaelis-Menten enzymatic reaction.

PubMed

Bakalis, Evangelos; Kosmas, Marios; Papamichael, Emmanouel M

2012-11-01

The Henry-Michaelis-Menten (HMM) mechanism of enzymatic reaction is studied by means of perturbation theory in the reaction rate constant k (2) of product formation. We present analytical solutions that provide the concentrations of the enzyme (E), the substrate (S), as well as those of the enzyme-substrate complex (C), and the product (P) as functions of time. For k (2) small compared to k (-1), we properly describe the entire enzymatic activity from the beginning of the reaction up to longer times without imposing extra conditions on the initial concentrations E ( o ) and S ( o ), which can be comparable or much different.
Inhibition of purine phosphoribosyltransferases of Ehrlich ascites-tumour cells by 6-mercaptopurine

PubMed Central

Atkinson, M. R.; Murray, A. W.

1965-01-01

1. The formation of adenosine 5′-phosphate, guanosine 5′-phosphate and inosine 5′-phosphate from [8-14C]adenine, [8-14C]guanine and [8-14C]hypoxanthine respectively in the presence of 5-phosphoribosyl pyrophosphate and an extract from Ehrlich ascites-tumour cells was assayed by a method involving liquid-scintillation counting of the radioactive nucleotides on diethylaminoethylcellulose paper. The results obtained with guanine were confirmed by a spectrophotometric assay which was also used to assay the conversion of 6-mercaptopurine and 5-phosphoribosyl pyrophosphate into 6-thioinosine 5′-phosphate in the presence of 6-mercaptopurine phosphoribosyltransferase from these cells. 2. At pH 7·8 and 25° the Michaelis constants for adenine, guanine and hypoxanthine were 0·9 μm, 2·9 μm and 11·0 μm in the assay with radioactive purines; the Michaelis constant for guanine in the spectrophotometric assay was 2·6 μm. At pH 7·9 the Michaelis constant for 6-mercaptopurine was 10·9 μm. 3. 25 μm-6-Mercaptopurine did not inhibit adenine phosphoribosyltransferase. 6-Mercaptopurine is a competitive inhibitor of guanine phosphoribosyltransferase (Ki 4·7 μm) and hypoxanthine phosphoribosyltransferase (Ki 8·3 μm). Hypoxanthine is a competitive inhibitor of guanine phosphoribosyltransferase (Ki 3·4 μm). 4. Differences in kinetic parameters and in the distribution of phosphoribosyltransferase activities after electrophoresis in starch gel indicate that different enzymes are involved in the conversion of adenine, guanine and hypoxanthine into their nucleotides. 5. From the low values of Ki for 6-mercaptopurine, and from published evidence that ascites-tumour cells require supplies of purines from the host tissues, it is likely that inhibition of hypoxanthine and guanine phosphoribosyltransferases by free 6-mercaptopurine is involved in the biological activity of this drug. PMID:14342250
Quantification of biodegradation for o-xylene and naphthalene using first order decay models, Michaelis-Menten kinetics and stable carbon isotopes.

PubMed

Blum, Philipp; Hunkeler, Daniel; Weede, Matthias; Beyer, Christof; Grathwohl, Peter; Morasch, Barbara

2009-04-01

At a former wood preservation plant severely contaminated with coal tar oil, in situ bulk attenuation and biodegradation rate constants for several monoaromatic (BTEX) and polyaromatic hydrocarbons (PAH) were determined using (1) classical first order decay models, (2) Michaelis-Menten degradation kinetics (MM), and (3) stable carbon isotopes, for o-xylene and naphthalene. The first order bulk attenuation rate constant for o-xylene was calculated to be 0.0025 d(-1) and a novel stable isotope-based first order model, which also accounted for the respective redox conditions, resulted in a slightly smaller biodegradation rate constant of 0.0019 d(-1). Based on MM-kinetics, the o-xylene concentration decreased with a maximum rate of k(max)=0.1 microg/L/d. The bulk attenuation rate constant of naphthalene retrieved from the classical first order decay model was 0.0038 d(-1). The stable isotope-based biodegradation rate constant of 0.0027 d(-1) was smaller in the reduced zone, while residual naphthalene in the oxic part of the plume further downgradient was degraded at a higher rate of 0.0038 d(-1). With MM-kinetics a maximum degradation rate of k(max)=12 microg/L/d was determined. Although best fits were obtained by MM-kinetics, we consider the carbon stable isotope-based approach more appropriate as it is specific for biodegradation (not overall attenuation) and at the same time accounts for the dominant electron-accepting process. For o-xylene a field based isotope enrichment factor epsilon(field) of -1.4 could be determined using the Rayleigh model, which closely matched values from laboratory studies of o-xylene degradation under sulfate-reducing conditions.
Affinity of microbial transglutaminase to αs1-, β-, and acid casein under atmospheric and high pressure conditions.

PubMed

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

2009-05-27

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

NASA Astrophysics Data System (ADS)

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

1996-06-01

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

ERIC Educational Resources Information Center

Alberty, Robert A.

2008-01-01

Rapid-equilibrium rate equations for enzyme-catalyzed reactions are especially useful because if experimental data can be fit by these simpler rate equations, the Michaelis constants can be interpreted as equilibrium constants. However, for some reactions it is necessary to use the more complicated steady-state rate equations. Thermodynamics is…
Kinetic study of the oxidation of 4-hydroxyanisole catalyzed by tyrosinase.

PubMed

Espín, J C; Varón, R; Tudela, J; García-Cánovas, F

1997-05-01

Despite the importance of the substrate 4-hydroxyanisole in melanoma therapy, the kinetics of its oxidation catalyzed by tyrosinase has never been properly characterized. This approach is reported here for the first time. The applicability to 4-hydroxyanisole of the reaction mechanism of tyrosinase previously proposed for other monophenols has been corroborated. The Michaelis constant for the oxidation of 4-hydroxyanisole catalyzed by mushroom tyrosinase was (62 +/- 1.5) microM at pH 7 and increased when the pH decreased, reaching a value of (195 +/- 5) microM at pH 5.5. However the maximum steady-state rate, whose value was (0.54 +/- 0.01) microM/min, did not change with the pH. The apparent catalytic constant was (184 +/- 5) s-1, around twenty three times higher than that previously described for L-tyrosine (8 s-1).
Modification of kinetic parameters of glycogen phosphorylase from mantle tissue of Mytilus galloprovincialis by a phosphorylation mechanism.

PubMed

San Juan Serrano, F; Fernández González, M; Sánchez López, J L; García Martín, L O

1995-09-01

Initial rate and affinity studies on mantle Mytilus phosphorylase a were carried out in order to find possible differences in its kinetic properties with respect to phosphorylase b. Phosphorylase a was not stimulated for any AMP concentrations. Michaelis constants (Km) are 0.05 mg/ml glycogen, 1.15 mM inorganic phosphate and 1.50 mM glucose-1-phosphate. The Kms for the substrates, in the direction of glycogen breakdown, are enhanced by non-saturating concentrations of cosubstrate, without reducing the apparent maximum velocity. First order and hyperbolic kinetics and values of the allosteric constant smaller than 2 were observed. These results suggest a catalytic mechanism different to that shown for mantle Mytilus phosphorylase b.
Bioconjugation of zirconium uridine monophosphate: application to myoglobin direct electrochemistry.

PubMed

Qiao, Yuanbiao; Jian, Fangfang; Bai, Qian

2008-03-14

Porous nano-granule of zirconium uridine monophosphate, Zr(UMP)2.H2O is, for the first time, synthesized under mild experimental conditions and applied to the bioconjugation of myoglobin (Mb) to realize its direct electron transfer. UV-vis and resonance Raman spectroscopies prove that Mb in the Zr(UMP)2.H2O film maintains its secondary structure similar to the native state. The conjugation film of the Mb-Zr(UMP)2.H2O on the glassy carbon (GC) electrode gives a well-defined and quasi-reversible cyclic voltammogram, which reflects the direct electron transfer of the heme Fe III/Fe II couple of Mb. On the basis of the satisfying bioelectrocatalysis of the nano-conjugation of Mb and genetic substrate, a kind of mediator-free biosensor for H2O2 is developed. The linear range for H2O2 detection is estimated to be 3.92-180.14 microM. The apparent Michaelis-Menten constant (Km) and the detection limit based on the signal-to-noise ratio of 3 are found to be 196.1 microM and 1.52 microM, respectively. Both the apparent Michaelis-Menten constant and the detection limit herein are much lower than currently reported values from other Mb films. This kind of sensor possesses excellent stability, long-term life (more than 20 days) and good reproducibility.
Purification and Thermal Dependence of Glutathione Reductase from Two Forage Legume Species 1

PubMed Central

Kidambi, Saranga P.; Mahan, James R.; Matches, Arthur G.

1990-01-01

Alfalfa (Medicago sativa L.) and sainfoin (Onobrychis viciifolia Scop.) are forage legumes that differ in their responses to high and low temperature stresses. Thermal limitations on the function of glutathione reductase (EC 1.6.4.2) could adversely affect the ability of the plant to cope with adverse temperatures. Our objectives were to (a) purify glutathione reductase from `Cimarron' alfalfa and `PI 212241' sainfoin and (b) investigate the intraspecies variation in the thermal dependency of glutathione reductase from each of three cultivars of alfalfa and two cultivars and an introduction of sainfoin. Glutathione reductase was purified 1222-and 1948-fold to a specific activity of 281 and 273 units per milligram of protein, from one species each of alfalfa and sainfoin, respectively. The relative molecular mass of the protein was approximately 140 kilodaltons with subunits of 57 and 37 kilodaltons under denaturing conditions. The activation energies were approximately 50 kilojoules per mole for both species. Over a 5 to 45°C temperature gradient, large variation among species and genotypes within species was found for: (a) the minimum apparent Michaelis constant (0.6-2.1 micromoles of NADPH), (b) the temperature at which the minimum apparent Michaelis constant was observed (10-25°C), and (c) the thermal kinetic windows (6-19°C width). Future studies will focus on relating the thermal dependence of the Michaelis constant of the glutathione reductases and plant growth rates and forage quality of these species throughout the growing season. PMID:16667283

Kinetic Parameters for the Noncatalyzed and Enzyme-Catalyzed Mutarotation of Glucose Using a Blood Glucometer

ERIC Educational Resources Information Center

Hardee, John R.; Delgado, Bryan; Jones, Wray

2011-01-01

The kinetic parameters for the conversion of alpha-D-glucose to beta-D-glucose were measured using a blood glucometer. The reaction order, rate constant, and Arrhenius activation energy are reported for the noncatalyzed reaction and turnover number and Michaelis constant are reported for the reaction catalyzed by porcine kidney mutarotase. The…
Ortho effects in quantitative structure-activity relationships for acetylcholinesterase inhibition by aryl carbamates.

PubMed

Lin, Gialih; Liu, Yu-Chen; Lin, Yan-Fu; Wu, Yon-Gi

2004-10-01

Ortho-substituted phenyl-N-butyl carbamates (1-9) are characterized as "pseudo-pseudo-substrate" inhibitors of acetylcholinesterase. Since the inhibitors protonate at pH 7.0 buffer solution, the virtual inhibition constants (K'is) of the protonated inhibitors are calculated from the equation, - logK'i = - logKi - logKb. The logarithms of the inhibition constant (Ki), the carbamylation constant (k(c)), and the bimolecular inhibition constant (k(i)) for the enzyme inhibitions by carbamates 1-9 are multiply linearly correlated with the Hammett para-substituent constant (sigma(p)), the Taft-Kutter-Hansch ortho steric constant (E(S)), and the Swan-Lupton ortho polar constant (F). Values of rho, delta, and f for the - logKi-, logk(c)-, and logk(i)-correlations are -0.6, -0.16, 0.7; 0.11, 0.03, -0.3; and - 0.5, - 0.12, 0.4, respectively. The Ki step further divides into two steps: 1) the pre-equilibrium protonation of the inhibitors, Kb step and 2) formation of a negatively charged enzyme-inhibitor Michaelis-Menten complex--virtual inhibition, K'i step. The Ki step has little ortho steric enhancement effect; moreover, the k(c)step is insensitive to the ortho steric effect. The f value of 0.7 for the Ki step indicates that ortho electron-withdrawing substituents of the inhibitors accelerate the inhibition reactions from the ortho polar effect; however, the f value of -0.3 for the k(c)step implies that ortho electron-withdrawing substituents of the inhibitors lessen the inhibition reactions from the ortho polar effect.
Kinetic analysis of a Michaelis-Menten mechanism in which the enzyme is unstable.

PubMed Central

Garrido-del Solo, C; García-Cánovas, F; Havsteen, B H; Varón-Castellanos, R

1993-01-01

A kinetic analysis of the Michaelis-Menten mechanism is made for the cases in which the free enzyme, or the enzyme-substrate complex, or both, are unstable, either spontaneously or as a result of the addition of a reagent. The explicit time-course equations of all of the species involved has been derived under conditions of limiting enzyme concentration. The validity of these equations has been checked by using numerical simulations. An experimental design and a kinetic data analysis allowing the evaluation of the parameters and kinetic constants are recommended. PMID:8373361
Differential substrate behaviour of phenol and aniline derivatives during oxidation by horseradish peroxidase: kinetic evidence for a two-step mechanism.

PubMed

Gilabert, María Angeles; Hiner, Alexander N P; García-Ruiz, Pedro Antonio; Tudela, José; García-Molina, Francisco; Acosta, Manuel; García-Cánovas, Francisco; Rodríguez-López, José Neptuno

2004-06-01

The catalytic constant (k(cat)) and the second-order association constant of compound II with reducing substrate (k(5)) of horseradish peroxidase C (HRPC) acting on phenols and anilines have been determined from studies of the steady-state reaction velocities (V(0) vs. [S(0)]). Since k(cat)=k(2)k(6)/k(2)+k(6), and k(2) (the first-order rate constant for heterolytic cleavage of the oxygen-oxygen bond of hydrogen peroxide during compound I formation) is known, it has been possible to calculate the first-order rate constant for the transformation of each phenol or aniline by HRPC compound II (k(6)). The values of k(6) are quantitatively correlated to the sigma values (Hammett equation) and can be rationalized by an aromatic substrate oxidation mechanism in which the substrate donates an electron to the oxyferryl group in HRPC compound II, accompanied by two proton additions to the ferryl oxygen atom, one from the substrate and the other the protein or solvent. k(6) is also quantitatively correlated to the experimentally determined (13)C-NMR chemical shifts (delta(1)) and the calculated ionization potentials, E (HOMO), of the substrates. Similar dependencies were observed for k(cat) and k(5). From the kinetic analysis, the absolute values of the Michaelis constants for hydrogen peroxide and the reducing substrates (K(M)(H(2)O(2)) and K(M)(S)), respectively, were obtained.
Determination of the Michaelis-Menten kinetics and the genes expression involved in phyto-degradation of cyanide and ferri-cyanide.

PubMed

Yu, Xiao-Zhang; Zhang, Xue-Hong

2016-07-01

Hydroponic experiments were conducted with different species of plants (rice, maize, soybean and willow) exposed to ferri-cyanide to investigate the half-saturation constant (K M ) and the maximal metabolic capacity (v max ) involved in phyto-assimilation. Three varieties for each testing species were collected from different origins. Measured concentrations show that the uptake rates responded biphasically to ferri-cyanide treatments by showing increases linearly at low and almost constant at high concentrations from all treatments, indicating that phyto-assimilation of ferri-cyanide followed the Michaelis-Menten kinetics. Using non-linear regression, the highest v max was by rice, followed by willows. The lowest v max was found for soybean. All plants, except maize (DY26) and rice (XJ12), had a similar K M value, suggesting the same enzyme was active in phyto-assimilation of ferri-cyanide. Transcript level, by real-time quantitative PCR, of enzymes involved in degradation of cyanides showed that the analyzed genes were differently expressed during different cyanides exposure. The expression of CAS and ST genes responded positively to KCN exposure, suggesting that β-CAS and ST pathways were two possible pathways for cyanide detoxification in rice. The transcript level of NIT and ASPNASE genes also showed a remarkable up-regulation to KCN, implying the contribution to the pool of amino acid aspartate, which is an end product of CN metabolism. Up-regulation of GS genes suggests that acquisition of ammonium released from cyanide degradation may be an additional nitrogen source for plant nutrition. Results also revealed that the expressions of these genes, except for GS, were relatively constant during iron cyanide exposure, suggesting that they are likely metabolized by plants through a non-defined pathway rather than the β-CAS pathway.
Phosphatidylglycerol synthesis in castor bean endosperm. [Ricinus communis

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

Moore, T.S. Jr.

1974-01-01

The synthesis of phosphatidylglycerol in castor bean (Ricinus communis var. Hale) endosperm tissue was found to be located in both the endoplasmic reticulum and mitochondrial fractions separated on sucrose density gradients. The enzyme of both fractions attained maximum activity at 5 mM Mn/sup 2 +/, 0.075 percent Triton X-100, and pH 7.3. The addition of dithiothreitol produced little effect, but sulfhydryl inhibitors reduced activity in both systems. Cytidine diphosphate-diglyceride exhibited an apparent Michaelis constant for the endoplasmic reticulum enzyme of 2.8 ..mu..M and for the mitochondrial enzyme of 2.0 ..mu..M; the maximum reaction rate was achieved at about 20 ..mu..M.more » For the second substrate, glycerol-phosphate, the apparent Michaelis constant for both fractions was about 50 ..mu..M and maximum velocity was reached at 400 ..mu..M. The specific activity of the mitochondrial enzyme was generally twice that of the endoplasmic reticulum.« less
Microscale Measurements of Michaelis-Menten Constants of Neuraminidase with Nanogel Capillary Electrophoresis for the Determination of the Sialic Acid Linkage.

PubMed

Gattu, Srikanth; Crihfield, Cassandra L; Holland, Lisa A

2017-01-03

Phospholipid nanogels enhance the stability and performance of the exoglycosidase enzyme neuraminidase and are used to create a fixed zone of enzyme within a capillary. With nanogels, there is no need to covalently immobilize the enzyme, as it is physically constrained. This enables rapid quantification of Michaelis-Menten constants (K M ) for different substrates and ultimately provides a means to quantify the linkage (i.e., 2-3 versus 2-6) of sialic acids. The fixed zone of enzyme is inexpensive and easily positioned in the capillary to support electrophoresis mediated microanalysis using neuraminidase to analyze sialic acid linkages. To circumvent the limitations of diffusion during static incubation, the incubation period is reproducibly achieved by varying the number of forward and reverse passes the substrate makes through the stationary fixed zone using in-capillary electrophoretic mixing. A K M value of 3.3 ± 0.8 mM (V max , 2100 ± 200 μM/min) was obtained for 3'-sialyllactose labeled with 2-aminobenzoic acid using neuraminidase from Clostridium perfringens that cleaves sialic acid monomers with an α2-3,6,8,9 linkage, which is similar to values reported in the literature that required benchtop analyses. The enzyme cleaves the 2-3 linkage faster than the 2-6, and a K M of 2 ± 1 mM (V max , 400 ± 100 μM/min) was obtained for the 6'-sialyllactose substrate. An alternative neuraminidase selective for 2-3 sialic acid linkages generated a K M value of 3 ± 2 mM (V max , 900 ± 300 μM/min) for 3'-sialyllactose. With a knowledge of V max , the method was applied to a mixture of 2-3 and 2-6 sialyllactose as well as 2-3 and 2-6 sialylated triantennary glycan. Nanogel electrophoresis is an inexpensive, rapid, and simple alternative to current technologies used to distinguish the composition of 3' and 6' sialic acid linkages.
The Power of Integrating Kinetic Isotope Effects into the Formalism of the Michaelis-Menten Equation

PubMed Central

Klinman, Judith P.

2014-01-01

The final arbiter of enzyme mechanism is the ability to establish and test a kinetic mechanism. Isotope effects play a major role in expanding the scope and insight derived from the Michaelis-Menten equation. The integration of isotope effects into the formalism of the Michaelis-Menten equation began in the 1970s and has continued to this day. This review discusses a family of eukaryotic copper proteins that includes dopamine β-monooxygenase, tyramine β-monooxygenase, and peptidylglycine α-amidating enzyme, responsible for the synthesis of the neuro-active compounds, norepinephrine, octopamine and C-terminally carboxamidated peptides, respectively. Highlighted are results that show how combining kinetic isotope effects with initial rate parameters permits an evaluation of: (i) the order of substrate binding to multi-substrate enzymes; (ii) the magnitude of individual rate constants in complex, multi-step reactions; (iii) the identification of chemical intermediates; and (iv) the role of non-classical (tunneling) behavior in C–H activation. PMID:23937475
Transient competitive complexation in biological kinetic isotope fractionation explains non-steady isotopic effects: Theory and application to denitrification in soils

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

Maggi, F.M.; Riley, W.J.

2009-06-01

The theoretical formulation of biological kinetic reactions in isotopic applications often assume first-order or Michaelis-Menten-Monod kinetics under the quasi-steady-state assumption to simplify the system kinetics. However, isotopic e ects have the same order of magnitude as the potential error introduced by these simpli cations. Both formulations lead to a constant fractionation factor which may yield incorrect estimations of the isotopic effect and a misleading interpretation of the isotopic signature of a reaction. We have analyzed the isotopic signature of denitri cation in biogeochemical soil systems by Menyailo and Hungate [2006], where high {sup 15}N{sub 2}O enrichment during N{sub 2}O productionmore » and inverse isotope fractionation during N{sub 2}O consumption could not be explained with first-order kinetics and the Rayleigh equation, or with the quasi-steady-state Michaelis-Menten-Monod kinetics. When the quasi-steady-state assumption was relaxed, transient Michaelis-Menten-Monod kinetics accurately reproduced the observations and aided in interpretation of experimental isotopic signatures. These results may imply a substantial revision in using the Rayleigh equation for interpretation of isotopic signatures and in modeling biological kinetic isotope fractionation with first-order kinetics or quasi-steady-state Michaelis-Menten-Monod kinetics.« less
Preparation and characterization of a dextran-amylase conjugate.

PubMed

Marshall, J J

1976-07-01

Bacillus amyloliquefaciens alpha-amylase was attached to dextran after activation of the polysaccharide by using a modification of the cyanogen bromide method. The soluble dextran-amylase conjugate was purified by molecular-sieve chromatography. The conjugated enzyme has greater stability than the unmodified enzyme at low pH values, during heat treatment, and on removal of calcium ions with a chelating agent. Attachment of dextran to alpha-amylase did not alter the Michaelis constant of the enzyme acting on starch. The polysaccharide-enzyme conjugate probably consists of a cross-linked aggregate of many dextran and many enzyme molecules, in which a proportion of the enzyme molecules, although not inactivated, are unable to express their activity, except after dextranase treatment.
DEVELOPMENT OF A PHYSIOLOGICALLY BASED PHARMACOKINETIC MODEL FOR DELTAMETHRIN IN DEVELOPING SPRAGUE-DAWLEY RATS

EPA Science Inventory

This work describes the development of a physiologically based pharmacokinetic (PBPK) model of deltamethrin, a type II pyrethroid, in the developing male Sprague-Dawley rat. Generalized Michaelis-Menten equations were used to calculate metabolic rate constants and organ weights ...
An approach to bioassessment of water quality using diversity measures based on species accumulative curves.

PubMed

Xu, Guangjian; Zhang, Wei; Xu, Henglong

2015-02-15

Traditional community-based bioassessment is time-consuming because they rely on full species-abundance data of a community. To improve bioassessment efficiency, the feasibility of the diversity measures based on species accumulative curves for bioassessment of water quality status was studied based on a dataset of microperiphyton fauna. The results showed that: (1) the species accumulative curves well fitted the Michaelis-Menten equation; (2) the β- and γ-diversity, as well as the number of samples to 50% of the maximum species number (Michaelis-Menten constant K), can be statistically estimated based on the formulation; (3) the rarefied α-diversity represented a significant negative correlation with the changes in the nutrient NH4-N; and (4) the estimated β-diversity and the K constant were significantly positively related to the concentration of NH4-N. The results suggest that the diversity measures based on species accumulative curves might be used as a potential bioindicator of water quality in marine ecosystems. Copyright © 2014 Elsevier Ltd. All rights reserved.
DEVELOPMENT OF A PHYSIOLOGICALLY BASED PHARMACOKINETIC MODEL FOR DELTAMETHRIN IN ADULT AND DEVELOPING SPRAGUE-DAWLEY RATS

EPA Science Inventory

This work describes the development of a physiologically based pharmacokinetic (PBPK) model of deltamethrin, a type II pyrethroid, in the developing male Sprague-Dawley rat. Generalized Michaelis-Menten equations were used to calculate metabolic rate constants and organ weights ...
Effects of multi-frequency power ultrasound on the enzymolysis of corn gluten meal: Kinetics and thermodynamics study.

PubMed

Jin, Jian; Ma, Haile; Qu, Wenjuan; Wang, Kai; Zhou, Cunshan; He, Ronghai; Luo, Lin; Owusu, John

2015-11-01

The effects of multi-frequency power ultrasound (MPU) pretreatment on the kinetics and thermodynamics of corn gluten meal (CGM) were investigated in this research. The apparent constant (KM), apparent break-down rate constant (kA), reaction rate constants (k), energy of activation (Ea), enthalpy of activation (ΔH), entropy of activation (ΔS) and Gibbs free energy of activation (ΔG) were determined by means of the Michaelis-Menten equation, first-order kinetics model, Arrhenius equation and transition state theory, respectively. The results showed that MPU pretreatment can accelerate the enzymolysis of CGM under different enzymolysis conditions, viz. substrate concentration, enzyme concentration, pH, and temperature. Kinetics analysis revealed that MPU pretreatment decreased the KM value by 26.1% and increased the kA value by 7.3%, indicating ultrasound pretreatment increased the affinity between enzyme and substrate. In addition, the values of k for ultrasound pretreatment were increased by 84.8%, 41.9%, 28.9%, and 18.8% at the temperature of 293, 303, 313 and 323 K, respectively. For the thermodynamic parameters, ultrasound decreased Ea, ΔH and ΔS by 23.0%, 24.3% and 25.3%, respectively, but ultrasound had little change in ΔG value in the temperature range of 293-323 K. In conclusion, MPU pretreatment could remarkably enhance the enzymolysis of CGM, and this method can be applied to protein proteolysis industry to produce peptides. Copyright © 2015 Elsevier B.V. All rights reserved.
Kinetics of Ethylene and Ethylene Oxide in Subcellular Fractions of Lungs and Livers of Male B6C3F1 Mice and Male Fischer 344 Rats and of Human Livers

PubMed Central

Csanády, György András; Kessler, Winfried; Klein, Dominik; Pankratz, Helmut; Pütz, Christian; Richter, Nadine; Filser, Johannes Georg

2011-01-01

Ethylene (ET) is metabolized in mammals to the carcinogenic ethylene oxide (EO). Although both gases are of high industrial relevance, only limited data exist on the toxicokinetics of ET in mice and of EO in humans. Metabolism of ET is related to cytochrome P450-dependent mono-oxygenase (CYP) and of EO to epoxide hydrolase (EH) and glutathione S-transferase (GST). Kinetics of ET metabolism to EO and of elimination of EO were investigated in headspace vessels containing incubations of subcellular fractions of mouse, rat, or human liver or of mouse or rat lung. CYP-associated metabolism of ET and GST-related metabolism of EO were found in microsomes and cytosol, respectively, of each species. EH-related metabolism of EO was not detectable in hepatic microsomes of rats and mice but obeyed saturation kinetics in hepatic microsomes of humans. In ET-exposed liver microsomes, metabolism of ET to EO followed Michaelis-Menten-like kinetics. Mean values of Vmax [nmol/(min·mg protein)] and of the apparent Michaelis constant (Km [mmol/l ET in microsomal suspension]) were 0.567 and 0.0093 (mouse), 0.401 and 0.031 (rat), and 0.219 and 0.013 (human). In lung microsomes, Vmax values were 0.073 (mouse) and 0.055 (rat). During ET exposure, the rate of EO production decreased rapidly. By modeling a suicide inhibition mechanism, rate constants for CYP-mediated catalysis and CYP inactivation were estimated. In liver cytosol, mean GST activities to EO expressed as Vmax/Km [μl/(min·mg protein)] were 27.90 (mouse), 5.30 (rat), and 1.14 (human). The parameters are most relevant for reducing uncertainties in the risk assessment of ET and EO. PMID:21785163
Competition between roots and microorganisms for nitrogen: mechanisms and ecological relevance

NASA Astrophysics Data System (ADS)

Kuzyakov, Yakov; Xu, Xingliang

2014-05-01

Demand of all living organisms on the same nutrients forms the basis for interspecific competition between plants and microorganisms in soils. This competition is especially strong in the rhizosphere. To evaluate competitive and mutualistic interactions between plants and microorganisms and to analyse ecological consequences of these interactions, we analysed 424 data pairs from 41 15N-labelling studies that investigated 15N redistribution between roots and microorganisms. Calculated Michaelis-Menten kinetics based on Km (Michaelis constant) and Vmax (maximum uptake capacity) values from 77 studies on the uptake of nitrate, ammonia, and amino acids by roots and microorganisms clearly showed that, shortly after nitrogen (N) mobilization from soil organic matter and litter, microorganisms take up most N. Lower Km values of microorganisms suggest that they are especially efficient at low N concentrations, but can also acquire more N at higher N concentrations (Vmax) compared with roots. Because of the unidirectional flow of nutrients from soil to roots, plants are the winners for N acquisition in the long run. Therefore, despite strong competition between roots and microorganisms for N, a temporal niche differentiation reflecting their generation times leads to mutualistic relationships in the rhizosphere. This temporal niche differentiation is highly relevant ecologically because it: protects ecosystems from N losses by leaching during periods of slow or no root uptake; continuously provides roots with available N according to plant demand; and contributes to the evolutionary development of mutualistic interactions between roots and microorganisms.
Use of an uncertainty analysis for genome-scale models as a prediction tool for microbial growth processes in subsurface environments.

PubMed

Klier, Christine

2012-03-06

The integration of genome-scale, constraint-based models of microbial cell function into simulations of contaminant transport and fate in complex groundwater systems is a promising approach to help characterize the metabolic activities of microorganisms in natural environments. In constraint-based modeling, the specific uptake flux rates of external metabolites are usually determined by Michaelis-Menten kinetic theory. However, extensive data sets based on experimentally measured values are not always available. In this study, a genome-scale model of Pseudomonas putida was used to study the key issue of uncertainty arising from the parametrization of the influx of two growth-limiting substrates: oxygen and toluene. The results showed that simulated growth rates are highly sensitive to substrate affinity constants and that uncertainties in specific substrate uptake rates have a significant influence on the variability of simulated microbial growth. Michaelis-Menten kinetic theory does not, therefore, seem to be appropriate for descriptions of substrate uptake processes in the genome-scale model of P. putida. Microbial growth rates of P. putida in subsurface environments can only be accurately predicted if the processes of complex substrate transport and microbial uptake regulation are sufficiently understood in natural environments and if data-driven uptake flux constraints can be applied.
Quantitative Comparison of Human Parainfluenza Virus Hemagglutinin-Neuraminidase Receptor Binding and Receptor Cleavage

PubMed Central

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

2013-01-01

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

PubMed

Saha, Shibu; Arya, Sunil K; Singh, S P; Sreenivas, K; Malhotra, B D; Gupta, Vinay

2009-03-15

Nanoporous cerium oxide (CeO(2)) thin film deposited onto platinum (Pt) coated glass plate using pulsed laser deposition (PLD) has been utilized for immobilization of glucose oxidase (GOx). Atomic force microscopy studies reveal the formation of nanoporous surface morphology of CeO(2) thin film. Response studies carried out using differential pulsed voltammetry (DPV) and optical measurements show that the GOx/CeO(2)/Pt bio-electrode shows linearity in the range of 25-300 mg/dl of glucose concentration. The low value of Michaelis-Menten constant (1.01 mM) indicates enhanced enzyme affinity of GOx to glucose. The observed results show promising application of the nanoporous CeO(2) thin film for glucose sensing application without any surface functionalization or mediator.
Occurrence of dead core in catalytic particles containing immobilized enzymes: analysis for the Michaelis-Menten kinetics and assessment of numerical methods.

PubMed

Pereira, Félix Monteiro; Oliveira, Samuel Conceição

2016-11-01

In this article, the occurrence of dead core in catalytic particles containing immobilized enzymes is analyzed for the Michaelis-Menten kinetics. An assessment of numerical methods is performed to solve the boundary value problem generated by the mathematical modeling of diffusion and reaction processes under steady state and isothermal conditions. Two classes of numerical methods were employed: shooting and collocation. The shooting method used the ode function from Scilab software. The collocation methods included: that implemented by the bvode function of Scilab, the orthogonal collocation, and the orthogonal collocation on finite elements. The methods were validated for simplified forms of the Michaelis-Menten equation (zero-order and first-order kinetics), for which analytical solutions are available. Among the methods covered in this article, the orthogonal collocation on finite elements proved to be the most robust and efficient method to solve the boundary value problem concerning Michaelis-Menten kinetics. For this enzyme kinetics, it was found that the dead core can occur when verified certain conditions of diffusion-reaction within the catalytic particle. The application of the concepts and methods presented in this study will allow for a more generalized analysis and more accurate designs of heterogeneous enzymatic reactors.

Characterization of the human cytochrome P450 enzymes involved in the metabolism of dihydrocodeine

PubMed Central

Kirkwood, L. C.; Nation, R. L.; Somogyi, A. A.

1997-01-01

Aims Using human liver microsomes from donors of the CYP2D6 poor and extensive metabolizer genotypes, the role of individual cytochromes P-450 in the oxidative metabolism of dihydrocodeine was investigated. Methods The kinetics of formation of N- and O-demethylated metabolites, nordihydrocodeine and dihydromorphine, were determined using microsomes from six extensive and one poor metabolizer and the effects of chemical inhibitors selective for individual P-450 enzymes of the 1A, 2A, 2C, 2D, 2E and 3A families and of LKM1 (anti-CYP2D6) antibodies were studied. Results Nordihydrocodeine was the major metabolite in both poor and extensive metabolizers. Kinetic constants for N-demethylation derived from the single enzyme Michaelis-Menten model did not differ between the two groups. Troleandomycin and erythromycin selectively inhibited N-demethylation in both extensive and poor metabolizers. The CYP3A inducer, α-naphthoflavone, increased N-demethylation rates. The kinetics of formation of dihydromorphine in both groups were best described by a single enzyme Michaelis-Menten model although inhibition studies in extensive metabolizers suggested involvement of two enzymes with similar Km values. The kinetic constants for O-demethylation were significantly different in extensive and poor metabolizers. The extensive metabolizers had a mean intrinsic clearance to dihydromorphine more than ten times greater than the poor metabolizer. The CYP2D6 chemical inhibitors, quinidine and quinine, and LKM1 antibodies inhibited O-demethylation in extensive metabolizers; no effect was observed in microsomes from a poor metabolizer. Conclusions CYP2D6 is the major enzyme mediating O-demethylation of dihydrocodeine to dihydromorphine. In contrast, nordihydrocodeine formation is predominantly catalysed by CYP3A. PMID:9431830
Diffusion Limitations in Root Uptake of Cadmium and Zinc, But Not Nickel, and Resulting Bias in the Michaelis Constant1[W][OA

PubMed Central

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

2012-01-01

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

PubMed

Kuzyakov, Yakov; Xu, Xingliang

2013-05-01

Demand of all living organisms on the same nutrients forms the basis for interspecific competition between plants and microorganisms in soils. This competition is especially strong in the rhizosphere. To evaluate competitive and mutualistic interactions between plants and microorganisms and to analyse ecological consequences of these interactions, we analysed 424 data pairs from 41 (15)N-labelling studies that investigated (15)N redistribution between roots and microorganisms. Calculated Michaelis-Menten kinetics based on K(m) (Michaelis constant) and V(max) (maximum uptake capacity) values from 77 studies on the uptake of nitrate, ammonia, and amino acids by roots and microorganisms clearly showed that, shortly after nitrogen (N) mobilization from soil organic matter and litter, microorganisms take up most N. Lower K(m) values of microorganisms suggest that they are especially efficient at low N concentrations, but can also acquire more N at higher N concentrations (V(max)) compared with roots. Because of the unidirectional flow of nutrients from soil to roots, plants are the winners for N acquisition in the long run. Therefore, despite strong competition between roots and microorganisms for N, a temporal niche differentiation reflecting their generation times leads to mutualistic relationships in the rhizosphere. This temporal niche differentiation is highly relevant ecologically because it: protects ecosystems from N losses by leaching during periods of slow or no root uptake; continuously provides roots with available N according to plant demand; and contributes to the evolutionary development of mutualistic interactions between roots and microorganisms. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.
Studies on long chain cis- and trans-acyl-CoA esters and Acyl-CoA dehydrogenase from rat heart mitochondria.

PubMed

Korsrud, G O; Conacher, H B; Jarvis, G A; Beare-Rogers, J L

1977-02-01

The beta-oxidation of long chain fatty acids was investigated in a preparation of rat heart mitochondria. The acyl-CoA esters of the cis and trans isomers of delta9-hexadecenoic, delta9-octadecenoic, delta11-eicosenoic, and delta13-docosenoic acids were prepared. Rates of the acyl-CoA reaction were determined with an extract from rat heart mitochondria. The apparent Michaelis constant (Km) and maximum velocity (Vmax) were calculated for each substrate. In general, apparent Vmax values decreased with increasing chain length of the monoenoic substrates. Reduced activity of acyl-CoA dehydrogenase with long chain acyl-CoA esters could have contributed to accumulation of lipids in hearts of rats fed diets containing long chain fatty acids.
Sulfated, low-molecular-weight lignins are potent inhibitorsof plasmin, in addition to thrombin and factor Xa: Novel opportunity for controlling complex pathologies.

PubMed

Henry, Brian L; Abdel Aziz, May; Zhou, Qibing; Desai, Umesh R

2010-03-01

Recently we prepared sulfated, low-molecular-weight lignins (LMWLs) to mimic the biological activities of heparin and heparan sulfate. Chemo-enzymatically prepared sulfated LMWLs represent a library of diverse non-sugar, aromatic molecules with structures radically different from the heparins, and have been found to potently inhibit thrombin and factor Xa. To assess their effect on the fibrinolytic system, we studied the interaction of LMWLs with human plasmin. Enzyme inhibition studies indicate that the three sulfated LMWLs studied inhibit plasmin with IC50 values in the range of 0.24 and 1.3 mM, which are marginally affected in the presence of antithrombin. Similarly, plasmin degradation of polymeric fibrin is also inhibited by sulfated LMWLs. Michaelis-Menten kinetic studies indicate that maximal velocity of hydrolysis of chromogenic substrates decreases nearly 70% in the presence of LMWLs, while the effect on Michaelis constant is dependent on the nature of the substrate. Competitive binding studies indicate that the sulfated LMWLs compete with full-length heparin. Comparison with thrombin-heparin crystal structure identifies an anionic region on plasmin as a plausible sulfated LMWL binding site. Overall, the chemo-enzymatic origin coupled with coagulation and fibrinolysis inhibition properties of sulfated LMWLs present novel opportunities for designing new pharmaceutical agents that regulate complex pathologies in which both systems are known to play important roles such as disseminated intravascular coagulation.
Real-Time Enzyme Kinetics by Quantitative NMR Spectroscopy and Determination of the Michaelis-Menten Constant Using the Lambert-W Function

ERIC Educational Resources Information Center

Her, Cheenou; Alonzo, Aaron P.; Vang, Justin Y.; Torres, Ernesto; Krishnan, V. V.

2015-01-01

Enzyme kinetics is an essential part of a chemistry curriculum, especially for students interested in biomedical research or in health care fields. Though the concept is routinely performed in undergraduate chemistry/biochemistry classrooms using other spectroscopic methods, we provide an optimized approach that uses a real-time monitoring of the…
Metabolic capabilities of cytochrome P450 enzymes in Chinese liver microsomes compared with those in Caucasian liver microsomes

PubMed Central

Yang, Junling; He, Minxia M; Niu, Wei; Wrighton, Steven A; Li, Li; Liu, Yang; Li, Chuan

2012-01-01

AIM The most common causes of variability in drug response include differences in drug metabolism, especially when the hepatic cytochrome P450 (CYP) enzymes are involved. The current study was conducted to assess the differences in CYP activities in human liver microsomes (HLM) of Chinese or Caucasian origin. METHODS The metabolic capabilities of CYP enzymes in 30 Chinese liver microsomal samples were compared with those of 30 Caucasian samples utilizing enzyme kinetics. Phenacetin O-deethylation, coumarin 7-hydroxylation, bupropion hydroxylation, amodiaquine N-desethylation, diclofenac 4′-hydroxylation (S)-mephenytoin 4′-hydroxylation, dextromethorphan O-demethylation, chlorzoxazone 6-hydroxylation and midazolam 1′-hydroxylation/testosterone 6β-hydroxylation were used as probes for activities of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A, respectively. Mann-Whitney U test was used to assess the differences. RESULTS The samples of the two ethnic groups were not significantly different in cytochrome-b5 concentrations but were significantly different in total CYP concentrations and NADPH-P450 reductase activity (P < 0.05). Significant ethnic differences in intrinsic clearance were observed for CYP1A2, CYP2C9, CYP2C19 and CYP2E1; the median values of the Chinese group were 54, 58, 26, and 35% of the corresponding values of the Caucasian group, respectively. These differences were associated with differences in Michaelis constant or maximum velocity. Despite negligible difference in intrinsic clearance, the Michaelis constant of CYP2B6 appeared to have a significant ethnic difference. No ethnic difference was observed for CYP2A6, CYP2C8, CYP2D6 and CYP3A. CONCLUSIONS These data extend our knowledge on the ethnic differences in CYP enzymes and will have implications for drug discovery and drug therapy for patients from different ethnic origins. PMID:21815912
Effects of missense mutations in sortase A gene on enzyme activity in Streptococcus mutans.

PubMed

Zhuang, P L; Yu, L X; Tao, Y; Zhou, Y; Zhi, Q H; Lin, H C

2016-04-11

Streptococcus mutans (S. mutans) is the major aetiological agent of dental caries, and the transpeptidase Sortase A (SrtA) plays a major role in cariogenicity. The T168G and G470A missense mutations in the srtA gene may be linked to caries susceptibility, as demonstrated in our previous studies. This study aimed to investigate the effects of these missense mutations of the srtA gene on SrtA enzyme activity in S. mutans. The point mutated recombinant S.mutans T168G and G470A sortases were expressed in expression plasmid pET32a. S. mutans UA159 sortase coding gene srtA was used as the template for point mutation. Enzymatic activity was assessed by quantifying increases in the fluorescence intensity generated when a substrate Dabcyl-QALPNTGEE-Edans was cleaved by SrtA. The kinetic constants were calculated based on the curve fit for the Michaelis-Menten equation. SrtA△N40(UA159) and the mutant enzymes, SrtA△N40(D56E) and SrtA△N40(R157H), were expressed and purified. A kinetic analysis showed that the affinity of SrtA△N40(D56E) and SrtA△N40(R157H) remained approximately equal to the affinity of SrtA△N40(UA159), as determined by the Michaelis constant (K m ). However, the catalytic rate constant (k cat ) and catalytic efficiency (k cat /K m ) of SrtA△N40(D56E) were reduced compared with those of SrtA△N40(R157H) and SrtA△N40(UA159), whereas the k cat and k cat /K m values of SrtA△N40(R157H) were slightly lower than those of SrtA△N40(UA159). The findings of this study indicate that the T168G missense mutation of the srtA gene results in a significant reduction in enzymatic activity compared with S. mutans UA159, suggesting that the T168G missense mutation of the srtA gene may be related to low cariogenicity.
A new amperometric enzyme electrode for alcohol determination.

PubMed

Gülce, H; Gülce, A; Kavanoz, M; Coşkun, H; Yildiz, A

2002-06-01

A new enzyme electrode for the determination of alcohols was developed by immobilizing alcohol oxidase in polvinylferrocenium matrix coated on a Pt electrode surface. The amperometric response due to the electrooxidation of enzymatically generated H(2)O(2) was measured at a constant potential of +0.70 V versus SCE. The effects of substrate, buffer and enzyme concentrations, pH and temperature on the response of the electrode were investigated. The optimum pH was found to be pH 8.0 at 30 degrees C. The steady-state current of this enzyme electrode was reproducible within +/-5.0% of the relative error. The sensitivity of the enzyme electrode decreased in the following order: methanol>ethanol>n-butanol>benzyl alcohol. The linear response was observed up to 3.7 mM for methanol, 3.0 mM for ethanol, 6.2 mM for n-butanol, and 5.2 mM for benzyl alcohol. The apparent Michaelis-Menten constant (K(Mapp)) value and the activation energy, E(a), of this immobilized enzyme system were found to be 5.78 mM and 38.07 kJ/mol for methanol, respectively.
Kinetics of Bacterial Growth on Chlorinated Aliphatic Compounds

PubMed Central

van den Wijngaard, Arjan J.; Wind, Richèle D.; Janssen, Dick B.

1993-01-01

With the pure bacterial cultures Ancylobacter aquaticus AD20 and AD25, Xanthobacter autotrophicus GJ10, and Pseudomonas sp. strain AD1, Monod kinetics was observed during growth in chemostat cultures on 1,2-dichloroethane (AD20, AD25, and GJ10), 2-chloroethanol (AD20 and GJ10), and 1,3-dichloro-2-propanol (AD1). Both the Michaelis-Menten constants (Km) of the first catabolic (dehalogenating) enzyme and the Monod half-saturation constants (Ks) followed the order 2-chloroethanol, 1,3-dichloro-2-propanol, epichlorohydrin, and 1,2-dichloroethane. The Ks values of strains GJ10, AD20, and AD25 for 1,2-dichloroethane were 260, 222, and 24 μM, respectively. The low Ks value of strain AD25 was correlated with a higher haloalkane dehalogenase content of this bacterium. The growth rates of strains AD20 and GJ10 in continuous cultures on 1,2-dichloroethane were higher than the rates predicted from the kinetics of the haloalkane dehalogenase and the concentration of the enzyme in the cells. The results indicate that the efficiency of chlorinated compound removal is indeed influenced by the kinetic properties and cellular content of the first catabolic enzyme. The cell envelope did not seem to act as a barrier for permeation of 1,2-dichloroethane. PMID:16348981
Phenytoin pharmacokinetics in critically ill trauma patients.

PubMed

Boucher, B A; Rodman, J H; Jaresko, G S; Rasmussen, S N; Watridge, C B; Fabian, T C

1988-12-01

Preliminary data have suggested that phenytoin systemic clearance may increase during initial therapy in critically ill patients. The objectives for this study were to model the time-variant phenytoin clearance and evaluate concomitant changes in protein binding and urinary metabolite elimination. Phenytoin was given as an intravenous loading dose of 15 mg/kg followed by an initial maintenance dose of 6 mg/kg/day in 10 adult critically ill trauma patients. Phenytoin bound and unbound plasma concentrations were determined in 10 patients and urinary excretion of the metabolite p-hydroxyphenyl phenylhydantoin (p-HPPH) was measured in seven patients for 7 to 14 days. A Michaelis-Menten one-compartment model incorporating a time-variant maximal velocity (Vmax) was sufficient to describe the data and superior to a conventional time-invariant Michaelis-Menten model. Vmax for the time-variant model was defined as V'max + Vmax delta (1 - e(-kindt)). Vmax infinity is the value for Vmax when t is large. The median values (ranges) for the parameters were Km = 4.8 (2.6 to 20) mg/L, Vmax infinity = 1348 (372 to 4741) mg/day, and kind = 0.0115 (0.0045 to 0.132) hr-1. Phenytoin free fraction increased in a majority of patients during the study period, with a binding ratio inversely related to albumin. Measured urinary p-HPPH data were consistent with the proposed model. A loading and constant maintenance dose of phenytoin frequently yielded a substantial, clinically significant fall in plasma concentrations with a pattern of apparently increasing clearance that may be a consequence of changes in protein binding, induction of metabolism, or the influence of stress on hepatic metabolic capacity.
Molecular modeling of the inhibition of enzyme PLA2 from snake venom by dipyrone and 1-phenyl-3-methyl-5-pyrazolone

NASA Astrophysics Data System (ADS)

Silva, S. L. Da; Comar, M., Jr.; Oliveira, K. M. T.; Chaar, J. S.; Bezerra, E. R. M.; Calgarotto, A. K.; Baldasso, P. A.; Veber, C. L.; Villar, J. A. F. P.; Oliveira, A. R. M.; Marangoni, S.

Phospholipases A2 (PLA2) are enzymes that trigger the degradation cascade of the arachidonic acid, leading to the formation of pro-inflammatory eicosanoids. The selective inhibition of PLA2s is crucial in the search for a more efficient anti-inflammatory drug with fewer side effects than the drugs currently used. Hence, we studied the influences caused by two pyrazolonic inhibitors: dipyrone (DIP) and 1-phenyl-3-methyl-5-pyrazolone (PMP) on the kinetic behavior of PLA2 from Crotalus adamanteus venom. Molecular modeling results, by DFT and MM approaches, showed that DIP is strongly associated to the active site of PLA2 through three hydrogen bonds, whereas PMP is associated to the enzyme just through hydrophobic interactions. In addition, only PMP presents an intramolecular hydrogen bond that make difficult the formation of more efficient interactions with PLA2. These results help in the understanding of the experimental observations. Experimentally, the results showed that PLA2 from C. adamanteus present a typical Michaelian behavior. In addition, the calculated kinetic parameters showed that, in the presence of DIP or PMP, the maximum enzymatic velocity (VMAX) value was kept constant, whereas the Michaelis constant (KM) values increased and the inhibition constant (KI) decreased, indicating competitive inhibition. These results show that the phenyl-pyrazolonic structures might help in the development and design of new drugs able to selectively inhibit PLA2.
Enzyme inhibition studies by integrated Michaelis-Menten equation considering simultaneous presence of two inhibitors when one of them is a reaction product.

PubMed

Bezerra, Rui M F; Pinto, Paula A; Fraga, Irene; Dias, Albino A

2016-03-01

To determine initial velocities of enzyme catalyzed reactions without theoretical errors it is necessary to consider the use of the integrated Michaelis-Menten equation. When the reaction product is an inhibitor, this approach is particularly important. Nevertheless, kinetic studies usually involved the evaluation of other inhibitors beyond the reaction product. The occurrence of these situations emphasizes the importance of extending the integrated Michaelis-Menten equation, assuming the simultaneous presence of more than one inhibitor because reaction product is always present. This methodology is illustrated with the reaction catalyzed by alkaline phosphatase inhibited by phosphate (reaction product, inhibitor 1) and urea (inhibitor 2). The approach is explained in a step by step manner using an Excel spreadsheet (available as a template in Appendix). Curve fitting by nonlinear regression was performed with the Solver add-in (Microsoft Office Excel). Discrimination of the kinetic models was carried out based on Akaike information criterion. This work presents a methodology that can be used to develop an automated process, to discriminate in real time the inhibition type and kinetic constants as data (product vs. time) are achieved by the spectrophotometer. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.
A binomial stochastic kinetic approach to the Michaelis-Menten mechanism

NASA Astrophysics Data System (ADS)

Lente, Gábor

2013-05-01

This Letter presents a new method that gives an analytical approximation of the exact solution of the stochastic Michaelis-Menten mechanism without computationally demanding matrix operations. The method is based on solving the deterministic rate equations and then using the results as guiding variables of calculating probability values using binomial distributions. This principle can be generalized to a number of different kinetic schemes and is expected to be very useful in the evaluation of measurements focusing on the catalytic activity of one or a few individual enzyme molecules.
The unique kinetic behavior of the very large NAD-dependent glutamate dehydrogenase from Janthinobacterium lividum.

PubMed

Kawakami, Ryushi; Oyama, Masaki; Sakuraba, Haruhiko; Ohshima, Toshihisa

2010-01-01

The kinetics of a very large NAD-dependent glutamate dehydrogenase from Janthinobacterium lividum showed positive cooperativity toward alpha-ketoglutarate and NADH, and the Michaelis-Menten type toward ammonium chloride in the absence of the catalytic activator, L-aspartate. An increase in the maximum activity accompanied the decrease in the S(0.5) values for alpha-ketoglutarate and NADH with the addition of L-aspartate, and the kinetic response for alpha-ketoglutarate changed completely to a typical Michaelis-Menten type in the presence of 10 mM L-aspartate.
Catalytic properties of IgMs with amylolytic activity isolated from patients with multiple sclerosis.

PubMed

Ivanen, Dina R; Kulminskaya, Anna A; Shabalin, Konstantin A; Isaeva-Ivanova, Luydmila V; Ershova, Nadezhda A; Saveliev, Andrew N; Nevinsky, Gregory A; Neustroev, Kirill N

2004-08-01

Recently, amylolytic activity was detected in IgMs isolated from the sera of the patients with multiple sclerosis. All purified samples of IgM were electrophoretically homogenous and did not contain any co-purified a-amylase and a-glucosidase activities, in accordance with a set of criteria developed for abzymes. The amylolytic activity of abzymes was studied in the hydrolysis of p-nitrophenyl a-D-maltooligosaccharides with different degrees of polymerization from 1 to 8 by TLC and reverse-phase HPLC techniques. All IgM samples isolated from 54 patients with clinically definite multiple sclerosis demonstrated hydrolytic activity towards the above artificial substrates. The Michaelis constant values (Km) in the hydrolysis of p-nitrophenyl a-D-maltoheptaoside were in the range of 10 p-nitrophenyl or p-nitrophenyl a-D-glucosides, thus indicating the presence of an a-D-glucosidase activity. For a number of the investigated samples, specific amylolytic activity increased depending on the length of substrates (from p-nitrophenyl maltopentaoside to p-nitrophenyl maltohexaoside); for other IgMs, the opposite dependence was observed. All IgMs studied did not exhibit any other glycoside hydrolase activities toward p-nitrophenyl glycoside substrates. Abzyme fractions from different donors demonstrated catalytic heterogeneity in Michaelis-Menten parameters and different modes of action in the hydrolysis of p-nitrophenyl maltooligosaccharides. Enzymatic properties of the IgMs tested varied from human a-amylases. All investigated abzyme samples did not show transglycosylating ability.
Using Optical Oxygen Sensors and Injection Experiments to Determine in situ Microbial Rate Constants for Methane Oxidation and Heterotrophic Respiration in a Boreal Bog and Fen

NASA Astrophysics Data System (ADS)

Waldo, N.; Moorberg, C.; Waldrop, M. P.; Turetsky, M. R.; Neumann, R. B.

2015-12-01

Wetlands are the largest natural source of methane to the atmosphere, and play a key role in feedback cycles to climate change. In recognition of this, many researchers are developing process-based models of wetland methane emissions at various scales. In these models, the three key biogeochemical reactions are methane production, methane oxidation, and heterotrophic respiration, and they are modeled using Michaelis-Menten kinetics. The majority of Michaelis-Menten rate constants used in models are based on experiments involving slurries of peat incubated in vials. While these slurries provide a highly controlled setting, they are different from in situ conditions in multiple ways; notably they lack live plants and the centimeter-scale heterogeneities that exist in the field. To determine rate constants in a system more representative of in situ conditions, we extracted peat cores intact from a bog and fen located in the Bonanza Creek Experimental Forest near Fairbanks, Alaska and part of the Alaska Peatland Experiment (APEX) research program. Into those cores we injected water with varying concentrations of methane and oxygen at multiple depths. We used planar oxygen sensors installed on the peat cores to collect high resolution, two dimensional oxygen concentration data during the injections and used oxygen consumption rates under various conditions to calculate rate constants. Results were compared to a similar but smaller set of injection experiments conducted against planar oxygen sensors installed in the bog. Results will inform parametrization of microbial processes in wetland models, improving estimates of methane emissions both under current climate conditions and in the future.
Exploration of two-enzyme coupled catalysis system using scanning electrochemical microscopy.

PubMed

Wu, Zeng-Qiang; Jia, Wen-Zhi; Wang, Kang; Xu, Jing-Juan; Chen, Hong-Yuan; Xia, Xing-Hua

2012-12-18

In biological metabolism, a given metabolic process usually occurs via a group of enzymes working together in sequential pathways. To explore the metabolism mechanism requires the understanding of the multienzyme coupled catalysis systems. In this paper, an approach has been proposed to study the kinetics of a two-enzyme coupled reaction using SECM combining numerical simulations. Acetylcholine esterase and choline oxidase are immobilized on cysteamine self-assembled monolayers on tip and substrate gold electrodes of SECM via electrostatic interactions, respectively. The reaction kinetics of this two-enzyme coupled system upon various separation distance precisely regulated by SECM are measured. An overall apparent Michaelis-Menten constant of this enzyme cascade is thus measured as 2.97 mM at an optimal tip-substrate gap distance of 18 μm. Then, a kinetic model of this enzyme cascade is established for evaluating the kinetic parameters of individual enzyme by using the finite element method. The simulated results demonstrate the choline oxidase catalytic reaction is the rate determining step of this enzyme cascade. The Michaelis-Menten constant of acetylcholine esterase is evaluated as 1.8 mM. This study offers a promising approach to exploring mechanism of other two-enzyme coupled reactions in biological system and would promote the development of biosensors and enzyme-based logic systems.
Glucose biosensing using glassy carbon electrode modified with polyhydroxy-C60, glucose oxidase and ionic-liquid.

PubMed

Yang, Tian; Yang, Xiao-Lu; Zhang, Yu-Shuai; Xiao, BaoLin; Hong, Jun

2014-01-01

Direct electrochemistry of glucose oxidase (GOD) was achieved when an ionic liquid/GOD-Polyhydroxy-C60 functional membrane was confined on a glassy carbon electrode (GCE). The cyclic voltammograms (CVs) of the modified GCE showed a pair of redox peaks with a formal potential (E°') of - 329 ± 2 mV. The heterogeneous electron transfer constant (k(s)) was 1.43 s-1. The modified GCE response to glucose was linear in the range from 0.02 to 2.0 mM. The detection limit was 1 μM. The apparent Michaelis-Menten constant (K(m)(app)) was 1.45 mM.
Simple method for preparing glucose biosensor based on in-situ polypyrrole cross-linked chitosan/glucose oxidase/gold bionanocomposite film.

PubMed

Şenel, Mehmet

2015-03-01

A film of chitosan-polypyrrole-gold nanoparticles was fabricated by in-situ chemical synthesis method and its application in glucose biosensor was investigated. The obtained biosensor exhibited a high and reproducible sensitivity of 0.58μA/mM, response time ~4s, linear dynamic range from 1 to 20mM, correlation coefficient of R(2)=0.9981, and limit of detection (LOD), based on S/N ratio (S/N=3) of 0.068mM. A value of 1.83mM for the apparent Michaelis-Menten constant was obtained. The resulting bio-nanocomposite provided a suitable environment for the enzyme to retain its bioactivity at considerably extreme conditions, and the decorated gold nanoparticles in the bio-nanocomposite offer good affinity to enzyme. Copyright © 2014. Published by Elsevier B.V.

The temperature-dependence of adenylate cyclase from baker's yeast.

PubMed Central

Londesborough, J; Varimo, K

1979-01-01

The Michaelis constant of membrane-bound adenylate cyclase increased from 1.1 to 1.8 mM between 7 and 38 degrees C (delta H = 13 kJ/mol). Over this temperature range, the maximum velocity increased 10-fold, and the Arrhenius plot was nearly linear, with an average delta H* of 51 kJ/mol. The temperature-dependence of the reaction rate at 2 mM-ATP was examined in more detail: for Lubrol-dispersed enzyme, Arrhenius plots were nearly linear with average delta H* values of 45 and 68 kJ/mol, respectively, for untreated and gel-filtered enzymes; for membrane-bound enzyme, delta H changed from 40 kJ/mol above about 21 degrees C to 62 kJ/mol below 21 degrees C, but this behaviour does not necessarily indicate an abrupt, lipid-induced, transition in the reaction mechanism. PMID:391221
Enhancing Activity and Stability of Uricase from Lactobacillus plantarum by Zeolite immobilization

NASA Astrophysics Data System (ADS)

Iswantini, D.; Nurhidayat, N.; Sarah

2017-03-01

Lactobacillus plantarum has been known be able to produce uricase for uric acid biosensor. Durability and stability of L. plantarum in generating uricase enzyme was low. Hence, we tried to enhance its durability and stability by immobilizing it onto activated 250 mg zeolite at room temperature using 100 μL L.plantarum suspension and 2.87 mM uric acid, while Michaelis-Menten constant (KM) and Vmax were obtained at 6.7431 mM and 0.9171 µA consecutively, and the linearity range was 0.1-3.3 mM (R2 = 0.9667). Limit of detection (LOD) and limit of quantification (LOQ) value of the measurement were 0.4827 mM and 1.6092 mM respectively. Biosensor stability treatment was carried out in two different treatments, using the same electrode and using disposable electrode. The disposable electrode stability showed better result based on repeated measurements, but stability was still need improvement.
Polyaniline-carboxymethyl cellulose nanocomposite for cholesterol detection.

PubMed

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

2010-10-01

Cholesterol oxidase (ChOx) has been covalently immobilized onto polyaniline-carboxymethyl cellulose (PANI-CMC) nanocomposite film deposited onto indium-tin-oxide (ITO) coated glass plate using glutaraldehyde as a cross-linker. Fourier transform infrared (FTIR) spectroscopic and electrochemical studies have been used to characterize the PANI-CMC/ITO nanocomposite electrode and ChOx/PANI-CMC/ITO bioelectrode. Scanning electron microscopy (SEM) studies reveal the formation of PANI-CMC nanocomposite fibers of size approximately 150 nm in diameter. The ChOx/PANI-CMC/ITO bioelectrode exhibits linearity as 0.5-22 mM, detection limit as 1.31 mM, sensitivity as 0.14 mA/mM cm2, response time as 10 s and shelf-life of about 10 weeks when bioelectrode is stored at 4 degrees C. The low value of Michaelis-Menten constant (K(m)) obtained as 2.71 mM reveals high affinity of immobilized ChOx for PANI-CMC/ITO nanocomposite electrode.
Transient kinetic studies of pH-dependent hydrolyses by exo-type carboxypeptidase P on a 27-MHz quartz crystal microbalance.

PubMed

Furusawa, Hiroyuki; Takano, Hiroki; Okahata, Yoshio

2008-02-15

pH-Dependent kinetic parameters (k(on), k(off), and k(cat)) of protein (myoglobin) hydrolyses catalyzed by exo-enzyme (carboxypeptidase P, CPP) were obtained by using a protein-immobilized quartz crystal microbalance (QCM) in acidic aqueous solutions. The formation of the enzyme-substrate (ES) complex (k(on)), the decay of the ES complex (k(off)), and the formation of the product (k(cat)) could be analyzed by transient kinetics as mass changes on the QCM plate. The Kd (k(off)/k(on)) value was different from the Michaelis constant Km calculated from (k(off) + k(cat))/k(on) due to k(cat) > k(off). The rate-determining step was the binding step (k(on), and the catalytic rate k(cat) was faster than other k(on) and k(off) values. In the range of pH 2.5-5.0, values of k(on) gradually increased with decreasing pH showing a maximum at pH 3.7, values of k(off) were independent of pH, and k(cat) increased gradually with decreasing pH. As a result, the apparent rate constant (k(cat)/Km) showed a maximum at pH 3.7 and gradually increased with decreasing pH. The optimum pH at 3.7 of k(on) is explained by the optimum binding ability of CPP to the COOH terminus of the substrate with hydrogen bonds. The increase of k(cat) at the lower pH correlated with the decrease of alpha-helix contents of the myoglobin substrate on the QCM.
Quantification of in vivo metabolic kinetics of hyperpolarized pyruvate in rat kidneys using dynamic 13C MRSI.

PubMed

Xu, Tao; Mayer, Dirk; Gu, Meng; Yen, Yi-Fen; Josan, Sonal; Tropp, James; Pfefferbaum, Adolf; Hurd, Ralph; Spielman, Daniel

2011-10-01

With signal-to-noise ratio enhancements on the order of 10,000-fold, hyperpolarized MRSI of metabolically active substrates allows the study of both the injected substrate and downstream metabolic products in vivo. Although hyperpolarized [1-(13)C]pyruvate, in particular, has been used to demonstrate metabolic activities in various animal models, robust quantification and metabolic modeling remain important areas of investigation. Enzyme saturation effects are routinely seen with commonly used doses of hyperpolarized [1-(13)C]pyruvate; however, most metrics proposed to date, including metabolite ratios, time-to-peak of metabolic products and single exchange rate constants, fail to capture these saturation effects. In addition, the widely used small-flip-angle excitation approach does not correctly model the inflow of fresh downstream metabolites generated proximal to the target slice, which is often a significant factor in vivo. In this work, we developed an efficient quantification framework employing a spiral-based dynamic spectroscopic imaging approach. The approach overcomes the aforementioned limitations and demonstrates that the in vivo (13)C labeling of lactate and alanine after a bolus injection of [1-(13)C]pyruvate is well approximated by saturatable kinetics, which can be mathematically modeled using a Michaelis-Menten-like formulation, with the resulting estimated apparent maximal reaction velocity V(max) and apparent Michaelis constant K(M) being unbiased with respect to critical experimental parameters, including the substrate dose, bolus shape and duration. Although the proposed saturatable model has a similar mathematical formulation to the original Michaelis-Menten kinetics, it is conceptually different. In this study, we focus on the (13)C labeling of lactate and alanine and do not differentiate the labeling mechanism (net flux or isotopic exchange) or the respective contribution of various factors (organ perfusion rate, substrate transport kinetics, enzyme activities and the size of the unlabeled lactate and alanine pools) to the labeling process. Copyright © 2011 John Wiley & Sons, Ltd.
Immobilization of polyphenol oxidase in conducting copolymers and determination of phenolic compounds in wines with enzyme electrodes.

PubMed

Kiralp, Senem; Toppare, Levent; Yağci, Yusuf

2003-11-01

Polyphenol oxidase (PPO) was immobilized in copolymers of thiophene functionalized menthyl monomer (MM) with pyrrole. Immobilization of enzyme was performed via entrapment in conducting copolymers during electrochemical polymerization of pyrrole. Maximum reaction rates, Michaelis-Menten constants and temperature, pH and operational stabilities of enzyme electrodes were investigated. Total amount of phenolic compounds in red wines of Turkey were analyzed by using these electrodes.
A highly efficient nano-cluster artificial peroxidase and its direct electrochemistry on a nano complex modified glassy carbon electrode.

PubMed

Hong, Jun; Wang, Wei; Huang, Kun; Yang, Wei-Yun; Zhao, Ying-Xue; Xiao, Bao-Lin; Gao, Yun-Fei; Moosavi-Movahedi, Zainab; Ghourchian, Hedayatollah; Moosavi-Movahedi, Ali Akbar

2012-01-01

A nano-cluster with highly efficient peroxide activity was constructed based on nafion (NF) and cytochrome c (Cyt c). UV-Vis spectrometry and transmission electron microscopy (TEM) methods were utilized for characterization of the nano-structured enzyme or artificial peroxidase (AP). The nano-cluster was composed of a Chain-Ball structure, with an average ball size of about 40 nm. The Michaelis-Menten (K(m)) and catalytic rate (k(cat)) constants of the AP were determined to be 2.5 ± 0.4 µM and 0.069 ± 0.001 s(-1), respectively, in 50 mM PBS at pH 7.0. The catalytic efficiency of the AP was evaluated to be 0.028 ± 0.005 µM(-1) s(-1), which was 39 ± 5% as efficient as the native horseradish peroxidase (HRP). The AP was also immobilized on a functional multi-wall carbon nanotube (MWNCTs)-gold colloid nanoparticles (AuNPs) nano-complex modified glassy carbon (GC) electrode. The cyclic voltammetry of AP on the nano complex modified GC electrode showed a pair of well-defined redox peaks with a formal potential (E°') of -45 ± 2 mV (vs. Ag/AgCl) at a scan rate of 0.05 V/s. The heterogeneous electron transfer rate constant (k(s)) was evaluated to be 0.65 s(-1). The surface concentration of electroactive AP on GC electrode (Γ) was 7 × 10(-10) mol cm(-2). The apparent Michaelis-Menten constant (K(m)(app)) was 0.23 nM.
Purification and characterization of NADP-dependent 7 beta-hydroxysteroid dehydrogenase from Peptostreptococcus productus strain b-52.

PubMed

Masuda, N; Oda, H; Tanaka, H

1983-01-04

An NADP-dependent 7 beta-hydroxysteroid dehydrogenase was purified 11.5-fold over the activity in crude cell extracts prepared from Peptostreptococcus productus strain b-52, by using Sephadex G-200 and DEAE-cellulose column chromatography. 7 beta-Dehydrogenation was the sole transformation of bile acids catalyzed by the partially purified enzyme. The enzyme preparation (spec. act. 2.781 IU per mg protein) had an optimum pH of 9.8. Lineweaver-Burk plots showed a Michaelis constant (Km) value of 0.05 mM for 3 alpha, 7 beta-dihydroxy-5 beta-cholanoic acid whereas higher values were obtained with 3 alpha,7 beta-dihydroxy-5 beta-cholanoyl glycine (0.20 mM), and 3 alpha,7 beta-dihydroxy-5 beta-cholanoyl taurine (0.26 mM). NADP but not NAD could function as an electron acceptor, and had a Km value of 0.30 mM. A molecular weight of 64000 was determined by SDS-polyacrylamide gel electrophoresis. The addition of 0.4 mM of either bile acid to the growth medium suppressed not only cell growth, but also the enzyme yield.
Synthesis, Surface Modification and Optical Properties of Thioglycolic Acid-Capped ZnS Quantum Dots for Starch Recognition at Ultralow Concentration

NASA Astrophysics Data System (ADS)

Tayebi, Mahnoush; Tavakkoli Yaraki, Mohammad; Ahmadieh, Mahnaz; Mogharei, Azadeh; Tahriri, Mohammadreza; Vashaee, Daryoosh; Tayebi, Lobat

2016-11-01

In this research, water-soluble thioglycolic acid-capped ZnS quantum dots (QDs) are synthesized by the chemical precipitation method. The prepared QDs are characterized using x-ray diffraction and transmission electron microscopy. Results revealed that ZnS QDs have a 2.73 nm crystallite size, cubic zinc blende structure, and spherical morphology with a diameter less than 10 nm. Photoluminescence (PL) spectroscopy is performed to determine the presence of low concentrations of starch. Four emission peaks are observed at 348 nm, 387 nm, 422 nm, and 486 nm and their intensities are quenched by increasing concentration of starch. PL intensity variations in the studied concentrations range (0-100 ppm) are best described by a Michaelis-Menten model. The Michaelis constant ( K m) for immobilized α-amylase in this system is about 101.07 ppm. This implies a great tendency for the enzyme to hydrolyze the starch as substrate. Finally, the limit of detection is found to be about 6.64 ppm.
Diatomite Modified Immobilized Delftia sp. for the Bio-Abiotic Removal of Antibiotics Amoxicillin in the Aqueous System

NASA Astrophysics Data System (ADS)

Gao, Lijuan; Sun, Jing; Guan, Kai; Shen, Tingting; Wang, Xikui

2017-05-01

Diatomite modified sodium alginate (Si/SA) immobilized Delftia sp. A2(2011) (STT01) was applied to degrade amoxicillin. The immobilized pellets provided a direct and visual probe for the degradation process due to their intrinsic bright colour. The results demonstrated that 100% of amoxicillin and 68.5% of CODcr removal were achieved after 72 h, comparing with the cases of sodium alginate (SA) system (81.2%, 46.9%) and the free cells system (60.5%, 35.5%). The degradation kinetics was in good agreement with Michaelis-Menten equation. The maximum rate (Vm ) and Michaelis constant (Km ) were calculated as 9.09 mg L-1 h-1 and 228 mg L-1, respectively. The results further revealed that diatomite not only acted as immobilization support to improve the mechanical strength and lifetime of the pellets but also as absorbent to promote the treatment efficiency. Therefore, both enzymatic catalysis and chemisorption were responsible for the removal of amoxicillin.
Utilization of integrated Michaelis-Menten equations for enzyme inhibition diagnosis and determination of kinetic constants using Solver supplement of Microsoft Office Excel.

PubMed

Bezerra, Rui M F; Fraga, Irene; Dias, Albino A

2013-01-01

Enzyme kinetic parameters are usually determined from initial rates nevertheless, laboratory instruments only measure substrate or product concentration versus reaction time (progress curves). To overcome this problem we present a methodology which uses integrated models based on Michaelis-Menten equation. The most severe practical limitation of progress curve analysis occurs when the enzyme shows a loss of activity under the chosen assay conditions. To avoid this problem it is possible to work with the same experimental points utilized for initial rates determination. This methodology is illustrated by the use of integrated kinetic equations with the well-known reaction catalyzed by alkaline phosphatase enzyme. In this work nonlinear regression was performed with the Solver supplement (Microsoft Office Excel). It is easy to work with and track graphically the convergence of SSE (sum of square errors). The diagnosis of enzyme inhibition was performed according to Akaike information criterion. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.
Screening of traditional antidiabetic medicinal plants of Mauritius for possible alpha-amylase inhibitory effects in vitro.

PubMed

Kotowaroo, M I; Mahomoodally, M F; Gurib-Fakim, A; Subratty, A H

2006-03-01

In this study, seven exotic/indigenous medicinal plants of Mauritius, namely Coix lacryma-jobi (Poaceae), Aegle marmelos (Rutaceae), Artocarpus heterophyllus (Moraceae), Vangueria madagascariensis (Rubiaceae), Azadirachta indica (Meliaceae), Eriobotrya japonica (Rosaceae) and Syzigium cumini (Myrtaceae) were studied for possible effects on starch breakdown by alpha-amylase in vitro. The results showed that only Artocarpus heterophyllus significantly (p < 0.05) inhibited alpha-amylase activity in vitro. To confirm the observed effects, a further biochemical assay was undertaken to investigate the effects of Artocarpus heterophyllus on alpha-amylase activity using rat plasma in vitro. It was found that the aqueous leaf extract significantly (p < 0.05) inhibited alpha-amylase activity in rat plasma. The highest inhibitory activity (27.20 +/- 5.00%) was observed at a concentration of 1000 microg/mL. However, in both cases dose dependency was not observed. Enzyme kinetic studies using the Michaelis-Menten and Lineweaver-Burk equations were performed to establish the type of inhibition involved. In the presence of the plant extract the maximal velocity (Vmax) remained constant (1/150 g / L/s) whereas the Michaelis-Menten constant (Km) increased by 5.79 g / L, indicating that the aqueous leaf extract of Artocarpus heterophyllus behaved as a competitive inhibitor. Results from the present study tend to indicate that Artocarpus heterophyllus could act as a 'starch blocker' thereby reducing post-prandial glucose peaks. Copyright 2006 John Wiley & Sons, Ltd.
[Effects of methomyl on acetylcholinesterase in erythrocyte membrane and various brain areas].

PubMed

Zhao, Fei; Li, Tao; Zhang, Changchun; Xu, Yiping; Xu, Hangong; Shi, Nian

2015-06-01

To study the toxicity of methomyl to acetylcholinesterase (AChE) in different regions. The optimal temperature and time for measurement of AChE activity were determined in vitro. The dose- and time-response relationships of methomyl with AChE activity in human erythrocyte membrane, rat erythrocyte membrane, cortical synapses, cerebellar synapses, hippocampal synapses, and striatal synapses were evaluated. The half maximal inhibitory concentration (IC50) and bimolecular rate constant (K) of methomyl for AChE activity in different regions were calculated, and the type of inhibition of AChE activity by methomyl was determined. AChE achieved the maximum activity at 370 °C, and the optimal time to determine initial reaction velocity was 0-17 min. There were dose- and time-response relationships between methomyl and AChE activity in the erythrocyte membrane and various brain areas. The IC50 value of methomyl for AChE activity in human erythrocyte membrane was higher than that in rat erythrocyte membrane, while the Ki value of methomyl for AChE activity in rat erythrocyte membrane was higher than that in human erythrocyte membrane. Among synapses in various brain areas, the striatum had the highest IC50 value, followed by the cerebellum, cerebral cortex, and hippocampus, while the cerebral cortex had the highest Ki value, followed by the hippocampus, striatum, and cerebellum. Lineweaver-Burk diagram demonstrated that with increasing concentration of methomyl, the maximum reaction velocity (Vmax) of AChE decreased, and the Michaelis constant (Km) remained the same. Methomyl is a reversible non-competitive inhibitor of AChE. AChE of rat erythrocyte membrane is more sensitive to methomyl than that of human erythrocyte membrane; the cerebral cortical synapses have the most sensitive AChE to methomyl among synapses in various brain areas.
Properties of lubrol-extracted uridine diphosphate glucuronyltransferase.

PubMed

Howland, R D; Burkhalter, A; Trevor, A J; Hegeman, S; Shirachi, D Y

1971-12-01

1. A partially purified UDP-glucuronyltransferase was obtained by extracting rat liver microsomal preparations with Lubrol, a non-ionic detergent. 2. The soluble enzyme catalysed conjugation of both o-aminophenol and p-nitrophenol and was extremely stable when compared with untreated microsomal preparations. 3. The characteristics of the conjugation of the two phenols were found to differ with respect to pH optimum, bivalent cation requirement and Michaelis constants, suggesting that more than one enzyme is involved in the conjugation reaction.
Properties of Lubrol-extracted uridine diphosphate glucuronyltransferase

PubMed Central

Howland, R. D.; Burkhalter, A.; Trevor, A. J.; Hegeman, S.; Shirachi, D. Y.

1971-01-01

1. A partially purified UDP-glucuronyltransferase was obtained by extracting rat liver microsomal preparations with Lubrol, a non-ionic detergent. 2. The soluble enzyme catalysed conjugation of both o-aminophenol and p-nitrophenol and was extremely stable when compared with untreated microsomal preparations. 3. The characteristics of the conjugation of the two phenols were found to differ with respect to pH optimum, bivalent cation requirement and Michaelis constants, suggesting that more than one enzyme is involved in the conjugation reaction. PMID:5144269
The use of gas chromatographic-mass spectrometric-computer systems in pharmacokinetic studies.

PubMed

Horning, M G; Nowlin, J; Stafford, M; Lertratanangkoon, K; Sommer, K R; Hill, R M; Stillwell, R N

1975-10-29

Pharmacokinetic studies involving plasma, urine, breast milk, saliva and liver homogenates have been carried out by selective ion detection with a gas chromatographic-mass spectrometric-computer system operated in the chemical ionization mode. Stable isotope labeled drugs were used as internal standards for quantification. The half-lives, the concentration at zero time, the slope (regression coefficient), the maximum velocity of the reaction and the apparent Michaelis constant of the reaction were determined by regression analysis, and also by graphic means.
Ternary borate-nucleoside complex stabilization by Ribonuclease A demonstrates phosphate mimicry

PubMed Central

Gabel, Scott A.; London, Robert E.

2010-01-01

Phosphate esters play a central role in cellular energetics, biochemical activation, signal transduction and conformational switching. The structural homology of the borate anion with phosphate, combined with its ability to spontaneously esterify hydroxyl groups, suggested that phosphate-ester recognition sites on proteins might exhibit significant affinity for non-enzymatically formed borate esters. 11B NMR studies and activity measurements on ribonuclease A in the presence of borate and several cytidine analogs demonstrate the formation of a stable ternary RNase A•3′-deoxycytidine-2′-borate ternary complex that mimics the complex formed between RNase A and a 2′-cytidine monophosphate (2′-CMP) inhibitor. Alternatively, no slowly exchanging borate resonance is observed for a ternary RNase A, borate, 2′-deoxycytidine mixture, demonstrating the critical importance of the 2′-hydroxyl group for complex formation. Titration of the ternary complex with 2′-CMP shows that it can displace the bound borate ester with a binding constant that is close to the reported inhibition constant of RNase A by 2′CMP. RNase A binding of a cyclic cytidine-2′,3′-borate ester, which is a structural homolog of the cytidine-2′,3′-cyclic phosphate substrate, could also be demonstrated. The apparent dissociation constant for the cytidine-2′,3′-borate•RNase A complex is 0.8 mM, which compares with a Michaelis constant of 11 mM for cCMP at pH 7, indicating considerably stronger binding. However, the value is 1000-fold larger than the reported dissociation constant of the RNase A complex with uridine-vanadate. These results are consistent with recent reports suggesting that in situ formation of borate esters that mimic the corresponding phosphate esters support enzyme catalysis. PMID:17957392
Classical Michaelis-Menten and system theory approach to modeling metabolite formation kinetics.

PubMed

Popović, Jovan

2004-01-01

When single doses of drug are administered and kinetics are linear, techniques, which are based on the compartment approach and the linear system theory approach, in modeling the formation of the metabolite from the parent drug are proposed. Unlike the purpose-specific compartment approach, the methodical, conceptual and computational uniformity in modeling various linear biomedical systems is the dominant characteristic of the linear system approach technology. Saturation of the metabolic reaction results in nonlinear kinetics according to the Michaelis-Menten equation. The two compartment open model with Michaelis-Menten elimination kinetics is theorethicaly basic when single doses of drug are administered. To simulate data or to fit real data using this model, one must resort to numerical integration. A biomathematical model for multiple dosage regimen calculations of nonlinear metabolic systems in steady-state and a working example with phenytoin are presented. High correlation between phenytoin steady-state serum levels calculated from individual Km and Vmax values in the 15 adult epileptic outpatients and the observed levels at the third adjustment of phenytoin daily dose (r=0.961, p<0.01) were found.
Theoretical model of the effect of potassium on the uptake of radiocesium by rice.

PubMed

Fujimura, Shigeto; Ishikawa, Junko; Sakuma, Yuuki; Saito, Takashi; Sato, Mutsuto; Yoshioka, Kunio

2014-12-01

After the accident at the Fukushima Dai-ichi Nuclear Power Plant owned by Tokyo Electric Power Company on 11 March 2011, potassium was applied to fields in the Tohoku and Kanto areas of Japan to reduce radiocesium uptake by crops. Despite the intense studies relating to the effect of potassium application on availability of radiocesium in the soil, physiological changes of radiocesium uptake by crops in response to K(+) concentration around roots remains elusive. In the present study, we developed physiological models describing the effect of K(+) on the uptake of radiocesium by rice. Two Cs(+):K(+) competition models were evaluated using a wide range of data obtained from pot and field experiments: the model assuming a uniformity in the gene expression of K(+) transporter (Model I) and the model assuming the increase in the gene expression of K(+) transporter in response to K(+) concentration below threshold (Model II). The root-mean-square deviation between the measured and estimated values was larger in Model I than in Model II. Residuals were positively correlated with K(+) in Model I but showed no deflection in Model II. These results indicate that Model II explains the effect of K(+) on the uptake of radiocesium better than Model I. Model II may provide the appropriate countermeasures in inhibiting the transfer of radiocesium from soil to crop. The effect of changes in the variables in Model II on the relationship between available K(+) in soil and (137)Cs uptake by plant was simulated. An increase in available (137)Cs(+) in soil enhanced the response of (137)Cs uptake to K(+). The effects of Michaelis-Menten constant for Cs(+) were the inverse of the (137)Cs(+) effect. The effect of Michaelis-Menten constant for K(+) showed the same tendency as that of (137)Cs(+), but the effect was much less than that of (137)Cs(+). An increase in the threshold of K(+) below which the gene expression of K(+) transporter increases enhanced the response of (137)Cs uptake to K(+) in the high-K(+) range. Copyright © 2014 Elsevier Ltd. All rights reserved.
Thermal and single frequency counter-current ultrasound pretreatments of sodium caseinate: enzymolysis kinetics and thermodynamics, amino acids composition, molecular weight distribution and antioxidant peptides.

PubMed

Abdualrahman, Mohammed Adam Y; Ma, Haile; Zhou, Cunshan; Yagoub, Abu ElGasim A; Hu, Jiali; Yang, Xue

2016-12-01

Due to the disadvantages of traditional enzymolysis, pretreatments are crucial to enhance protein enzymolysis. Enzymolysis kinetics and thermodynamics, amino acids composition, molecular weight distribution, fluorescence spectroscopy and antioxidant activity of thermal (HT) and single frequency counter-current ultrasound (SCFU) pretreated sodium caseinate (NaCas) were studied. Enzymolysis of untreated NaCas (control) improved significantly (P < 0.05) by SFCU and followed by HT. Values of the Michaelis-Menten constant (K M ) of SFCU and HT were 0.0212 and 0.0250, respectively. HT and SFCU increased (P < 0.05) the reaction rate constant (k) by 38.64 and 90.91%, respectively at 298 K. k values decreased with increasing temperature. The initial activation energy (46.39 kJ mol -1 ) reduced (P < 0.05) by HT (39.66 kJ mol -1 ) and further by SFCU (33.42 kJ mol -1 ). SFCU-pretreated NaCas hydrolysates had the highest contents of hydrophobic, aromatic, positively and negatively charged amino acids. Medium-sized peptides (5000-1000 Da) are higher in SFCU (78.11%) than HT and the control. SFCU induced molecular unfolding of NaCas proteins. Accordingly, SFCU-pretreated NaCas hydrolysate exhibited the highest scavenging activity on DPPH and hydroxyl radicals, reducing power, and iron chelating ability. SFCU pretreatment would be a useful tool for production of bioactive peptides from NaCas hydrolysate. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Electrochemical Quantification of the Antioxidant Capacity of Medicinal Plants Using Biosensors

PubMed Central

Rodríguez-Sevilla, Erika; Ramírez-Silva, María-Teresa; Romero-Romo, Mario; Ibarra-Escutia, Pedro; Palomar-Pardavé, Manuel

2014-01-01

The working area of a screen-printed electrode, SPE, was modified with the enzyme tyrosinase (Tyr) using different immobilization methods, namely entrapment with water-soluble polyvinyl alcohol (PVA), cross-linking using glutaraldehyde (GA), and cross-linking using GA and human serum albumin (HSA); the resulting electrodes were termed SPE/Tyr/PVA, SPE/Tyr/GA and SPE/Tyr/HSA/GA, respectively. These biosensors were characterized by means of amperometry and EIS techniques. From amperometric evaluations, the apparent Michaelis-Menten constant, Km′, of each biosensor was evaluated while the respective charge transfer resistance, Rct, was assessed from impedance measurements. It was found that the SPE/Tyr/GA had the smallest Km′ (57 ± 7) μM and Rct values. This electrode also displayed both the lowest detection and quantification limits for catechol quantification. Using the SPE/Tyr/GA, the Trolox Equivalent Antioxidant Capacity (TEAC) was determined from infusions prepared with “mirto” (Salvia microphylla), “hHierba dulce” (Lippia dulcis) and “salve real” (Lippia alba), medicinal plants commonly used in Mexico. PMID:25111237
Electrochemical quantification of the antioxidant capacity of medicinal plants using biosensors.

PubMed

Rodríguez-Sevilla, Erika; Ramírez-Silva, María-Teresa; Romero-Romo, Mario; Ibarra-Escutia, Pedro; Palomar-Pardavé, Manuel

2014-08-08

The working area of a screen-printed electrode, SPE, was modified with the enzyme tyrosinase (Tyr) using different immobilization methods, namely entrapment with water-soluble polyvinyl alcohol (PVA), cross-linking using glutaraldehyde (GA), and cross-linking using GA and human serum albumin (HSA); the resulting electrodes were termed SPE/Tyr/PVA, SPE/Tyr/GA and SPE/Tyr/HSA/GA, respectively. These biosensors were characterized by means of amperometry and EIS techniques. From amperometric evaluations, the apparent Michaelis-Menten constant, Km', of each biosensor was evaluated while the respective charge transfer resistance, Rct, was assessed from impedance measurements. It was found that the SPE/Tyr/GA had the smallest Km' (57 ± 7) µM and Rct values. This electrode also displayed both the lowest detection and quantification limits for catechol quantification. Using the SPE/Tyr/GA, the Trolox Equivalent Antioxidant Capacity (TEAC) was determined from infusions prepared with "mirto" (Salvia microphylla), "hHierba dulce" (Lippia dulcis) and "salve real" (Lippia alba), medicinal plants commonly used in Mexico.
Characterization of procaine metabolism as probe for the butyrylcholinesterase enzyme investigation by simultaneous determination of procaine and its metabolite using capillary electrophoresis with electrochemiluminescence detection.

PubMed

Yuan, Jipei; Yin, Jianyuan; Wang, Erkang

2007-06-22

Capillary electrophoresis with electrochemiluminescene detection was used to characterize procaine hydrolysis as a probe for butyrylcholinesterase by in vitro procaine metabolism in plasma with butyrylcholinesterase acting as bioscavenger. Procaine and its metabolite N,N-diethylethanolamine were separated at 16 kV and then detected at 1.25 V in the presence of 5.0 mM Ru(bpy)(3)2+, with the detection limits of 2.4x10(-7) and 2.0x10(-8) mol/L (S/N=3), respectively. The Michaelis constant Km value was 1.73x10(-4) mol/L and the maximum velocity Vmax was 1.62x10(-6) mol/L/min. Acetylcholine bromide and choline chloride presented inhibition effects on the enzymatic cleavage of procaine, with the 50% inhibition concentration (IC50) of 6.24x10(-3) and 2.94x10(-4) mol/L.
Degradation kinetics and metabolites in continuous biodegradation of isoprene.

PubMed

Srivastva, Navnita; Singh, Ram S; Upadhyay, Siddh N; Dubey, Suresh K

2016-04-01

The kinetic parameters of isoprene biodegradation were studied in a bioreactor, comprising of bioscrubber and polyurethane foam packed biofilter in series and inoculated with Pseudomonas sp., using a Michaelis-Menten type model. The maximum elimination capacity, ECmax; substrate constant, Ks and ECmax/Ks values for bioscrubber were found to be 666.7 g m(-3) h(-1), 9.86 g m(-3) and 67.56 h(-1), respectively while those for biofilter were 3333 g m(-3) h(-1), 13.96 g m(-3) and 238.7 h(-1), respectively. The biofilter section exhibited better degradation efficiency compared to the bioscrubber unit. Around 62-75% of the feed isoprene got converted to carbon dioxide, indicating the efficient capability of bacteria to mineralize isoprene. The FTIR and GC-MS analyses of degradation products indicated oxidative cleavage of unsaturated bond of isoprene. These results were used for proposing a plausible degradation pathway for isoprene. Copyright © 2016 Elsevier Ltd. All rights reserved.
Substrate specificity and pH dependence of homogeneous wheat germ acid phosphatase.

PubMed

Van Etten, R L; Waymack, P P

1991-08-01

The broad substrate specificity of a homogeneous isoenzyme of wheat germ acid phosphatase (WGAP) was extensively investigated by chromatographic, electrophoretic, NMR, and kinetic procedures. WGAP exhibited no divalent metal ion requirement and was unaffected upon incubation with EDTA or o-phenanthroline. A comparison of two catalytically homogeneous isoenzymes revealed little difference in substrate specificity. The specificity of WGAP was established by determining the Michaelis constants for a wide variety of substrates. p-Nitrophenyl phosphate, pyrophosphate, tripolyphosphate, and ATP were preferred substrates while lesser activities were seen toward sugar phosphates, trimetaphosphate, phosphoproteins, and (much less) phosphodiesters. An extensive table of Km and Vmax values is given. The pathway for the hydrolysis of trimetaphosphate was examined by colorimetric and 31P NMR methods and it was found that linear tripolyphosphate is not a free intermediate in the enzymatic reaction. In contrast to literature reports, homogeneous wheat germ acid phosphatase exhibits no measurable carboxylesterase activity, nor does it hydrolyze phenyl phosphonothioate esters or phytic acid at significant rates.
Quantifying stream nutrient uptake from ambient to saturation with instantaneous tracer additions

NASA Astrophysics Data System (ADS)

Covino, T. P.; McGlynn, B. L.; McNamara, R.

2009-12-01

Stream nutrient tracer additions and spiraling metrics are frequently used to quantify stream ecosystem behavior. However, standard approaches limit our understanding of aquatic biogeochemistry. Specifically, the relationship between in-stream nutrient concentration and stream nutrient spiraling has not been characterized. The standard constant rate (steady-state) approach to stream spiraling parameter estimation, either through elevating nutrient concentration or adding isotopically labeled tracers (e.g. 15N), provides little information regarding the stream kinetic curve that represents the uptake-concentration relationship analogous to the Michaelis-Menten curve. These standard approaches provide single or a few data points and often focus on estimating ambient uptake under the conditions at the time of the experiment. Here we outline and demonstrate a new method using instantaneous nutrient additions and dynamic analyses of breakthrough curve (BTC) data to characterize the full relationship between spiraling metrics and nutrient concentration. We compare the results from these dynamic analyses to BTC-integrated, and standard steady-state approaches. Our results indicate good agreement between these three approaches but we highlight the advantages of our dynamic method. Specifically, our new dynamic method provides a cost-effective and efficient approach to: 1) characterize full concentration-spiraling metric curves; 2) estimate ambient spiraling metrics; 3) estimate Michaelis-Menten parameters maximum uptake (Umax) and the half-saturation constant (Km) from developed uptake-concentration kinetic curves, and; 4) measure dynamic nutrient spiraling in larger rivers where steady-state approaches are impractical.
Activation of pollen tube callose synthase by detergents. Evidence for different mechanisms of action.

PubMed Central

Li, H; Bacic, A; Read, S M

1997-01-01

In pollen tubes of Nicotiana alata, a membrane-bound, Ca(2+)-independent callose synthase (CalS) is responsible for the biosynthesis of the (1,3)-beta-glucan backbone of callose, the main cell wall component. Digitonin increases CalS activity 3- to 4-fold over a wide range of concentrations, increasing the maximum initial velocity without altering the Michaelis constant for UDP-glucose. The CalS activity that requires digitonin for assay (the latent CalS activity) is not inhibited by the membrane-impermeant, active site-directed reagent UDP-pyridoxal when the reaction is conducted in the absence of digitonin. This is consistent with digitonin increasing CalS activity by the permeabilization of membrane vesicles. A second group of detergents, including 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS), Zwittergent 3-16, and 1-alpha-lysolecithin, activate pollen tube CalS 10- to 15-fold, but only over a narrow range of concentrations just below their respective critical micellar concentrations. This activation could not be attributed to any particular chemical feature of these detergents. CHAPS increases maximum initial velocity and decreases the Michaelis constant for UDP-glucose and activates CalS even in the presence of permeabilizing concentrations of digitonin. Inhibition studies with UDP-pyridoxal indicate that activation by CHAPS occurs by recruitment of previously inactive CalS molecules to the pool of active enzyme. The activation of pollen tube CalS by these detergents therefore resembles activation of the enzyme by trypsin. PMID:9276948
Influence of ultrasound pretreatment on enzymolysis kinetics and thermodynamics of sodium hydroxide extracted proteins from tea residue.

PubMed

Ayim, Ishmael; Ma, Haile; Alenyorege, Evans Adingba; Ali, Zeshan; Donkor, Prince Ofori

2018-03-01

The effect of ultrasound pretreatment using Single Frequency Counter Current Ultrasound (SFCCU) on the enzymolysis of tea residue protein (TRP) extracted with sodium hydroxide was investigated. The concentration of TRP hydrolysate, enzymolysis kinetics and thermodynamic parameters after SFCCU pretreatment were determined and compared with traditional enzymolysis. The results indicated that both ultrasound assisted and traditional enzymolysis conformed to first-order kinetics within the limits of the studied parameters. Temperature and sonication had affirmative effect on the enzymolysis of TRP with temperature yielding greater impact. Michaelis constant ( K M ) in ultrasonic pretreated enzymolysis decreased by 32.7% over the traditional enzymolysis. The highest polypeptide concentration of 24.12 mg ml -1 was obtained with the lowest energy requirement at improved conditions of 50 g L -1 of TRP, alcalase concentration of 2000 U g -1 , time of 10 min and temperature of 50 °C for the ultrasonic treated enzymolysis. The values of reaction rate constant ( k ) for TRP enzymolysis increased by 78, 40, 82 and 60% at 20, 30, 40 and 50 °C, respectively. The thermodynamic properties comprising activation energy ( Ea ), change in enthalpy (∆H ) and entropy (∆S ) were reduced by ultrasound pretreatment whereas Gibbs free energy (∆G ) was increased.
Allometric scaling in-vitro

NASA Astrophysics Data System (ADS)

Ahluwalia, Arti

2017-02-01

About two decades ago, West and coworkers established a model which predicts that metabolic rate follows a three quarter power relationship with the mass of an organism, based on the premise that tissues are supplied nutrients through a fractal distribution network. Quarter power scaling is widely considered a universal law of biology and it is generally accepted that were in-vitro cultures to obey allometric metabolic scaling, they would have more predictive potential and could, for instance, provide a viable substitute for animals in research. This paper outlines a theoretical and computational framework for establishing quarter power scaling in three-dimensional spherical constructs in-vitro, starting where fractal distribution ends. Allometric scaling in non-vascular spherical tissue constructs was assessed using models of Michaelis Menten oxygen consumption and diffusion. The models demonstrate that physiological scaling is maintained when about 5 to 60% of the construct is exposed to oxygen concentrations less than the Michaelis Menten constant, with a significant concentration gradient in the sphere. The results have important implications for the design of downscaled in-vitro systems with physiological relevance.
Subsite mapping of enzymes. Application of the depolymerase computer model to two alpha-amylases.

PubMed Central

Allen, J D; Thoma, J A

1976-01-01

In the preceding paper (Allen and Thoma, 1976) we developed a depolymerase computer model, which uses a minimization routine to establish a subsite map for a depolymerase. In the present paper we show how the model is applied to experimental data for two alpha-amylases. Michaelis parameters and bond-cleavage frequencies for substrates of chain lengths up to twelve glucosyl units have been reported for Bacillus amyloliquefaciens, and a subsite map has been proposed for this enzyme [Thoma et al. (1971) J. Biol. Chem. 246, 5621-5635]. By applying the computer model to the experimental data, we have arrived at a ten-subsite map. We find that a significant improvement in this map is achieved by allowing the hydrolytic rate coefficient to vary as a function of the number of occupied subsites comprising the enzyme-binding region. The bond-cleavage frequencies, the enzyme is found to have eight subsites. A partial subsite map is arrived at, but the entire binding region cannot be mapped because Michaelis parameters are complicated by transglycosylation reactions. The hydrolytic rate coefficients for this enzyme are not constant. PMID:999630
Effects of dimethyl sulfoxide, temperature, and sodium chloride on the activity of human matrix metalloproteinase 7 (matrilysin).

PubMed

Oneda, H; Inouye, K

2000-11-01

Effects of dimethyl sulfoxide (DMSO), temperature, and sodium chloride on the matrilysin-catalyzed hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2, 4-dinitrophenyl)-L-2,3-diamino-propionyl]-L-Ala-L-Arg-NH(2) [MOCAc-PLGL(Dpa)AR] were examined. DMSO inhibited the matrilysin activity competitively with the inhibitor constant (K(i)) of 0. 59+/-0.04 M, and the binding between them was endothermic and entropy-driven. The binding of matrilysin with MOCAc-PLGL(Dpa)AR was also found to be entropy-driven. The matrilysin activity was increased in a biphasic exponential fashion with increasing concentration of NaCl, and was 5.3 times higher in the presence of 4 M NaCl than that in its absence. The first and second phases were separated at 0.5 M NaCl, and the activation at x M NaCl compared with the activity in the absence of NaCl was expressed as 2.1(x) at [NaCl] < 0.5 M and 1.4(x) at [NaCl] > 0.5 M. The activation was brought about solely through a decrease in the Michaelis constant (K(m)), and the catalytic constant (k(cat)) was not much altered. This suggests that the decrease in the electrostatic interaction and the increase in the hydrophobic interaction between matrilysin and the substrate might enhance the enzyme activity by reducing the K(m) value.
Choice Defines Value: A Predictive Modeling Competition in Health Preference Research.

PubMed

Jakubczyk, Michał; Craig, Benjamin M; Barra, Mathias; Groothuis-Oudshoorn, Catharina G M; Hartman, John D; Huynh, Elisabeth; Ramos-Goñi, Juan M; Stolk, Elly A; Rand, Kim

2018-02-01

To identify which specifications and approaches to model selection better predict health preferences, the International Academy of Health Preference Research (IAHPR) hosted a predictive modeling competition including 18 teams from around the world. In April 2016, an exploratory survey was fielded: 4074 US respondents completed 20 out of 1560 paired comparisons by choosing between two health descriptions (e.g., longer life span vs. better health). The exploratory data were distributed to all teams. By July, eight teams had submitted their predictions for 1600 additional pairs and described their analytical approach. After these predictions had been posted online, a confirmatory survey was fielded (4148 additional respondents). The victorious team, "Discreetly Charming Econometricians," led by Michał Jakubczyk, achieved the smallest χ 2 , 4391.54 (a predefined criterion). Its primary scientific findings were that different models performed better with different pairs, that the value of life span is not constant proportional, and that logit models have poor predictive validity in health valuation. The results demonstrated the diversity and potential of new analytical approaches in health preference research and highlighted the importance of predictive validity in health valuation. Copyright © 2018 International Society for Pharmacoeconomics and Outcomes Research (ISPOR). Published by Elsevier Inc. All rights reserved.
Effective immobilization of glucose oxidase on chitosan submicron particles from gladius of Todarodes pacificus for glucose sensing.

PubMed

Anusha, J R; Fleming, Albin T; Kim, Hee-Je; Kim, Byung Chul; Yu, Kook-Hyun; Raj, C Justin

2015-08-01

An effective enzymatic glucose biosensor was developed by immobilizing glucose oxidase on chitosan submicron particles synthesized from the gladius of Todarodes pacificus (GCSP). The chemically synthesized chitosan from gladius was pulverized to submicron particles by ball milling technique, which was further characterized and compared with the standard chitosan (SCS). The degree of deacetylation of GCSP was determined using FTIR spectroscopy which was comparable to the value of standard chitosan. The glucose oxidase (GOx) was immobilized over GCSP on porous zinc oxide/platinum nanoparticle (ZnO/Pt) based electrode. The morphological and structural properties of the electrodes were analyzed using scanning electron microscopy and X-ray diffraction analysis. The glucose sensing behavior of electrode was estimated using electrochemical analysis and showed an excellent analytical performance. The electrode ZnO/Pt/GCSP conjugated with GOx displayed high sensitivity (88.76 μA mM(-1) cm(-2)) with low detection limit in short response time. In addition, the very low value of Michaelis-Menten constant for GCSP based electrode contributes a better affinity of the electrode surface towards glucose oxidase. Copyright © 2015 Elsevier B.V. All rights reserved.
Antioxidant and Inhibitory Effects of Saponin Extracts from Dianthus basuticus Burtt Davy on Key Enzymes Implicated in Type 2 Diabetes In vitro.

PubMed

Nafiu, Mikhail Olugbemiro; Ashafa, Anofi Omotayo Tom

2017-01-01

Dianthus basuticus is a plant of South African origin with various acclaimed pharmaceutical potentials. This study explored the antioxidant and antidiabetic activities of saponin extract from D. basuticus in vitro . Antioxidant activity of saponin was evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and nitric oxide (*NO)-free radical scavenging activity while antidiabetic potentials were measured by the α-amylase and α-glucosidase inhibitory activities of the saponin extract. The results showed that the saponin extract, compared with quercetin, displayed better DPPH (IC 50 = 6.95 mg/ml) and NO (IC 50 = 3.31 mg/ml) radical scavenging capabilities. Similarly, the saponin extracts elicited stronger α-glucosidase (IC 50 = 3.80 mg/ml) and moderate α-amylase (IC 50 = 4.18 mg/ml) inhibitory activities as compared to acarbose. Saponin exhibited a competitive mode of inhibition on α-amylase with same maximum velocity (Vmax) of 0.0093 mM/min for saponin compared with control 0.0095 mM/min and different the Michaelis constant (Km) values of 2.6 × 10 -6 mM and 2.1 × 10 -5 mM, respectively, while for α-glucosidase, the inhibition was uncompetitive, Vmax of 0.027 mM/min compared with control 0.039 mM/min and Km values of 1.02 × 10 -6 mM and 1.38 × 10 -6 mM, respectively. The gas chromatography-mass spectrometric analysis revealed the presence of bioactive like β- and α-amyrin, 3-O-methyl-D-glucose, methyl commate, and olean-12-en-3-beta-ol. Overall, the data suggested that the saponin extract from D. basuticus has potentials as natural antioxidants and antidiabetics. Saponin extract from Dianthus basuticus displayed promising antidiabetic and antioxidant activitySaponin competitively and uncompetitively inhibited a-amylase and a-glucosidase, respectivelyThe stronger inhibition of α-glucosidase and moderate inhibition of α-amylase by saponin extract from D. basuticus is promising good antidiabetes compared with existing drugs with associated side effects. Abbreviations used: DPPH: 2,2-diphenyl-1-picrylhydrazyl, Km: The Michaelis constant, Vmax: Maximum velocity, ROS: Reactive oxygen species, NIDDM: Non-insulin-dependent diabetes mellitus, UFS: University of the Free State, GC-MS: Gas chromatography-mass spectrometric, MS: Mass spectrometry, NIST: National Institute of Standards and Technology, DNS: 3,5-dinitrosalicylic acid, NO: Nitric oxide, RNS: Reactive nitrogen species, PNPG: p-Nitrophenyl-α-D-glucopyranoside.
Antioxidant and Inhibitory Effects of Saponin Extracts from Dianthus basuticus Burtt Davy on Key Enzymes Implicated in Type 2 Diabetes In vitro

PubMed Central

Nafiu, Mikhail Olugbemiro; Ashafa, Anofi Omotayo Tom

2017-01-01

Context: Dianthus basuticus is a plant of South African origin with various acclaimed pharmaceutical potentials. Aims: This study explored the antioxidant and antidiabetic activities of saponin extract from D. basuticus in vitro. Materials and Methods: Antioxidant activity of saponin was evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and nitric oxide (*NO)-free radical scavenging activity while antidiabetic potentials were measured by the α-amylase and α-glucosidase inhibitory activities of the saponin extract. Results: The results showed that the saponin extract, compared with quercetin, displayed better DPPH (IC50 = 6.95 mg/ml) and NO (IC50 = 3.31 mg/ml) radical scavenging capabilities. Similarly, the saponin extracts elicited stronger α-glucosidase (IC50 = 3.80 mg/ml) and moderate α-amylase (IC50 = 4.18 mg/ml) inhibitory activities as compared to acarbose. Saponin exhibited a competitive mode of inhibition on α-amylase with same maximum velocity (Vmax) of 0.0093 mM/min for saponin compared with control 0.0095 mM/min and different the Michaelis constant (Km) values of 2.6 × 10-6 mM and 2.1 × 10-5 mM, respectively, while for α-glucosidase, the inhibition was uncompetitive, Vmax of 0.027 mM/min compared with control 0.039 mM/min and Km values of 1.02 × 10-6 mM and 1.38 × 10-6 mM, respectively. The gas chromatography-mass spectrometric analysis revealed the presence of bioactive like β- and α-amyrin, 3-O-methyl-D-glucose, methyl commate, and olean-12-en-3-beta-ol. Conclusion: Overall, the data suggested that the saponin extract from D. basuticus has potentials as natural antioxidants and antidiabetics. SUMMARY Saponin extract from Dianthus basuticus displayed promising antidiabetic and antioxidant activitySaponin competitively and uncompetitively inhibited a-amylase and a-glucosidase, respectivelyThe stronger inhibition of α-glucosidase and moderate inhibition of α-amylase by saponin extract from D. basuticus is promising good antidiabetes compared with existing drugs with associated side effects. Abbreviations used: DPPH: 2,2-diphenyl-1-picrylhydrazyl, Km: The Michaelis constant, Vmax: Maximum velocity, ROS: Reactive oxygen species, NIDDM: Non-insulin-dependent diabetes mellitus, UFS: University of the Free State, GC-MS: Gas chromatography-mass spectrometric, MS: Mass spectrometry, NIST: National Institute of Standards and Technology, DNS: 3,5-dinitrosalicylic acid, NO: Nitric oxide, RNS: Reactive nitrogen species, PNPG: p-Nitrophenyl-α-D-glucopyranoside. PMID:29200716
Effects of growth rate, cell size, motion, and elemental stoichiometry on nutrient transport kinetics

PubMed Central

Skibinski, David O. F.

2018-01-01

Nutrient acquisition is a critical determinant for the competitive advantage for auto- and osmohetero- trophs alike. Nutrient limited growth is commonly described on a whole cell basis through reference to a maximum growth rate (Gmax) and a half-saturation constant (KG). This empirical application of a Michaelis-Menten like description ignores the multiple underlying feedbacks between physiology contributing to growth, cell size, elemental stoichiometry and cell motion. Here we explore these relationships with reference to the kinetics of the nutrient transporter protein, the transporter rate density at the cell surface (TRD; potential transport rate per unit plasma-membrane area), and diffusion gradients. While the half saturation value for the limiting nutrient increases rapidly with cell size, significant mitigation is afforded by cell motion (swimming or sedimentation), and by decreasing the cellular carbon density. There is thus potential for high vacuolation and high sedimentation rates in diatoms to significantly decrease KG and increase species competitive advantage. Our results also suggest that Gmax for larger non-diatom protists may be constrained by rates of nutrient transport. For a given carbon density, cell size and TRD, the value of Gmax/KG remains constant. This implies that species or strains with a lower Gmax might coincidentally have a competitive advantage under nutrient limited conditions as they also express lower values of KG. The ability of cells to modulate the TRD according to their nutritional status, and hence change the instantaneous maximum transport rate, has a very marked effect upon transport and growth kinetics. Analyses and dynamic models that do not consider such modulation will inevitably fail to properly reflect competitive advantage in nutrient acquisition. This has important implications for the accurate representation and predictive capabilities of model applications, in particular in a changing environment. PMID:29702650
Diabetic Hyperglycemia: Link to Impaired Glucose Transport in Pancreatic β Cells

NASA Astrophysics Data System (ADS)

Unger, Roger H.

1991-03-01

Glucose uptake into pancreatic β cells by means of the glucose transporter GLUT-2, which has a high Michaelis constant, is essential for the normal insulin secretory response to hyperglycemia. In both autoimmune and nonautoimmune diabetes, this glucose transport is reduced as a consequence of down-regulation of the normal β-cell transporter. In autoimmune diabetes, circulating immunoglobulins can further impair this glucose transport by inhibiting functionally intact transporters. Insights into mechanisms of the unresponsiveness of β cells to hyperglycemia may improve the management and prevention of diabetes.
Malate dehydrogenase isozymes in the longnose dace, Rhinichthys cataractae.

PubMed

Starzyk, R M; Merritt, R B

1980-08-01

The interspecies homology of dace supernatant (A2,AB,B2) and mitochondrial (C2) malate dehydrogenase isozymes has been established through cell fractionation and tissue distribution studies. Isolated supernatant malate dehydrogenase (s-MDH) isozymes show significant differences in Michaelis constants for oxaloacetate and in pH optima. Shifts in s-MDH isozyme pH optima with temperature may result in immediate compensation for increase in ectotherm body pH with decrease in temperature, but duplicate s-MDH isozymes are probably maintained through selection for tissue specific regulation of metabolism.
A novel hydrogen peroxide biosensor based on hemoglobin-collagen-CNTs composite nanofibers.

PubMed

Li, J; Mei, H; Zheng, W; Pan, P; Sun, X J; Li, F; Guo, F; Zhou, H M; Ma, J Y; Xu, X X; Zheng, Y F

2014-06-01

In this paper, carbon nanotubes (CNTs) were successfully incorporated in the composite composed of hemoglobin (Hb) and collagen using co-electrospinning technology. The formed Hb-collagen-CNTs composite nanofibers possessed distinct advantage of three-dimensional porous structure, biocompatibility and excellent stability. The Hb immobilized in the electrospun nanofibers retained its natural structure and the heterogeneous electron transfer rate constant (ks) of the direct electron transfer between Hb and electrodes was 5.3s(-1). In addition, the electrospun Hb-collagen-CNTs nanofibers modified electrodes showed good electrocatalytic properties toward H2O2 with a detection limit of 0.91μM (signal-to-noise ratio of 3) and the apparent Michaelis-Menten constant (Km(app)) of 32.6μM. Copyright © 2014 Elsevier B.V. All rights reserved.
Effect of quantifying peptide release on ruminal protein degradation determined using the inhibitor in vitro system.

PubMed

Colombini, S; Broderick, G A; Clayton, M K

2011-04-01

The aim of this work was to compare use of an o-phthaldialdehyde (OPA) colorimetric assay (OPA-C), which responds to both free AA and peptides, with an OPA fluorimetric assay (OPA-F), which is insensitive to peptides, to quantify rates of ruminal protein degradation in the inhibitor in vitro system using Michaelis-Menten saturation kinetics. Four protein concentrates (expeller-extracted soybean meal, ESBM; 2 solvent-extracted soybean meals, SSBM1 and SSBM2; and casein) were incubated in a ruminal in vitro system treated with hydrazine and chloramphenicol to inhibit microbial uptake of protein degradation products. Proteins were weighed to give a range of N concentrations (from 0.15 to 3 mg of N/mL of inoculum) and incubated with 10 mL of ruminal inoculum and 5 mL of buffer; fermentations were stopped after 2 h by adding trichloroacetic acid (TCA). Proteins were analyzed for buffer-soluble N and buffer extracts were treated with TCA to determine N degraded at t=0 (FD0). The TCA supernatants were analyzed for ammonia (phenol-hypochlorite assay), total AA (TAA; OPA-F), and TAA plus oligopeptides (OPA-C) by flow injection analysis. Velocity of protein degradation was computed from extent of release of 1) ammonia plus free TAA or 2) ammonia plus free TAA and peptides. Rate of degradation (kd) was quantified using nonlinear regression of the integrated Michaelis-Menten equation. The parameters Km (Michaelis constant) and kd (Vmax/Km), where Vmax=maximum velocity, were estimated directly; kd values were adjusted (Akd) for the fraction FD0 using the equation Akd=kd-FD0/2. The OPA-C assay yielded faster degradation rates due to the contribution of peptides to the fraction degraded (overall mean=0.280/h by OPA-C and 0.219/h by OPA-F). Degradation rates for SSBM samples (0.231/h and 0.181/h) and ESBM (0.086/h) obtained by the OPA-C assay were more rapid than rates reported by the National Research Council (NRC). Both assays indicated that the 2 SSBM differed in rumen-undegradable protein (RUP) content; the more slowly degraded SSBM had RUP content (35% by OPA-C) similar to that reported by the NRC. The RUP content of ESBM (42% by OPA-C) was lower than the NRC value. Preliminary studies with 4 additional protein concentrates confirmed that accounting for peptide formation increased degradation rate; however, a trend for an interaction between assay and protein source suggested that peptide release made a smaller contribution to rate for more slowly degraded proteins. The OPA-C assay is a simple and reliable method to quantify formation of small peptides. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Effect of bacterial growth rate on bacteriophage population growth rate.

PubMed

Nabergoj, Dominik; Modic, Petra; Podgornik, Aleš

2018-04-01

It is important to understand how physiological state of the host influence propagation of bacteriophages (phages), due to the potential higher phage production needs in the future. In our study, we tried to elucidate the effect of bacterial growth rate on adsorption constant (δ), latent period (L), burst size (b), and bacteriophage population growth rate (λ). As a model system, a well-studied phage T4 and Escherichia coli K-12 as a host was used. Bacteria were grown in a continuous culture operating at dilution rates in the range between 0.06 and 0.98 hr -1 . It was found that the burst size increases linearly from 8 PFU·cell -1 to 89 PFU·cell -1 with increase in bacteria growth rate. On the other hand, adsorption constant and latent period were both decreasing from 2.6∙10 -9 ml·min -1 and 80 min to reach limiting values of 0.5 × 10 -9 ml·min -1 and 27 min at higher growth rates, respectively. Both trends were mathematically described with Michaelis-Menten based type of equation and reasons for such form are discussed. By applying selected equations, a mathematical equation for prediction of bacteriophage population growth rate as a function of dilution rate was derived, reaching values around 8 hr -1 at highest dilution rate. Interestingly, almost identical description can be obtained using much simpler Monod type equation and possible reasons for this finding are discussed. © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.
Enhancement stability and catalytic activity of immobilized α-amylase using bioactive phospho-silicate glass as a novel inorganic support.

PubMed

Ahmed, Samia A; Mostafa, Faten A; Ouis, Mona A

2018-06-01

α-Amylase enzyme was immobilized on bioactive phospho-silicate glass (PS-glass) as a novel inorganic support by physical adsorption and covalent binding methods using glutaraldehyde and poly glutaraldehyde as a spacer. Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM) studies confirmed the glass-enzyme linkage. Dissolution of PS-glass in acidic and neutral pH is higher than that of alkaline pH. Some immobilization variables were optimized using statistical factorial design (Central Composite Design). Optimized immobilization variables enhanced the immobilization yield (IY) from 27.9 to 79.9% (2.9-fold). It was found that the immobilized enzyme had higher optimum temperature, higher half-life time (t 1/2 ), lower activation energy (E a ), lower deactivation constant rate (k d ) and higher decimal reduction time (D-values) within the temperature range of 40-60°C. Differential scanning calorimetry analysis (DSC) confirmed the thermalstability of the immobilized enzyme. The immobilized enzyme was stable at a wide pH range (5.0-8.0). Kinetic studies of starch hydrolysis demonstrated that immobilized enzyme had lower Michaelis constant (K m ), maximum velocity (V max ) and catalytic efficiency (V max /K m ) values. The storage stability and reusability of the immobilized enzyme were found to be about 74.7 and 62.5% of its initial activity after 28days and 11cycles, respectively. Enhanced α-amylase stabilities upon immobilization make it suitable for industrial application. Copyright © 2018 Elsevier B.V. All rights reserved.
In memory of Professor Leonor Michaelis in Nagoya: great contributions to biochemistry in Japan in the first half of the 20th century.

PubMed

Nagatsu, Toshiharu Toshi

2013-09-02

Leonor Michaelis spent the years of 1922-1926 as Professor of Biochemistry of the Aichi Medical College (now Graduate School of Medicine, Nagoya University) in Nagoya, Japan. Michaelis succeeded in gathering many bright young biochemists from all over Japan into his laboratory, and made tremendous contributions to the promotion of biochemistry in Japan. Michaelis was invited to many places in Japan to present lectures over those years. Kunio Yagi, who was Professor of Biochemistry at Nagoya University in the second half of the 20th century, succeeded in crystallizing the "Michaelis" enzyme-substrate complex. Historically, Michelis has had an enormous impact on biochemistry in Japan. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Torque-coupled thermodynamic model for FoF1 -ATPase

NASA Astrophysics Data System (ADS)

Ai, Guangkuo; Liu, Pengfei; Ge, Hao

2017-05-01

FoF1 -ATPase is a motor protein complex that utilizes transmembrane ion flow to drive the synthesis of adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and phosphate (Pi). While many theoretical models have been proposed to account for its rotary activity, most of them focus on the Fo or F1 portions separately rather than the complex as a whole. Here, we propose a simple but new torque-coupled thermodynamic model of FoF1 -ATPase. Solving this model at steady state, we find that the monotonic variation of each portion's efficiency becomes much more robust over a wide range of parameters when the Fo and F1 portions are coupled together, as compared to cases when they are considered separately. Furthermore, the coupled model predicts the dependence of each portion's kinetic behavior on the parameters of the other. Specifically, the power and efficiency of the F1 portion are quite sensitive to the proton gradient across the membrane, while those of the Fo portion as well as the related Michaelis constants for proton concentrations respond insensitively to concentration changes in the reactants of ATP synthesis. The physiological proton gradient across the membrane in the Fo portion is also shown to be optimal for the Michaelis constants of ADP and phosphate in the F1 portion during ATP synthesis. Together, our coupled model is able to predict key dynamic and thermodynamic features of the FoF1 -ATPase in vivo semiquantitatively, and suggests that such coupling approach could be further applied to other biophysical systems.
Direct electron transfer of Phanerochaete chrysosporium cellobiose dehydrogenase at platinum and palladium nanoparticles decorated carbon nanotubes modified electrodes.

PubMed

Bozorgzadeh, Somayyeh; Hamidi, Hassan; Ortiz, Roberto; Ludwig, Roland; Gorton, Lo

2015-10-07

In the present work, platinum and palladium nanoparticles (PtNPs and PdNPs) were decorated on the surface of multi-walled carbon nanotubes (MWCNTs) by a simple thermal decomposition method. The prepared nanohybrids, PtNPs-MWCNTs and PdNPs-MWCNTs, were cast on the surface of spectrographic graphite electrodes and then Phanerochaete chrysosporium cellobiose dehydrogenase (PcCDH) was adsorbed on the modified layer. Direct electron transfer between PcCDH and the nanostructured modified electrodes was studied using flow injection amperometry and cyclic voltammetry. The maximum current responses (Imax) and the apparent Michaelis-Menten constants (K) for the different PcCDH modified electrodes were calculated by fitting the data to the Michaelis-Menten equation and compared. The sensitivity towards lactose was 3.07 and 3.28 μA mM(-1) at the PcCDH/PtNPs-MWCNTs/SPGE and PcCDH/PdNPs-MWCNTs/SPGE electrodes, respectively, which were higher than those measured at the PcCDH/MWCNTs/SPGE (2.60 μA mM(-1)) and PcCDH/SPGE (0.92 μA mM(-1)). The modified electrodes were additionally tested as bioanodes for biofuel cell applications.
Brain glucose transport and phosphorylation under acute insulin-induced hypoglycemia in mice: an 18F-FDG PET study.

PubMed

Alf, Malte F; Duarte, João M N; Schibli, Roger; Gruetter, Rolf; Krämer, Stefanie D

2013-12-01

We addressed the questions of how cerebral glucose transport and phosphorylation change under acute hypoglycemia and what the underlying mechanisms of adaptation are. Quantitative (18)F-FDG PET combined with the acquisition of real-time arterial input function was performed on mice. Hypoglycemia was induced and maintained by insulin infusion. PET data were analyzed with the 2-tissue-compartment model for (18)F-FDG, and the results were evaluated with Michaelis-Menten saturation kinetics. Glucose clearance from plasma to brain (K1,glc) and the phosphorylation rate constant increased with decreasing plasma glucose (Gp), in particular at a Gp of less than 2.5 mmol/L. Estimated cerebral glucose extraction ratios taking into account an increased cerebral blood flow (CBF) at a Gp of less than 2 mmol/L were between 0.14 and 0.79. CBF-normalized K1,glc values were in agreement with saturation kinetics. Phosphorylation rate constants indicated intracellular glucose depletion at a Gp of less than 2-3 mmol/L. When brain regions were compared, glucose transport under hypoglycemia was lowest in the hypothalamus. Alterations in glucose transport and phosphorylation, as well as intracellular glucose depletion, under acute hypoglycemia can be modeled by saturation kinetics taking into account an increase in CBF. Distinct transport kinetics in the hypothalamus may be involved in its glucose-sensing function.
Kinetic characterization of oxyresveratrol as a tyrosinase substrate.

PubMed

Ortiz-Ruiz, Carmen Vanessa; Ballesta de Los Santos, Manuel; Berna, Jose; Fenoll, Jose; Garcia-Ruiz, Pedro Antonio; Tudela, Jose; Garcia-Canovas, Francisco

2015-11-01

Oxyresveratrol is a stilbenoid described as a powerful inhibitor of tyrosinase and proposed as skin-whitening and anti-browning agent. However, the enzyme is capable of acting on it, considering it as a substrate, as it has been proved in the case of its analogous resveratrol. Tyrosinase hydroxylates the oxyresveratrol to an o-diphenol and oxidizes the latter to an o-quinone, which finally isomerizes to p-quinone. For these reactions to take place the presence of the Eox (oxy-tyrosinase) form is necessary. The kinetic analysis of the proposed mechanism has allowed the kinetic characterization of this molecule as a substrate of tyrosinase, affording a catalytic constant of 5.39 ± 0.21 sec(-1) and a Michaelis constant of 8.65 ± 0.73 µM. © 2015 International Union of Biochemistry and Molecular Biology.
Direct, rapid, and label-free detection of enzyme-substrate interactions in physiological buffers using CMOS-compatible nanoribbon sensors.

PubMed

Mu, Luye; Droujinine, Ilia A; Rajan, Nitin K; Sawtelle, Sonya D; Reed, Mark A

2014-09-10

We demonstrate the versatility of Al2O3-passivated Si nanowire devices ("nanoribbons") in the analysis of enzyme-substrate interactions via the monitoring of pH change. Our approach is shown to be effective through the detection of urea in phosphate buffered saline (PBS), and penicillinase in PBS and urine, at limits of detection of <200 μM and 0.02 units/mL, respectively. The ability to extract accurate enzyme kinetics and the Michaelis-Menten constant (Km) from the acetylcholine-acetylcholinesterase reaction is also demonstrated.
Effect of linoleic-acid modified carboxymethyl chitosan on bromelain immobilization onto self-assembled nanoparticles

NASA Astrophysics Data System (ADS)

Tan, Yu-long; Liu, Chen-guang; Yu, Le-jun; Chen, Xi-guang

2008-06-01

Hydrogel nanoparticles could be prepared by using linoleic acid (LA) modified carboxymethyl chitosan (CMCS) after sonication. Bromelain could be loaded onto nanoparticles of LA-CMCS. Factors affecting the activity of the immobilized enzyme, including temperature, storage etc., were investigated in this study. The results showed that the stability of bromelain for heat and storage was improved after immobilization on nanoparticles. The Michaelis constant ( K m) of the immobilized enzyme was smaller than that of free enzyme, indicating that the immobilization could promote the stability of the enzyme and strengthen the affinity of the enzyme for the substrate.
Localized 1H NMR measurement of glucose consumption in the human brain during visual stimulation.

PubMed Central

Chen, W; Novotny, E J; Zhu, X H; Rothman, D L; Shulman, R G

1993-01-01

Spatially localized 1H NMR spectroscopy has been applied to measure changes in brain glucose concentration during 8-Hz photic stimulation. NMR spectroscopic measurements were made in a 12-cm3 volume centered on the calcarine fissure and encompassing the primary visual cortex. The average maximum change in glucose levels was 0.34 mumol.g-1 (n = 5) at 15 min; glucose level had turned toward resting level at 25 min. The glucose change was used to calculate the increase of glucose cerebral metabolic rate in the visual cortex region for individual subjects by using the Michaelis-Menten model of glucose transport on the assumption of constant transport kinetics. The glucose cerebral metabolic rate was calculated to increase over the nonstimulated rate by 22% during the first 15 min of photic stimulation. A model in which the glucose metabolic rate gradually decreases during stimulation was proposed as a possible explanation for the recovery of brain glucose and previously measured lactate concentrations to prestimulus values after 15 min. Images Fig. 1 PMID:8234332
Improvement of L(+)-Lactic Acid Production of Rhizopus Oryzae by Low-Energy Ions and Analysis of Its Mechanism

NASA Astrophysics Data System (ADS)

Ge, Chunmei; Yang, Yingge; Fan, Yonghong; Li, Wen; Pan, Renrui; Zheng, Zhiming; Yu, Zengliang

2008-02-01

The wild type strain Rhizopus oryzae PW352 was mutated by means of nitrogen ion implantation (15 keV, 7.8 × 1014 ~ 2.08 × 1015 ions/cm2) to find an industrial strain with a higher L(+)-lactic acid yield, and two mutants RE3303 and RF9052 were isolated. In order to discuss the mechanism primarily, Lactate Dehydrogenase of Rhizopus oryzae was studied. While the two mutants produced L(+)-lactic acid by 75% more than the wild strain did, their specific activity of Lactate Dehydrogenase was found to be higher than that in the wild strain. The optimum temperature of Lactate Dehydrogenase in Rhizopus oryzae RF9052 was higher. Compared to the wild strain, the Michaelis constant (Km) value of Lactate Dehydrogenase in the mutants was changed. All these changes show that L(+)-lactic acid production has a correlation with the specific activity of Lactate Dehydrogenase. The low-energy ions, implanted into the strain, may improve the specific activity of Lactate Dehydrogenase by influencing its gene structure and protein structure.
Enzymatic Transition States, Transition-State Analogs, Dynamics, Thermodynamics, and Lifetimes

PubMed Central

Schramm, Vern L.

2017-01-01

Experimental analysis of enzymatic transition-state structures uses kinetic isotope effects (KIEs) to report on bonding and geometry differences between reactants and the transition state. Computational correlation of experimental values with chemical models permits three-dimensional geometric and electrostatic assignment of transition states formed at enzymatic catalytic sites. The combination of experimental and computational access to transition-state information permits (a) the design of transition-state analogs as powerful enzymatic inhibitors, (b) exploration of protein features linked to transition-state structure, (c) analysis of ensemble atomic motions involved in achieving the transition state, (d) transition-state lifetimes, and (e) separation of ground-state (Michaelis complexes) from transition-state effects. Transition-state analogs with picomolar dissociation constants have been achieved for several enzymatic targets. Transition states of closely related isozymes indicate that the protein’s dynamic architecture is linked to transition-state structure. Fast dynamic motions in catalytic sites are linked to transition-state generation. Enzymatic transition states have lifetimes of femtoseconds, the lifetime of bond vibrations. Binding isotope effects (BIEs) reveal relative reactant and transition-state analog binding distortion for comparison with actual transition states. PMID:21675920
Selective synthesis of human milk fat-style structured triglycerides from microalgal oil in a microfluidic reactor packed with immobilized lipase

DOE PAGES

Wang, Jun; Liu, Xi; Wang, Xu -Dong; ...

2016-08-18

Human milk fat-style structured triacylglycerols were produced from microalgal oil in a continuous microfluidic reactor packed with immobilized lipase for the first time. A remarkably high conversion efficiency was demonstrated in the microreactor with reaction time being reduced by 8 times, Michaelis constant decreased 10 times, the lipase reuse times increased 2.25-fold compared to those in a batch reactor. In addition, the content of palmitic acid at sn-2 position (89.0%) and polyunsaturated fatty acids at sn-1, 3 positions (81.3%) are slightly improved compared to the product in a batch reactor. The increase of melting points (1.7 °C) and decrease ofmore » crystallizing point (3 °C) implied higher quality product was produced using the microfluidic technology. The main cost can be reduced from 212.3 to 14.6 per batch with the microreactor. Altogether, the microfluidic bioconversion technology is promising for modified functional lipids production allowing for cost-effective approach to produce high-value microalgal coproducts.« less
Selective synthesis of human milk fat-style structured triglycerides from microalgal oil in a microfluidic reactor packed with immobilized lipase

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

Wang, Jun; Liu, Xi; Wang, Xu -Dong

Human milk fat-style structured triacylglycerols were produced from microalgal oil in a continuous microfluidic reactor packed with immobilized lipase for the first time. A remarkably high conversion efficiency was demonstrated in the microreactor with reaction time being reduced by 8 times, Michaelis constant decreased 10 times, the lipase reuse times increased 2.25-fold compared to those in a batch reactor. In addition, the content of palmitic acid at sn-2 position (89.0%) and polyunsaturated fatty acids at sn-1, 3 positions (81.3%) are slightly improved compared to the product in a batch reactor. The increase of melting points (1.7 °C) and decrease ofmore » crystallizing point (3 °C) implied higher quality product was produced using the microfluidic technology. The main cost can be reduced from 212.3 to 14.6 per batch with the microreactor. Altogether, the microfluidic bioconversion technology is promising for modified functional lipids production allowing for cost-effective approach to produce high-value microalgal coproducts.« less
Kinetics study of invertase covalently linked to a new functional nanogel.

PubMed

Raj, Lok; Chauhan, Ghanshyam S; Azmi, Wamik; Ahn, J-H; Manuel, James

2011-02-01

Nanogels are promising materials as supports for enzyme immobilization. A new hydrogel comprising of methacrylic acid (MAAc) and N-vinyl pyrrolidone (N-VP) and ethyleneglycol dimethacrylate (EGDMA) was synthesized and converted to nanogel by an emulsification method. Nanogel was further functionalized by Curtius azide reaction for use as support for the covalent immobilization of invertase (Saccharomyces cerevisiae). As-prepared or invertase-immobilized nanogel was characterized by FTIR, XRD, TEM and nitrogen analysis. The characterization of both free and the immobilized-invertase were performed using a spectrophotometric method at 540 nm. The values of V(max), maximum reaction rate, (0.123 unit/mg), k(m), Michaelis constant (7.429 mol/L) and E(a), energy of activation (3.511 kj/mol) for the immobilized-invertase are comparable with those of the free invertase at optimum conditions (time 70 min, pH 6.0 and temperature 45°C). The covalent immobilization enhanced the pH and thermal stability of invertase. The immobilized biocatalyst was efficiently reused up to eight cycles. Copyright © 2010 Elsevier Ltd. All rights reserved.
Selective synthesis of human milk fat-style structured triglycerides from microalgal oil in a microfluidic reactor packed with immobilized lipase.

PubMed

Wang, Jun; Liu, Xi; Wang, Xu-Dong; Dong, Tao; Zhao, Xing-Yu; Zhu, Dan; Mei, Yi-Yuan; Wu, Guo-Hua

2016-11-01

Human milk fat-style structured triacylglycerols were produced from microalgal oil in a continuous microfluidic reactor packed with immobilized lipase for the first time. A remarkably high conversion efficiency was demonstrated in the microreactor with reaction time being reduced by 8 times, Michaelis constant decreased 10 times, the lipase reuse times increased 2.25-fold compared to those in a batch reactor. In addition, the content of palmitic acid at sn-2 position (89.0%) and polyunsaturated fatty acids at sn-1, 3 positions (81.3%) are slightly improved compared to the product in a batch reactor. The increase of melting points (1.7°C) and decrease of crystallizing point (3°C) implied higher quality product was produced using the microfluidic technology. The main cost can be reduced from $212.3 to $14.6 per batch with the microreactor. Overall, the microfluidic bioconversion technology is promising for modified functional lipids production allowing for cost-effective approach to produce high-value microalgal coproducts. Copyright © 2016 Elsevier Ltd. All rights reserved.
Biochemical characterization of Aspergillus oryzae recombinant α-l-rhamnosidase expressed in Pichia pastoris.

PubMed

Ishikawa, Mai; Shiono, Yoshihito; Koseki, Takuya

2017-12-01

An α-l-rhamnosidase-encoding gene from Aspergillus oryzae, which belongs to the glycoside hydrolase family 78, was cloned and expressed in Pichia pastoris. SDS-PAGE of the purified recombinant α-l-rhamnosidase protein revealed smeared bands with apparent molecular mass of 90-130 kDa. After N-deglycosylation, the recombinant enzyme showed a molecular mass of 70 kDa. The enzyme exhibited optimal activity at a pH of 5.0 and a temperature of 70 °C. Specific activity of the enzyme was higher toward hesperidin than toward naringin, which consist of α-1,6 and α-1,2 linkages, respectively. The activity was also higher toward hesperidin than toward rutin, which consist of 7-O- and 3-O-glycosyl linkages of flavonoids, respectively. Kinetic analysis of the enzyme showed that the Michaelis constant (K m ) was lowest toward rutin, moderate toward naringin, and higher toward p-nitrophenyl-α-l-rhamnopyranoside and hesperidin. Its high catalytic efficiency (k cat /K m ) toward rutin was results of its low K m value while its high catalytic efficiency toward hesperidin was results of a considerably high k cat value. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.
Lipase-nanoporous gold biocomposite modified electrode for reliable detection of triglycerides.

PubMed

Wu, Chao; Liu, Xueying; Li, Yufei; Du, Xiaoyu; Wang, Xia; Xu, Ping

2014-03-15

For triglycerides biosensor design, protein immobilization is necessary to create the interface between the enzyme and the electrode. In this study, a glassy carbon electrode (GCE) was modified with lipase-nanoporous gold (NPG) biocomposite (denoted as lipase/NPG/GCE). Due to highly conductive, porous, and biocompatible three-dimensional structure, NPG is suitable for enzyme immobilization. In cyclic voltammetry experiments, the lipase/NPG/GCE bioelectrode displayed surface-confined reaction in a phosphate buffer solution. Linear responses were obtained for tributyrin concentrations ranging from 50 to 250 mg dl(-1) and olive oil concentrations ranging from 10 to 200 mg dl(-1). The value of apparent Michaelis-Menten constant for tributyrin was 10.67 mg dl(-1) and the detection limit was 2.68 mg dl(-1). Further, the lipase/NPG/GCE bioelectrode had strong anti-interference ability against urea, glucose, cholesterol, and uric acid as well as a long shelf-life. For the detection of triglycerides in human serum, the values given by the lipase/NPG/GCE bioelectrode were in good agreement with those of an automatic biochemical analyzer. These properties along with a long self-life make the lipase/NPG/GCE bioelectrode an excellent choice for the construction of triglycerides biosensor. © 2013 Elsevier B.V. All rights reserved.
The influence of cyclomaltooligosaccharides (cyclodextrins) on the enzymatic decomposition of l-phenylalanine catalyzed by phenylalanine ammonia-lyase.

PubMed

Gubica, Tomasz; Pełka, Agnieszka; Pałka, Katarzyna; Temeriusz, Andrzej; Kańska, Marianna

2011-09-27

Cyclomaltohexaose (α-cyclodextrin) and cyclomaltoheptaose (β-cyclodextrin) as well as their four methyl ether derivatives, that is, hexakis(2,3-di-O-methyl)cyclomaltohexaose, hexakis(2,3,6-tri-O-methyl)cyclomaltohexaose, heptakis(2,3-di-O-methyl)cyclomaltoheptaose, and heptakis(2,3,6-tri-O-methyl)cyclomaltoheptaose were investigated as the additives in the course of enzymatic decomposition of l-phenylalanine catalyzed by phenylalanine ammonia-lyase. Only a few of those additives behaved like classical inhibitors of the enzymatic reaction under investigation because the values of the Michaelis constants that were obtained, as well as the maximum velocity values depended mostly atypically on the concentrations of those additives. In most cases cyclodextrins caused mixed inhibition, both competitive and noncompetitive, but they also acted as activators for selected concentrations. This atypical behaviour of cyclodextrins is caused by three different and independent effects. The inhibitory effect of cyclodextrins is connected with the decrease of substrate concentration and unfavourable influence on the flexibility of the enzyme molecules. On the other hand, the activating effect is connected with the decrease of product concentration (the product is an inhibitor of the enzymatic reaction under investigation). All these effects are caused by the ability of the cyclodextrins to form inclusion complexes. Copyright © 2011 Elsevier Ltd. All rights reserved.
Functional involvement of the organic cation transporter 2 (rOct2) in the renal uptake of organic cations in rats.

PubMed

Umehara, K-I; Iwatsubo, T; Noguchi, K; Kamimura, H

2008-01-01

This study examined the contribution made by organic cation transporters (hOCT/rOct) to the saturable component of the renal uptake of 1-methyl-4-phenylpyridinium, tetraethylammonium (TEA), cimetidine and metformin into rOct2-expressing HEK293 cells and rat kidney slices. All the test compounds accumulated in the rat kidney slices in a carrier-mediated manner. The Michaelis- Menten constant (K(m)) values for saturable uptake of TEA, cimetidine and metformin into rat kidney slices were relatively comparable with those for the rOct2-expressing HEK293 cells. In addition, the relative uptake activity values of TEA, cimetidine and metformin in rat kidney slices were similar to those in rOct2-expressing HEK293 cells. This suggests that the saturable components involved in the renal uptake of TEA, cimetidine and metformin are mediated mainly by rOct2. The saturable uptake profile of cationic compounds into rat kidney can be evaluated in both cDNA-expressing cells and rat kidney slices, as well as the transporter expression pattern. This approach can also be used to estimate the saturable uptake mechanism of cationic compounds into the human kidney when human kidney slices and hOCT2-expressing cells are used.

Maize Root Phytase (Purification, Characterization, and Localization of Enzyme Activity and Its Putative Substrate).

PubMed Central

Hubel, F.; Beck, E.

1996-01-01

Three phytase (EC 3.1.3.26) isoforms from the roots of 8-d-old maize (Zea mays L. var Consul) seedlings were separated from phosphatases and purified to near homogeneity. The molecular mass of the native protein was 71 kD, and the isoelectric points of the three isoforms were pH 5.0, 4.9, and 4.8. Each of the three isoforms consisted of two subunits with a molecular mass of 38 kD. The temperature and pH optima (40[deg]C, pH 5.0) of these three isoforms, as well as the apparent Michaelis constants for sodium inositol hexakisphosphate (phytate) (43, 25, and 24 [mu]M) as determined by the release of inorganic phosphate, were only slightly different. Phytate concentrations higher than 300 [mu]M were inhibitory to all three isoforms. In contrast, the dephosphorylation of 4-nitrophenyl phosphate was not inhibited by any substrate concentration, but the Michaelis constants for this substrate were considerably higher (137-157 [mu]M). Hydrolysis of phytate by the phytase isoforms is a nonrandom reaction. D/L-Inositol-1,2,3,4,5- pentakisphosphate was identified as the first and D/L-inositol-1,2,5,6-tetrakisphosphate as the second intermediate in phytate hydrolysis. Phytase activity was localized in root slices. Although phosphatase activity was present in the stele and the cortex of the primary root, phytase activity was confined to the endodermis. Phytate was identified as the putative native substrate in maize roots (45 [mu]g P g-1 dry matter). It was readily labeled upon supplying [32P]phosphate to the roots. PMID:12226456
Confocal analysis of hepatocellular long-chain fatty acid uptake.

PubMed

Elsing, C; Winn-Börner, U; Stremmel, W

1995-12-01

Transmembrane transport and cytosolic accumulation of fatty acids were investigated using confocal laser scanning microscopy (cLSM). A Zeiss LSM 310 system was used to determine the uptake of the fluorescent fatty acid derivative 12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3- diazol-4-yl)amino]octadecanoic acid (12-NBD stearate) (C18) in single rat hepatocytes. Uptake was a saturable process with a Michaelis-Menten constant value of 68 nM. Initial uptake velocity was dependent on extracellular presence of albumin and beta-lactoglobulin. Absence of albumin reduced uptake to 32 +/- 16% (P < 0.01) of control values. In the presence of unlabeled stearate, uptake of 12-NBD stearate was lowered to 49 +/- 12% (P < 0.01). Ion substitution experiments showed no sodium dependency of uptake. Increase in membrane potential led to a pronounced accumulation of the fatty acid derivative within the plasma membrane and in the adjacent cytoplasmic compartment, whereas membrane depolarization had no effect on uptake rates. In separate experiments line scans through representative hepatocytes were analyzed to generate "x-t" plots. 12-NBD stearate showed a fluorescence pattern with prominent staining of the area of the plasma membrane and the adjacent cytoplasm, dependent on the presence of extracellular albumin. For the hepatocellular cytosolic accumulation process of 12-NBD stearate a diffusion constant of 22.2 +/- 6.2 x 10(-9) cm2/s was calculated. In contrast to the long-chain fatty acid derivative 12-NBD stearate, short (C5)- and medium (C11)-chain fatty acids revealed no membrane interaction with hepatocytes. Erythrocytes also lacked a membrane interaction process for 12-NBD stearate. In conclusion, it was demonstrated that cLSM is capable of directly evaluating the cellular fatty acid uptake process at a subcellular level.
A mathematical model of liver metabolism: from steady state to dynamic

NASA Astrophysics Data System (ADS)

Calvetti, D.; Kuceyeski, A.; Somersalo, E.

2008-07-01

The increase in Type 2 diabetes and other metabolic disorders has led to an intense focus on the areas of research related to metabolism. Because the liver is essential in regulating metabolite concentrations that maintain life, it is especially important to have good knowledge of the functions within this organ. In silico mathematical models that can adequately describe metabolite concentrations, flux and transport rates in the liver in vivo can be a useful predictive tool. Fully dynamic models, which contain expressions for Michaelis-Menten reaction kinetics can be utilized to investigate different metabolic states, for example exercise, fed or starved state. In this paper we describe a two compartment (blood and tissue) spatially lumped liver metabolism model. First, we use Bayesian Flux Balance Analysis (BFBA) to estimate the values of flux and transport rates at steady state, which agree closely with values from the literature. These values are then used to find a set of Michaelis-Menten parameters and initial concentrations which identify a dynamic model that can be used for exploring different metabolic states. In particular, we investigate the effect of doubling the concentration of lactate entering the system via the hepatic artery and portal vein. This change in lactate concentration forces the system to a new steady state, where glucose production is increased.
Different enzyme kinetic models.

PubMed

Seibert, Eleanore; Tracy, Timothy S

2014-01-01

As described in Chapter 2 , a large number of enzymatic reactions can be adequately described by Michaelis-Menten kinetics. The Michaelis-Menten equation represents a rectangular hyperbola, with a y-asymptote at the V max value. In many cases, more complex kinetic models are required to explain the observed data. Atypical kinetic profiles are believed to arise from the simultaneous binding of multiple molecules within the active site of the enzyme (Tracy and Hummel, Drug Metab Rev 36:231-242, 2004). Several cytochromes P450 have large active sites that enable binding of multiple molecules (Wester et al. J Biol Chem 279:35630-35637, 2004; Yano et al. J Biol Chem 279:38091-38094, 2004). Thus, atypical kinetics are not uncommon in in vitro drug metabolism studies. This chapter covers enzyme kinetic reactions in which a single enzyme has multiple binding sites for substrates and/or inhibitors as well as reactions catalyzed by multiple enzymes.
The structure and mechanism of stem bromelain. Evaluation of the homogeneity of purified stem bromelain, determination of the molecular weight and kinetic analysis of the bromelain-catalysed hydrolysis of N-benzyloxycarbonyl-l-phenylalanyl-l-serine methyl ester

PubMed Central

Wharton, Christopher W.

1974-01-01

1. Purified stem bromelain (EC 3.4.22.4) was eluted from Sephadex G-100 as a single peak. The specific activity across the elution peak was approximately constant towards p-nitrophenyl hippurate but increased with elution volume with N2-benzoyl-l-arginine ethyl ester as substrate. 2. The apparent molecular weight, determined by elution analysis on Sephadex G-100, is 22500±1500, an anomalously low value. 3. Purified stem bromelain was eluted from CM-cellulose CM-32 as a single peak and behaved as a single species during column electrophoresis on Sephadex G-100. 4. Purified stem bromelain migrates as a single band during polyacrylamide-gel electrophoresis under a wide variety of conditions. 5. The molecular weight determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate is 28500±1000. 6. Sedimentation-velocity and equilibrium-ultracentrifugation experiments, under a variety of conditions, indicate that bromelain is an apparently homogeneous single peptide chain of mol.wt. 28400±1400. 7. The N-terminal amino acid composition is 0.64±0.04mol of valine and 0.36±0.04mol of alanine per mol of enzyme of mol.wt. 28500. (The amino acid recovery of the cyanate N-terminal amino acid analysis was standardized by inclusion of carbamoyl-norleucine at the cyclization stage.) 8. The pH-dependence of the Michaelis parameters of the bromelain-catalysed hydrolysis of N-benzyloxycarbonyl-l-phenylalanyl-l-serine methyl ester was determined. 9. The magnitude and pH-dependence of the Michaelis parameters have been interpreted in terms of the mechanism of the enzyme. 10. The enzyme is able to bind N-benzyloxycarbonyl-l-phenylalanyl-l-serine methyl ester relatively strongly but seems unable to make use of the binding energy to promote catalysis. PMID:4462742
Catalytic Activity of μ-Carbido-Dimeric Iron(IV) Octapropylporphyrazinate in the 3,5,7,2',4'-Pentahydroxyflavone Oxidation Reaction with tert-Butyl Hydroperoxide

NASA Astrophysics Data System (ADS)

Tyurin, D. V.; Zaitseva, S. V.; Kudrik, E. V.

2018-05-01

It is found for the first time that μ-carbido-dimeric iron(IV) octapropylporphyrazinate displays catalytic activity in the oxidation reaction of natural flavonol morin with tert-butyl hydroperoxide, with the catalyst being stable under conditions of the reaction. The kinetics of this reaction are studied. It is shown the reaction proceeds via tentative formation of a complex between the catalyst and the oxidant, followed by O‒O bond homolytic cleavage. The kinetics of the reaction is described in the coordinates of the Michaelis-Menten equation. A linear dependence of the apparent reaction rate constant on the concentration of the catalyst is observed, testifying to its participation in the limiting reaction step. The equilibrium constants and rates of interaction are found. A mechanism is proposed for the reaction on the basis of the experimental data.
Kinetics of ATP hydrolysis catalyzed by isolated TF1 and reconstituted TF0F1 ATPase.

PubMed

Rögner, M; Gräber, P

1986-09-01

The rate of ATP hydrolysis catalyzed by isolated TF1 and reconstituted TF0F1 was measured as a function of the ATP concentration in the presence of inhibitors [ADP, Pi and 3'-O-(1-naphthoyl)ATP]. ATP hydrolysis can be described by Michaelis-Menten kinetics with Km(TF1) = 390 microM and Km (TF0F1) = 180 microM. The inhibition constants are for ADP Ki(TF1) = 20 microM and Ki(TF0F1) = 100 microM, for 3'-O-(1-naphthoyl)ATP Ki(TF1) = 150 microM and Ki(TF0F1) = 3 microM, and for Pi Ki(TF1) = 60 mM. From these results it is concluded that upon binding of TF0 to TF1 the mechanism of ATP hydrolysis catalyzed by TF1 is not changed qualitatively; however, the kinetic constants differ quantitatively.
Papain hydrolysis of X-phenyl-N-methanesulfonyl glycinates: a quantitative structure-activity relationship and molecular graphics analysis.

PubMed

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

1984-02-15

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

PubMed

Wang, Ziquan; Tan, Xiangping; Lu, Guannan; Liu, Yanju; Naidu, Ravi; He, Wenxiang

2018-01-01

Soil phosphatase, which plays an important role in phosphorus cycling, is strongly inhibited by Arsenic (As). However, the inhibition mechanism in kinetics is not adequately investigated. In this study, we investigated the kinetic characteristics of soil acid phosphatase (ACP) in 14 soils with varied properties, and also explored how kinetic properties of soil ACP changed with different spiked As concentrations. The results showed that the Michaelis constant (K m ) and maximum reaction velocity (V max ) values of soil ACP ranged from 1.18 to 3.77mM and 0.025-0.133mMh -1 in uncontaminated soils. The kinetic parameters of soil ACP in different soils changed differently with As contamination. The K m remained unchanged and V max decreased with increase of As concentration in most acid and neutral soils, indicating a noncompetitive inhibition mechanism. However, in alkaline soils, the K m increased linearly and V max decreased with increase of As concentration, indicating a mixed inhibition mechanism that include competitive and noncompetitive. The competitive inhibition constant (K ic ) and noncompetitive inhibition constant (K iu ) varied among soils and ranged from 0.38 to 3.65mM and 0.84-7.43mM respectively. The inhibitory effect of As on soil ACP was mostly affected by soil organic matter and cation exchange capacity. Those factors influenced the combination of As with enzyme, which resulted in a difference of As toxicity to soil ACP. Catalytic efficiency (V max /K m ) of soil ACP was a sensitive kinetic parameter to assess the ecological risks of soil As contamination. Copyright © 2017 Elsevier Inc. All rights reserved.
Kinetics of bacterial phospholipase C activity at micellar interfaces: effect of substrate aggregate microstructure and a model for the kinetic parameters.

PubMed

Singh, Jasmeet; Ranganathan, Radha; Hajdu, Joseph

2008-12-25

Activity at micellar interfaces of bacterial phospholipase C from Bacillus cereus on phospholipids solubilized in micelles was investigated with the goal of elucidating the role of the interface microstructure and developing further an existing kinetic model. Enzyme kinetics and physicochemical characterization of model substrate aggregates were combined, thus enabling the interpretation of kinetics in the context of the interface. Substrates were diacylphosphatidylcholine of different acyl chain lengths in the form of mixed micelles with dodecyldimethylammoniopropanesulfonate. An early kinetic model, reformulated to reflect the interfacial nature of the kinetics, was applied to the kinetic data. A better method of data treatment is proposed, use of which makes the presence of microstructure effects quite transparent. Models for enzyme-micelle binding and enzyme-lipid binding are developed, and expressions incorporating the microstructural properties are derived for the enzyme-micelle dissociation constant K(s) and the interface Michaelis-Menten constant, K(M). Use of these expressions in the interface kinetic model brings excellent agreement between the kinetic data and the model. Numerical values for the thermodynamic and kinetic parameters are determined. Enzyme-lipid binding is found to be an activated process with an acyl chain length dependent free energy of activation that decreases with micelle lipid molar fraction with a coefficient of about -15RT and correlates with the tightness of molecular packing in the substrate aggregate. Thus, the physical insight obtained includes a model for the kinetic parameters that shows that these parameters depend on the substrate concentration and acyl chain length of the lipid. Enzyme-micelle binding is indicated to be hydrophobic and solvent mediated with a dissociation constant of 1.2 mM.
Free energy surface of the Michaelis complex of lactate dehydrogenase: a network analysis of microsecond simulations.

PubMed

Pan, Xiaoliang; Schwartz, Steven D

2015-04-30

It has long been recognized that the structure of a protein creates a hierarchy of conformations interconverting on multiple time scales. The conformational heterogeneity of the Michaelis complex is of particular interest in the context of enzymatic catalysis in which the reactant is usually represented by a single conformation of the enzyme/substrate complex. Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate with concomitant interconversion of two forms of the cofactor nicotinamide adenine dinucleotide (NADH and NAD(+)). Recent experimental results suggest that multiple substates exist within the Michaelis complex of LDH, and they show a strong variance in their propensity toward the on-enzyme chemical step. In this study, microsecond-scale all-atom molecular dynamics simulations were performed on LDH to explore the free energy landscape of the Michaelis complex, and network analysis was used to characterize the distribution of the conformations. Our results provide a detailed view of the kinetic network of the Michaelis complex and the structures of the substates at atomistic scales. They also shed light on the complete picture of the catalytic mechanism of LDH.
A novel H(2)O(2) amperometric biosensor based on gold nanoparticles/self-doped polyaniline nanofibers.

PubMed

Chen, Xiaojun; Chen, Zixuan; Zhu, Jinwei; Xu, Chenbin; Yan, Wei; Yao, Cheng

2011-10-01

A new kind of gold nanoparticles/self-doped polyaniline nanofibers (Au/SPAN) with grooves has been prepared for the immobilization of horseradish peroxidase (HRP) on the surface of glassy carbon electrode (GCE). The ratio of gold in the composite nanofibers was up to 64%, which could promote the conductivity and biocompatibility of SPAN and increase the immobilized amount of HRP molecules greatly. The electrode exhibits enhanced electrocatalytic activity in the reduction of H(2)O(2) in the presence of the mediator hydroquinone (HQ). The effects of concentration of HQ, solution pH and the working potential on the current response of the modified electrode toward H(2)O(2) were optimized to obtain the maximal sensitivity. The proposed biosensor exhibited a good linear response in the range from 10 to 2000 μM with a detection limit of 1.6 μM (S/N=3) under the optimum conditions. The response showed Michaelis-Menten behavior at larger H(2)O(2) concentrations, and the apparent Michaelis-Menten constant K(m) was estimated to be 2.21 mM. The detection of H(2)O(2) concentration in real sample showed acceptable accuracy with the traditional potassium permanganate titration. Copyright © 2011 Elsevier B.V. All rights reserved.
Adsorption of β-galactosidase on silica and aluminosilicate adsorbents

NASA Astrophysics Data System (ADS)

Atyaksheva, L. F.; Dobryakova, I. V.; Pilipenko, O. S.

2015-03-01

It is shown that adsorption of β-galactosidase of Aspergillus oryzae fungi on mesoporous and biporous silica and aluminosilicate adsorbents and the rate of the process grow along with the diameter of the pores of the adsorbent. It is found that the shape of the adsorption isotherms changes as well, depending on the texture of the adsorbent: the Michaelis constant rises from 0.3 mM for the enzyme in solution to 0.4-0.5 mM for the enzyme on a surface in the hydrolysis of o-nitrophenyl-β-D-galactopyranoside. It is concluded that β-galactosidase displays its maximum activity on the surface of biporous adsorbents.
Covalent immobilization of invertase on PAMAM-dendrimer modified superparamagnetic iron oxide nanoparticles

NASA Astrophysics Data System (ADS)

Uzun, K.; Çevik, E.; Şenel, M.; Sözeri, H.; Baykal, A.; Abasıyanık, M. F.; Toprak, M. S.

2010-10-01

In this study, polyamidoamine (PAMAM) dendrimer was synthesized on the surface of superparamagnetite nanoparticles to enhance invertase immobilization. The amount of immobilized enzyme on the surface-hyperbranched magnetite nanoparticle was up to 2.5 times (i.e., 250%) as much as that of magnetite nanoparticle modified with only amino silane. Maximum reaction rate ( V max) and Michaelis-Menten constant ( K m) were determined for the free and immobilized enzymes. Various characteristics of immobilized invertase such as; the temperature activity, thermal stability, operational stability, and storage stability were evaluated and results revealed that stability of the enzyme is improved upon immobilization.
Properties of the glycoprotein laccase immobilized by two methods.

PubMed

Froehner, S C; Eriksson, K

1975-01-01

Laccase (p-diphenol:oxygen oxidoreductase; EC 1.10.3.2) from Neurospora crassa has been immobilized by two different procedures: (1) Covalent attachment to Sepharose 4B activated with cyanogen bromide, and (2) Adsorption to Concanavalin A-Sepharose via the carbohydrate moiety. Except for small changes in the Michaelis-Menten constants, no differences were noted in the enzymological properties of the immobilized enzymes when compared to free enzyme. The carbohydrate moiety of laccase involved in the interaction with Concanavalin A does not appear to be closely associated with the active center since binding to the lectin has no effect on the enzymological parameters investigated.
Monitoring the enzymatic conversion of urea to ammonium by conventional or microchip capillary electrophoresis with contactless conductivity detection.

PubMed

Schuchert-Shi, Aiping; Hauser, Peter C

2008-05-15

Capillary electrophoresis with contactless conductivity detection was used to directly quantify the ammonium produced in the enzymatic conversion of urea with urease. This allowed the characterization of the reaction without having to use more elaborate indirect optical methods for quantification. The maximum rate of reaction, V(max), was determined as 5.1 mmol x mL(-1) x min(-1), and the Michaelis-Menten constant, K(m), was determined as 16 mM. Furthermore, the method was successfully applied to the determination of urea in clinical samples of human blood by using a conventional capillary and a microchip device.
Kinetic spectrophotometric method for trace determination of thiocyanate based on its inhibitory effect

NASA Astrophysics Data System (ADS)

Naik, Radhey M.; Kumar, Basant; Asthana, Abhas

2010-03-01

A kinetic spectrophotometric method for the determination of thiocyanate, based on its inhibitory effect on silver(I) catalyzed substitution of cyanide ion, by phenylhydrazine in hexacyanoferrate(II) is described. Thiocyanate ions form strong complexes with silver(I) catalyst which is used as the basis for its determination at trace level. The progress of reaction was monitored, spectrophotometrically, at 488 nm ( λmax of [Fe(CN) 5PhNHNH 2] 3-, complex) under the optimum reaction conditions at: 2.5 × 10 -3 M [Fe(CN) 6] 4-, 1.0 × 10 -3 M [PhNHNH 2], 8.0 × 10 -7 M [Ag +], pH 2.8 ± 0.02, ionic strength ( μ) 0.02 M (KNO 3) and temperature 30 ± 0.1 °C. A linear relationship obtained between absorbance (measured at 488 nm at different times) and inhibitor concentration, under specified conditions, has been used for the determination of [thiocyanate] in the range of 0.8-8.0 × 10 -8 M with a detection limit of 2 × 10 -9 M. The standard deviation and percentage error have been calculated and reported with each datum. A most plausible mechanistic scheme has been proposed for the reaction. The values of equilibrium constants for complex formation between catalyst-inhibitor ( KCI), catalyst-substrate ( Ks) and Michaelis-Menten constant ( Km) have been computed from the kinetic data. The influence of possible interference by major cations and anions on the determination of thiocyanate and their limits has been investigated.
Expression, purification and enzymatic characterization of Brugia malayi dihydrofolate reductase.

PubMed

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

2016-12-01

Brugia malayi (B. malayi) is one of the three causative agents of lymphatic filariasis, a neglected parasitic disease. Current literature suggests that dihydrofolate reductase is a potential drug target for the elimination of B. malayi. Here we report the recombinant expression and purification of a ∼20 kDa B. malayi dihydrofolate reductase (BmDHFR). A His6-tagged construct was expressed in E. coli and purified by affinity chromatography to yield active and homogeneous enzyme for steady-state kinetic characterization and inhibition studies. The catalytic activity kcat was found to be 1.4 ± 0.1 s(-1), the Michaelis Menten constant KM for dihydrofolate 14.7 ± 3.6 μM, and the equilibrium dissociation constant KD for NADPH 25 ± 24 nM. For BmDHFR, IC50 values for a six DHFR inhibitors were determined to be 3.1 ± 0.2 nM for methotrexate, 32 ± 22 μM for trimethoprim, 109 ± 34 μM for pyrimethamine, 154 ± 46 μM for 2,4-diaminoquinazoline, 771 ± 44 μM for cycloguanil, and >20,000 μM for 2,4-diaminopyrimidine. Our findings suggest that antifolate compounds can serve as inhibitors of BmDHFR. Copyright © 2016 Elsevier Inc. All rights reserved.
H2O2 sensing using HRP modified catalyst-free ZnO nanorods synthesized by RF sputtering

NASA Astrophysics Data System (ADS)

Srivastava, Amit; Kumar, Naresh; Singh, Priti; Singh, Sunil Kumar

2017-06-01

Catalyst-free ( 00 l) oriented ZnO nanorods (NRs) -based biosensor for the H2O2 sensing has been reported. The (002) oriented ZnO NRs as confirmed by X-ray diffraction were successfully grown on indium tin oxide (ITO) coated glass substrate by radio frequency (RF) sputtering technique without using any catalyst. Horseradish peroxidase (HRP) enzyme was immobilized on ZnO NRs by physical adsorption technique to prepare the biosensor. In this HRP/ZnO NR/ITO bioelectrode, nafion solution was added to form a tight membrane on surface. The prepared bioelectrode has been used for biosensing measurements by electrochemical analyzer. The electrochemical studies reveal that the prepared HRP/ZnO NR/ITO biosensor is highly sensitive to the detection of H2O2 over a linear range of 0.250-10 μM. The ZnO NR-based biosensor showed lower value of detection limit (0.125 μM) and higher sensitivity (13.40 µA/µM cm2) towards H2O2. The observed value of higher sensitivity attributed to larger surface area of ZnO nanostructure for effective loading of HRP besides its high electron communication capability. In addition, the biosensor also shows lower value of enzyme's kinetic parameter (Michaelis-Menten constant, K m) of 0.262 μM which indicates enhanced enzyme affinity of HRP to H2O2. The reported biosensor may be useful for various applications in biosensing, clinical, food, and beverage industry.
The Elementary Mass Action Rate Constants of P-gp Transport for a Confluent Monolayer of MDCKII-hMDR1 Cells

PubMed Central

Tran, Thuy Thanh; Mittal, Aditya; Aldinger, Tanya; Polli, Joseph W.; Ayrton, Andrew; Ellens, Harma; Bentz, Joe

2005-01-01

The human multi-drug resistance membrane transporter, P-glycoprotein, or P-gp, has been extensively studied due to its importance to human health and disease. Thus far, the kinetic analysis of P-gp transport has been limited to steady-state Michaelis-Menten approaches or to compartmental models, neither of which can prove molecular mechanisms. Determination of the elementary kinetic rate constants of transport will be essential to understanding how P-gp works. The experimental system we use is a confluent monolayer of MDCKII-hMDR1 cells that overexpress P-gp. It is a physiologically relevant model system, and transport is measured without biochemical manipulations of P-gp. The Michaelis-Menten mass action reaction is used to model P-gp transport. Without imposing the steady-state assumptions, this reaction depends upon several parameters that must be simultaneously fitted. An exhaustive fitting of transport data to find all possible parameter vectors that best fit the data was accomplished with a reasonable computation time using a hierarchical algorithm. For three P-gp substrates (amprenavir, loperamide, and quinidine), we have successfully fitted the elementary rate constants, i.e., drug association to P-gp from the apical membrane inner monolayer, drug dissociation back into the apical membrane inner monolayer, and drug efflux from P-gp into the apical chamber, as well as the density of efflux active P-gp. All three drugs had overlapping ranges for the efflux active P-gp, which was a benchmark for the validity of the fitting process. One novel finding was that the association to P-gp appears to be rate-limited solely by drug lateral diffusion within the inner monolayer of the plasma membrane for all three drugs. This would be expected if P-gp structure were open to the lipids of the apical membrane inner monolayer, as has been suggested by recent structural studies. The fitted kinetic parameters show how P-gp efflux of a wide range of xenobiotics has been maximized. PMID:15501934

Sequential capillary electrophoresis analysis using optically gated sample injection and UV/vis detection.

PubMed

Liu, Xiaoxia; Tian, Miaomiao; Camara, Mohamed Amara; Guo, Liping; Yang, Li

2015-10-01

We present sequential CE analysis of amino acids and L-asparaginase-catalyzed enzyme reaction, by combing the on-line derivatization, optically gated (OG) injection and commercial-available UV-Vis detection. Various experimental conditions for sequential OG-UV/vis CE analysis were investigated and optimized by analyzing a standard mixture of amino acids. High reproducibility of the sequential CE analysis was demonstrated with RSD values (n = 20) of 2.23, 2.57, and 0.70% for peak heights, peak areas, and migration times, respectively, and the LOD of 5.0 μM (for asparagine) and 2.0 μM (for aspartic acid) were obtained. With the application of the OG-UV/vis CE analysis, sequential online CE enzyme assay of L-asparaginase-catalyzed enzyme reaction was carried out by automatically and continuously monitoring the substrate consumption and the product formation every 12 s from the beginning to the end of the reaction. The Michaelis constants for the reaction were obtained and were found to be in good agreement with the results of traditional off-line enzyme assays. The study demonstrated the feasibility and reliability of integrating the OG injection with UV/vis detection for sequential online CE analysis, which could be of potential value for online monitoring various chemical reaction and bioprocesses. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
The key nickel enzyme of methanogenesis catalyses the anaerobic oxidation of methane.

PubMed

Scheller, Silvan; Goenrich, Meike; Boecher, Reinhard; Thauer, Rudolf K; Jaun, Bernhard

2010-06-03

Large amounts (estimates range from 70 Tg per year to 300 Tg per year) of the potent greenhouse gas methane are oxidized to carbon dioxide in marine sediments by communities of methanotrophic archaea and sulphate-reducing bacteria, and thus are prevented from escaping into the atmosphere. Indirect evidence indicates that the anaerobic oxidation of methane might proceed as the reverse of archaeal methanogenesis from carbon dioxide with the nickel-containing methyl-coenzyme M reductase (MCR) as the methane-activating enzyme. However, experiments showing that MCR can catalyse the endergonic back reaction have been lacking. Here we report that purified MCR from Methanothermobacter marburgensis converts methane into methyl-coenzyme M under equilibrium conditions with apparent V(max) (maximum rate) and K(m) (Michaelis constant) values consistent with the observed in vivo kinetics of the anaerobic oxidation of methane with sulphate. This result supports the hypothesis of 'reverse methanogenesis' and is paramount to understanding the still-unknown mechanism of the last step of methanogenesis. The ability of MCR to cleave the particularly strong C-H bond of methane without the involvement of highly reactive oxygen-derived intermediates is directly relevant to catalytic C-H activation, currently an area of great interest in chemistry.
Immobilization of glucose oxidase using CoFe2O4/SiO2 nanoparticles as carrier

NASA Astrophysics Data System (ADS)

Wang, Hai; Huang, Jun; Wang, Chao; Li, Dapeng; Ding, Liyun; Han, Yun

2011-04-01

Aminated-CoFe2O4/SiO2 magnetic nanoparticles (NPs) were prepared from primary silica particles using modified StÖber method. Glucose oxidase (GOD) was immobilized on CoFe2O4/SiO2 NPs via cross-linking with glutaraldehyde (GA). The optimal immobilization condition was achieved with 1% (v/v) GA, cross-linking time of 3 h, solution pH of 7.0 and 0.4 mg GOD (in 3.0 mg carrier). The immobilized GOD showed maximal catalytic activity at pH 6.5 and 40 °C. After immobilization, the GOD exhibited improved thermal, storage and operation stability. The immobilized GOD still maintained 80% of its initial activity after the incubation at 50 °C for 25 min, whereas free enzyme had only 20% of initial activity after the same incubation. After kept at 4 °C for 28 days, the immobilized and free enzyme retained 87% and 40% of initial activity, respectively. The immobilized GOD maintained approximately 57% of initial activity after reused 7 times. The KM (Michaelis-Menten constant) values for immobilized GOD and free GOD were 14.6 mM and 27.1 mM, respectively.
Direct immobilization of tyrosinase enzyme from natural mushrooms (Agaricus bisporus) on D-sorbitol cinnamic ester.

PubMed

Marín-Zamora, María Elisa; Rojas-Melgarejo, Francisco; García-Cánovas, Francisco; García-Ruiz, Pedro Antonio

2006-11-10

Mushroom tyrosinase was immobilized from an extract onto the totally cinnamoylated derivative of D-sorbitol by direct adsorption as a result of the intense hydrophobic interactions that took place. The immobilization pH value and mass of lyophilized mushrooms were important parameters that affected the immobilization efficiency, while the immobilization time and immobilization support concentration were not important in this respect. The extracted/immobilized enzyme could best be measured above pH 3.5 and the optimum measuring temperature was 55 degrees C. The apparent Michaelis constant using 4-tert-butylcatechol as substrate was 0.38+/-0.02 mM, which was lower than for the soluble enzyme from Sigma (1.41+/-0.20 mM). Immobilization stabilized the extracted enzyme against thermal inactivation and made it less susceptible to activity loss during storage. The operational stability was higher than in the case of the tyrosinase supplied by Sigma and immobilized on the same support. The results show that the use of p-nitrophenol as enzyme-inhibiting substrate during enzyme extraction and immobilization made the use of ascorbic acid unnecessary and is a suitable method for extracting and immobilizing the tyrosinase enzyme, providing good enzymatic activity and stability.
Calcium pump kinetics determined in single erythrocyte ghosts by microphotolysis and confocal imaging.

PubMed

Kubitscheck, U; Pratsch, L; Passow, H; Peters, R

1995-07-01

The activity of the plasma membrane calcium pump was measured in single cells. Human red blood cell ghosts were loaded with a fluorescent calcium indicator and either caged calcium and ATP (protocol A) or caged ATP and calcium (protocol B). In a suitably modified laser scanning microscope either calcium or ATP were released by a short UV light pulse. The time-dependent fluorescence intensity of the calcium indicator was then followed in single ghosts by repetitive confocal imaging. The fluorescence intensity was converted into calcium concentration, which in turn was used to derive the kinetic parameters of the calcium pump, the Michaelis-Menten constant Km, and the maximal transport rate vmax. Km and vmax values derived in this manner were 24 +/- 14 microM and 1.0 +/- 0.6 microM/(ghost s) for protocol A, and 4 +/- 3 microM and 1.0 +/- 0.6 microM/(ghost s) for protocol B, respectively. The difference between A and B is presumably caused by calmodulin, which is inactive in the experiments with protocol A. The possibilities to extend the new method to living nucleus-containing cells transiently transfected with mutants of the plasma membrane calcium pump are discussed.
A highly efficient microfluidic nano biochip based on nanostructured nickel oxide.

PubMed

Ali, Md Azahar; Solanki, Pratima R; Patel, Manoj K; Dhayani, Hemant; Agrawal, Ved Varun; John, Renu; Malhotra, Bansi D

2013-04-07

We present results of the studies relating to fabrication of a microfluidic biosensor chip based on nickel oxide nanorods (NRs-NiO) that is capable of directly measuring the concentration of total cholesterol in human blood through electrochemical detection. Using this chip we demonstrate, with high reliability and in a time efficient manner, the detection of cholesterol present in buffer solutions at clinically relevant concentrations. The microfluidic channel has been fabricated onto a nickel oxide nanorod-based electrode co-immobilized with cholesterol esterase (ChEt) and cholesterol oxidase (ChOx) that serves as the working electrode. Bare indium tin oxide served as the counter electrode. A Ag/AgCl wire introduced to the outlet of the microchannel acts as a reference electrode. The fabricated NiO nanorod-based electrode has been characterized using X-ray diffraction, Raman spectroscopy, HR-TEM, FT-IR, UV-visible spectroscopy and electrochemical techniques. The presented NRs-NiO based microfluidic sensor exhibits linearity in the range of 1.5-10.3 mM, a high sensitivity of 0.12 mA mM(-1) cm(-2) and a low value of 0.16 mM of the Michaelis-Menten constant (Km).
Bi-enzyme L-arginine-selective amperometric biosensor based on ammonium-sensing polyaniline-modified electrode.

PubMed

Stasyuk, Nataliya; Smutok, Oleh; Gayda, Galina; Vus, Bohdan; Koval'chuk, Yevgen; Gonchar, Mykhailo

2012-01-01

A novel L-arginine-selective amperometric bi-enzyme biosensor based on recombinant human arginase I isolated from the gene-engineered strain of methylotrophic yeast Hansenula polymorpha and commercial urease is described. The biosensing layer was placed onto a polyaniline-Nafion composite platinum electrode and covered with a calcium alginate gel. The developed sensor revealed a good selectivity to L-arginine. The sensitivity of the biosensor was 110 ± 1.3 nA/(mM mm(2)) with the apparent Michaelis-Menten constant (K(M)(app)) derived from an L-arginine (L-Arg) calibration curve of 1.27 ± 0.29 mM. A linear concentration range was observed from 0.07 to 0.6mM, a limit of detection being 0.038 mM and a response time - 10s. The developed biosensor demonstrated good storage stability. A laboratory prototype of the proposed amperometric biosensor was applied to the samples of three commercial pharmaceuticals ("Tivortin", "Cytrarginine", "Aminoplazmal 10% E") for L-Arg testing. The obtained L-Arg-content values correlated well with those declared by producers. Copyright © 2012 Elsevier B.V. All rights reserved.
Immobilization of dextranase from Chaetomium erraticum.

PubMed

Erhardt, Frank Alwin; Jördening, Hans-Joachim

2007-09-30

In order to facilitate the Co-Immobilization of dextransucrase and dextranase, various techniques for the immobilization of industrial endo-dextranase from Chaetomium erraticum (Novozymes A/S) were researched. Adsorption isotherms at various pH-values have been determined for bentonite (Montmorillonite), hydroxyapatite and Streamline DEAE. Using bentonite and hydroxyapatite, highest activity loads (12,000 Ug(-1); 2900 Ug(-1), respectively) can be achieved without a significant change of the apparent Michaelis-Menten constant K(M). For successful adsorption, enzyme to bentonite ratios greater than 0.4 (w/w) have to be used as lower ratios lead to 90% enzyme inactivation due to bentonite contact. In addition, covalent linkage using the activated oxiran carriers Eupergit C and Eupergit C250L as well as linkage with aminopropyl silica via metaperiodate activation of glycosyl moiety of dextranase are discussed. This is also the first report probing the structure of a matrix containing dextranase by use of substrate species with different molecular weights. From this we can observe a relationship between the porosity of Eupergit and dextran dependent activity. For the reactor concept using Co-Immobilisates, hydroxyapatite will be preferred to Eupergit because of its higher specific activity and dispersity.
Partial purification and characterization of polyphenol oxidase from banana (Musa sapientum L.) peel.

PubMed

Yang, C P; Fujita, S; Kohno, K; Kusubayashi, A; Ashrafuzzaman, M; Hayashi, N

2001-03-01

Polyphenol oxidase (EC 1.10.3.1, o-diphenol: oxygen oxidoreductase, PPO) of banana (Musa sapientum L.) peel was partially purified about 460-fold with a recovery of 2.2% using dopamine as substrate. The enzyme showed a single peak on Toyopearl HW55-S chromatography. However, two bands were detected by staining with Coomassie brilliant blue on PAGE: one was very clear, and the other was faint. Molecular weight for purified PPO was estimated to be about 41 000 by gel filtration. The enzyme quickly oxidized dopamine, and its Km value (Michaelis constant) for dopamine was 3.9 mM. Optimum pH was 6.5 and the PPO activity was quite stable in the range of pH 5-11 for 48 h. The enzyme had an optimum temperature at 30 degrees C and was stable up to 60 degrees C after heat treatment for 30 min. The enzyme activity was strongly inhibited by sodium diethyldithiocarbamate, potassium cyanide, L-ascorbic acid, and cysteine at 1 mM. Under a low buffer capacity, the enzyme was also strongly inhibited by citric acid and acetic acid at 10 mM.
DOE Office of Scientific and Technical Information (OSTI.GOV)

Toral-Barza, Lourdes; Zhang Weiguo; Lamison, Craig

The mammalian target of rapamycin (mTOR/TOR) is implicated in cancer and other human disorders and thus an important target for therapeutic intervention. To study human TOR in vitro, we have produced in large scale both the full-length TOR (289 kDa) and a truncated TOR (132 kDa) from HEK293 cells. Both enzymes demonstrated a robust and specific catalytic activity towards the physiological substrate proteins, p70 S6 ribosomal protein kinase 1 (p70S6K1) and eIF4E binding protein 1 (4EBP1), as measured by phosphor-specific antibodies in Western blotting. We developed a high capacity dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) for analysis of kinetic parameters. Themore » Michaelis constant (K {sub m}) values of TOR for ATP and the His6-S6K substrate were shown to be 50 and 0.8 {mu}M, respectively. Dose-response and inhibition mechanisms of several known inhibitors, the rapamycin-FKBP12 complex, wortmannin and LY294002, were also studied in DELFIA. Our data indicate that TOR exhibits kinetic features of those shared by traditional serine/threonine kinases and demonstrate the feasibility for TOR enzyme screen in searching for new inhibitors.« less
Solution Process Synthesis of High Aspect Ratio ZnO Nanorods on Electrode Surface for Sensitive Electrochemical Detection of Uric Acid

NASA Astrophysics Data System (ADS)

Ahmad, Rafiq; Tripathy, Nirmalya; Ahn, Min-Sang; Hahn, Yoon-Bong

2017-04-01

This study demonstrates a highly stable, selective and sensitive uric acid (UA) biosensor based on high aspect ratio zinc oxide nanorods (ZNRs) vertical grown on electrode surface via a simple one-step low temperature solution route. Uricase enzyme was immobilized on the ZNRs followed by Nafion covering to fabricate UA sensing electrodes (Nafion/Uricase-ZNRs/Ag). The fabricated electrodes showed enhanced performance with attractive analytical response, such as a high sensitivity of 239.67 μA cm-2 mM-1 in wide-linear range (0.01-4.56 mM), rapid response time (~3 s), low detection limit (5 nM), and low value of apparent Michaelis-Menten constant (Kmapp, 0.025 mM). In addition, selectivity, reproducibility and long-term storage stability of biosensor was also demonstrated. These results can be attributed to the high aspect ratio of vertically grown ZNRs which provides high surface area leading to enhanced enzyme immobilization, high electrocatalytic activity, and direct electron transfer during electrochemical detection of UA. We expect that this biosensor platform will be advantageous to fabricate ultrasensitive, robust, low-cost sensing device for numerous analyte detection.
Human and rat liver phenol sulfotransferase: structure-activity relationships for phenolic substrates.

PubMed

Campbell, N R; Van Loon, J A; Sundaram, R S; Ames, M M; Hansch, C; Weinshilboum, R

1987-12-01

Phenol sulfotransferase (PST) catalyzes the sulfate conjugation of many phenolic drugs. Human liver contains thermostable (TS) and thermolabile forms of PST. Ion exchange chromatography shows that two isozymes of TS PST (peaks I and II) are present in human liver preparations. Rat liver contains four forms of PST that can be separated by ion exchange chromatography. Quantitative structure-activity relationship (QSAR) analysis was used to study phenolic substrates for both human and rat liver PST. Thirty-six substituted phenols were tested as substrates for partially purified human liver TS PST peak I. QSAR analysis resulted in derivation of the following equation: log 1/Km = 0.92 (+/- 0.18)log P - 1.48 (+/- 0.38)MR'4 - 0.64 (+/- 0.41)MR3 + 1.04 (+/- 0.63)MR2 + 0.67(+/- 0.44) sigma- + 4.03 (+/- 0.42). In this equation Km is the Michaelis constant, P is the octanol-water partition coefficient, MR is the molar refractivity of substituents at the 2-, 3-, and 4-positions, and sigma- is the Hammett constant. Values of log 1/Km calculated with this equation were highly correlated with log 1/Km values (r = 0.950) that were observed experimentally. Nine phenols were also tested as substrates for partially purified human liver TS PST peak II. Log 1/Km values for these compounds were significantly correlated for the two isozymes of TS PST (r = 0.992, p less than 0.001). QSAR analysis was also used to derive equations that described the behavior of phenolic substrates for rat liver PST forms I and II. These equations differed substantially from the equation derived for compounds tested with human liver TS PST peak I. Therefore, the characteristics of the active sites of human liver TS PST peak I and rat liver PST forms I and II appear to differ. Application of these equations may make it possible to predict Km values of phenolic substrates for human liver TS PST and for rat liver PST forms I and II.
Purification and substrate specificity of polymorphic forms of esterase D from human erythrocytes.

PubMed Central

Scott, E M; Wright, R C

1978-01-01

Esterase D (EsD), purified from human erythrocytes and tested with a variety of substrates, hydrolyzed only triacetin, tributyrin, and certain soluble aryl esters of aliphatic acids. Esters of 4-methylumbelliferone were easily the best substrates. When the three genetically different isozymes were compared, the less common forms, EsD 2 and EsD 2-1, were less stable than EsD 1. With some substrates, the Michaelis constant of the EsD 2 form differed from that of the EsD 1 form. The EsD 2-1 hybrid form was usually, but not invariably, intermediate in properties. The physiologic significance of the genetic variability of this enzyme is unknown. PMID:623100
Kinetic studies of lipid oxidation induced by hemoglobin measured by consumption of dissolved oxygen in a liposome model system.

PubMed

Carvajal, Ana Karina; Rustad, Turid; Mozuraityte, Revilija; Storrø, Ivar

2009-09-09

The effect of hemoglobin (Hb) and lipid concentration, pH, temperature, and different antioxidants on heme-mediated lipid oxidation of liposomes from marine phospholipids was studied. The rate of lipid oxidation was measured by consumption of dissolved oxygen. Heme-mediated lipid oxidation at different Hb and lipid concentrations was modeled by Michaelis-Menten kinetics. The maximum rate (V(max)) for the reaction with cod and bovine Hb as a pro-oxidant was 66.2 +/- 3.4 and 56.6 +/- 3.4 microM/min, respectively. The Michaelis-Menten constant (K(m)) for the reaction with cod and bovine Hb was 0.67 +/- 0.09 and 1.2 +/- 0.2 microM, respectively. V(max) for the relationship between the oxygen uptake rate and lipid concentration was 43.2 +/- 1.5 microM/min, while the K(m) was 0.93 +/- 0.14 mg/mL. The effect of the temperature followed Arrhenius kinetics, and there was no significant difference in activation energy between cod and bovine Hb. The rate of lipid oxidation induced by bovine Hb was highest around pH 6. Ethylenediaminetetraacetic acid (EDTA) had no significant effect on heme-mediated lipid oxidation, but alpha-tocopherol and astaxanthin worked well as antioxidants. Kinetic differences were found between iron and Hb as pro-oxidants, and the efficacy of the antioxidants depended upon the pro-oxidant in the system.
Electrocatalytic Mechanism Involving Michaelis-Menten Kinetics at the Preparative Scale: Theory and Applicability to Photocurrents from a Photosynthetic Algae Suspension With Quinones.

PubMed

Longatte, Guillaume; Guille-Collignon, Manon; Lemaître, Frédéric

2017-10-06

In the past years, many strategies have been implemented to benefit from oxygenic photosynthesis to harvest photosynthetic electrons and produce a significant photocurrent. Therefore, electrochemical tools were considered and have globally relied on the electron transfer(s) between the photosynthetic chain and a collecting electrode. In this context, we recently reported the implementation of an electrochemical set-up at the preparative scale to produce photocurrents from a Chlamydomonas reinhardtii algae suspension with an appropriate mediator (2,6-DCBQ) and a carbon gauze as the working electrode. In the present work, we wish to describe a mathematical modeling of the recorded photocurrents to better understand the effects of the experimental conditions on the photosynthetic extraction of electrons. In that way, we established a general model of an electrocatalytic mechanism at the preparative scale (that is, assuming a homogenous bulk solution at any time and a constant diffusion layer, both assumptions being valid under forced convection) in which the chemical step involves a Michaelis-Menten-like behaviour. Dependences of transient and steady-state corresponding currents were analysed as a function of different parameters by means of zone diagrams. This model was tested to our experimental data related to photosynthesis. The corresponding results suggest that competitive pathways beyond photosynthetic harvesting alone should be taken into account. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
Use of CdSe/ZnS luminescent quantum dots incorporated within sol-gel matrix for urea detection.

PubMed

Duong, Hong Dinh; Rhee, Jong Il

2008-09-19

In this work, urea detection techniques based on the pH sensitivity of CdSe/ZnS QDs were developed using three types of sol-gel membranes: a QD-entrapped membrane, urease-immobilized membrane and double layer consisting of a QD-entrapped membrane and urease-immobilized membrane. The surface morphology of the sol-gel membranes deposited on the wells in a 24-well microtiter plate was investigated. The linear detection range of urea was in the range of 0-10mM with the three types of sol-gel membranes. The urea detection technique based on the double layer consisting of the QD-entrapped membrane and urease-immobilized membrane resulted in the highest sensitivity to urea due to the Michaelis-Menten kinetic parameters. That is, the Michaelis-Menten constant (K(m)=2.0745mM) of the free urease in the QD-entrapped membrane was about 4-fold higher than that (K(m)=0.549mM) of the immobilized urease in the urease-immobilized membrane and about 12-fold higher than that (K(m)=0.1698mM) of the immobilized urease in the double layer. The good stability of the three sol-gel membranes for urea sensing over 2 months showed that the use of sol-gel membranes immobilized with QDs or an enzyme is suitable for biomedical and environmental applications.
Crude Aloe vera Gel Shows Antioxidant Propensities and Inhibits Pancreatic Lipase and Glucose Movement In Vitro

PubMed Central

Taukoorah, Urmeela; Mahomoodally, M. Fawzi

2016-01-01

Aloe vera gel (AVG) is traditionally used in the management of diabetes, obesity, and infectious diseases. The present study aimed to investigate the inhibitory potential of AVG against α-amylase, α-glucosidase, and pancreatic lipase activity in vitro. Enzyme kinetic studies using Michaelis-Menten (K m) and Lineweaver-Burk equations were used to establish the type of inhibition. The antioxidant capacity of AVG was evaluated for its ferric reducing power, 2-diphenyl-2-picrylhydrazyl hydrate scavenging ability, nitric oxide scavenging power, and xanthine oxidase inhibitory activity. The glucose entrapment ability, antimicrobial activity, and total phenolic, flavonoid, tannin, and anthocyanin content were also determined. AVG showed a significantly higher percentage inhibition (85.56 ± 0.91) of pancreatic lipase compared to Orlistat. AVG was found to increase the Michaelis-Menten constant and decreased the maximal velocity (V max) of lipase, indicating mixed inhibition. AVG considerably inhibits glucose movement across dialysis tubes and was comparable to Arabic gum. AVG was ineffective against the tested microorganisms. Total phenolic and flavonoid contents were 66.06 ± 1.14 (GAE)/mg and 60.95 ± 0.97 (RE)/mg, respectively. AVG also showed interesting antioxidant properties. The biological activity observed in this study tends to validate some of the traditional claims of AVG as a functional food. PMID:26880905
Enzymatic carotenoid cleavage in star fruit (Averrhoa carambola).

PubMed

Fleischmann, Peter; Watanabe, Naoharu; Winterhalter, Peter

2003-05-01

This paper presents the first description of an enzyme fraction exhibiting carotenoid cleavage activity isolated from fruit skin of Averrhoa carambola. Partial purification of the enzyme could be achieved by acetone precipitation, ultrafiltration (300 kDa, 50 kDa), isoelectric focusing (pH 3-10) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (7.5%). In this way, an enzymatically active protein fraction was obtained, consisting of four proteins in the molecular weight range of between 12 and 90 kDa. Using beta-carotene as substrate, the enzyme activity was detected spectrophotometrically at 505 nm. The main reaction product, detected by GC analysis, was beta-ionone. This proves that the isolated enzymes are closely related to aroma metabolism and release of star fruit. The time constant of the reaction was 16.6 min, the Michaelis Constant K(m)=3.6 micromol 1(-1) and the maximum velocity V(max)=10.5 x 10(-3) micromol l(-1) s(-1) mg((Protein))(-1). The optimum temperature was 45 degrees C.
Preparation of biosensors by immobilization of polyphenol oxidase in conducting copolymers and their use in determination of phenolic compounds in red wine.

PubMed

Böyükbayram, A Elif; Kiralp, Senem; Toppare, Levent; Yağci, Yusuf

2006-10-01

Electrochemically produced graft copolymers of thiophene capped polytetrahydofuran (TPTHF1 and TPTHF2) and pyrrole were achieved by constant potential electrolysis using sodium dodecylsulfate (SDS) as the supporting electrolyte. Characterizations were based on Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Electrical conductivities were measured by the four-probe technique. Novel biosensors for phenolic compounds were constructed by immobilizing polyphenol oxidase (PPO) into conducting copolymers prepared by electropolymerization of pyrrole with thiophene capped polytetrahydrofuran. Kinetic parameters, maximum reaction rate (V(max)) and Michaelis-Menten constant (K(m)) and optimum conditions regarding temperature and pH were determined for the immobilized enzyme. Operational stability and shelf-life of the enzyme electrodes were investigated. Enzyme electrodes of polyphenol oxidase were used to determine the amount of phenolic compounds in two brands of Turkish red wines and found very useful owing to their high kinetic parameters and wide pH working range.
Inhibition by 6-mercaptopurine of purine phosphoribosyltransferases from Ehrlich ascites-tumour cells that are resistant to this drug

PubMed Central

Atkinson, M. R.; Murray, A. W.

1965-01-01

1. A strain of Ehrlich ascites-tumour cells that showed little inhibition of growth in the presence of 6-mercaptopurine accumulated less than 5% as much 6-thioinosine 5′-phosphate in vivo, in the presence of 6-mercaptopurine, as did the sensitive strain from which it was derived. 2. Specific activities of the phosphoribosyltransferases that convert adenine, guanine, hypoxanthine and 6-mercaptopurine into AMP, GMP, IMP and 6-thioinosine 5′-phosphate were similar in extracts of the resistant and the sensitive cells. 3. As found previously with sensitive cells, 6-mercaptopurine is a competitive inhibitor of guanine phosphoribosyltransferase and hypoxanthine phosphoribosyltransferase from the resistant cells and does not inhibit the adenine phosphoribosyltransferase from these cells. Michaelis constants and inhibitor constants of the purine phosphoribosyltransferases from resistant cells did not differ significantly from those measured with the corresponding enzymes from sensitive cells. 4. Resistance to 6-mercaptopurine in this case is probably not due to qualitative or quantitative changes in these transferases. PMID:14342251

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

PubMed

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

2010-12-01

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

PubMed

He, Yaping; Yang, Xiaohui; Han, Quan; Zheng, Jianbin

2017-06-23

A novel catechol (CA) biosensor was developed by embedding tyrosinase (Tyr) onto in situ electrochemical reduction graphene (EGR) on choline-functionalized gold nanoparticle (AuNPs-Ch) film. The results of UV-Vis spectra indicated that Tyr retained its original structure in the film, and an electrochemical investigation of the biosensor showed a pair of well-defined, quasi-reversible redox peaks with E pa = -0.0744 V and E pc = -0.114 V (vs. SCE) in 0.1 M, pH 7.0 sodium phosphate-buffered saline at a scan rate of 100 mV/s. The transfer rate constant k s is 0.66 s -1 . The Tyr-EGR/AuNPs-Ch showed a good electrochemical catalytic response for the reduction of CA, with the linear range from 0.2 to 270 μM and a detection limit of 0.1 μM (S/N = 3). The apparent Michaelis-Menten constant was estimated to be 109 μM.
[Kinetics of uptake of phosphates and nitrates by marine multicellular algae Gelidium latifolium (Grev.) Born. et Thur].

PubMed

Silkin, V A; Chubchikova, I N

2007-01-01

We studied nonstationary kinetics of the uptake of phosphates and nitrates by the red marine algae Gelidium latifolium (Grev.) Born et Thur. and calculated constants of the Michaelis-Menten equation for these elements. In the area of 0-3 microM, the kinetics of phosphate consumption had the following coefficients: maximum rate of uptake 0.8 micromol/(g x h), constant of half-saturation 1.745 microM. For nitrate nitrogen at 0-30 microM, an adaptive strategy of uptake kinetics was noted with change of the equation parameters with time: after 1 h, the maximum rate of uptake was 5.1 micromol/(g x h) and constant of half-saturation 19 gM, while within 2 h, the maximum rate of uptake significantly increased. This could be related to the synthesis of nitrate reductase. Coupled with the uptake of nitrates, nonstationary kinetics of the release of nitrates in the surrounding medium had a one-peak pattern: the maximum concentration of nitrites in the medium and the time of its achievement increased with the initial concentration of nitrates. The maximum concentration of nitrites was 6 to 14% of the initial concentration in the medium.
Explicit solution of integrated 1 - exp equation for predicting accumulation and decline of concentrations for drugs obeying nonlinear saturation kinetics.

PubMed

Keller, Frieder; Hartmann, Bertram; Czock, David

2009-12-01

To describe nonlinear, saturable pharmacokinetics, the Michaelis-Menten equation is frequently used. However, the Michaelis-Menten equation has no integrated solution for concentrations but only for the time factor. Application of the Lambert W function was proposed recently to obtain an integrated solution of the Michaelis-Menten equation. As an alternative to the Michaelis-Menten equation, a 1 - exp equation has been used to describe saturable kinetics, with the advantage that the integrated 1 - exp equation has an explicit solution for concentrations. We used the integrated 1 - exp equation to predict the accumulation kinetics and the nonlinear concentration decline for a proposed fictive drug. In agreement with the recently proposed method, we found that for the integrated 1 - exp equation no steady state is obtained if the maximum rate of change in concentrations (Vmax) within interval (Tau) is less than the difference between peak and trough concentrations (Vmax x Tau < C peak - C trough).
Redox-dependent substrate-cofactor interactions in the Michaelis-complex of a flavin-dependent oxidoreductase

NASA Astrophysics Data System (ADS)

Werther, Tobias; Wahlefeld, Stefan; Salewski, Johannes; Kuhlmann, Uwe; Zebger, Ingo; Hildebrandt, Peter; Dobbek, Holger

2017-07-01

How an enzyme activates its substrate for turnover is fundamental for catalysis but incompletely understood on a structural level. With redox enzymes one typically analyses structures of enzyme-substrate complexes in the unreactive oxidation state of the cofactor, assuming that the interaction between enzyme and substrate is independent of the cofactors oxidation state. Here, we investigate the Michaelis complex of the flavoenzyme xenobiotic reductase A with the reactive reduced cofactor bound to its substrates by X-ray crystallography and resonance Raman spectroscopy and compare it to the non-reactive oxidized Michaelis complex mimics. We find that substrates bind in different orientations to the oxidized and reduced flavin, in both cases flattening its structure. But only authentic Michaelis complexes display an unexpected rich vibrational band pattern uncovering a strong donor-acceptor complex between reduced flavin and substrate. This interaction likely activates the catalytic ground state of the reduced flavin, accelerating the reaction within a compressed cofactor-substrate complex.
Redox-dependent substrate-cofactor interactions in the Michaelis-complex of a flavin-dependent oxidoreductase

PubMed Central

Werther, Tobias; Wahlefeld, Stefan; Salewski, Johannes; Kuhlmann, Uwe; Zebger, Ingo; Hildebrandt, Peter; Dobbek, Holger

2017-01-01

How an enzyme activates its substrate for turnover is fundamental for catalysis but incompletely understood on a structural level. With redox enzymes one typically analyses structures of enzyme–substrate complexes in the unreactive oxidation state of the cofactor, assuming that the interaction between enzyme and substrate is independent of the cofactors oxidation state. Here, we investigate the Michaelis complex of the flavoenzyme xenobiotic reductase A with the reactive reduced cofactor bound to its substrates by X-ray crystallography and resonance Raman spectroscopy and compare it to the non-reactive oxidized Michaelis complex mimics. We find that substrates bind in different orientations to the oxidized and reduced flavin, in both cases flattening its structure. But only authentic Michaelis complexes display an unexpected rich vibrational band pattern uncovering a strong donor–acceptor complex between reduced flavin and substrate. This interaction likely activates the catalytic ground state of the reduced flavin, accelerating the reaction within a compressed cofactor–substrate complex.
The integrated Michaelis-Menten rate equation: déjà vu or vu jàdé?

PubMed

Goličnik, Marko

2013-08-01

A recent article of Johnson and Goody (Biochemistry, 2011;50:8264-8269) described the almost-100-years-old paper of Michaelis and Menten. Johnson and Goody translated this classic article and presented the historical perspective to one of incipient enzyme-reaction data analysis, including a pioneering global fit of the integrated rate equation in its implicit form to the experimental time-course data. They reanalyzed these data, although only numerical techniques were used to solve the model equations. However, there is also the still little known algebraic rate-integration equation in a closed form that enables direct fitting of the data. Therefore, in this commentary, I briefly present the integral solution of the Michaelis-Menten rate equation, which has been largely overlooked for three decades. This solution is expressed in terms of the Lambert W function, and I demonstrate here its use for global nonlinear regression curve fitting, as carried out with the original time-course dataset of Michaelis and Menten.
Improved peroxide biosensor based on Horseradish Peroxidase/Carbon Nanotube on a thiol-modified gold electrode.

PubMed

Kafi, A K M; Naqshabandi, M; Yusoff, Mashitah M; Crossley, Maxwell J

2018-06-01

A new 3-dimensional (3D) network of crosslinked Horseradish Peroxidase/Carbon Nanotube (HRP/CNT) on a thiol-modified Au surface has been described in order to build up the effective electrical wiring of the enzyme units with the electrode. The synthesized 3D HRP/CNT network has been characterized with cyclic voltammetry and amperometry which results the establishment of direct electron transfer between the redox active unit of HRP and the Au surface. Electrochemical measurements reveal that the high biological activity and stability is exhibited by the immobilized HRP and a quasi-reversible redox peak of the redox centre of HRP was observed at about -0.355 and -0.275V vs. Ag/AgCl. The electron transfer rate constant, K S and electron transfer co-efficient α were found as 0.57s -1 and 0.42, respectively. Excellent electrocatalytic activity for the reduction of H 2 O 2 was exhibited by the developed biosensor. The proposed biosensor modified with HRP/CNT 3D network displays a broader linear range and a lower detection limit for H 2 O 2 determination. The linear range is from 1.0×10 -7 to 1.2×10 -4 M with a detection limit of 2.2.0×10 -8 M at 3σ. The Michaelies-Menten constant Kapp M value is estimated to be 0.19mM. Moreover, this biosensor exhibits very high sensitivity, good reproducibility and long-time stability. Copyright © 2017 Elsevier Inc. All rights reserved.
Simultaneous measurement of glucose transport and utilization in the human brain

PubMed Central

Shestov, Alexander A.; Emir, Uzay E.; Kumar, Anjali; Henry, Pierre-Gilles; Seaquist, Elizabeth R.

2011-01-01

Glucose is the primary fuel for brain function, and determining the kinetics of cerebral glucose transport and utilization is critical for quantifying cerebral energy metabolism. The kinetic parameters of cerebral glucose transport, KMt and Vmaxt, in humans have so far been obtained by measuring steady-state brain glucose levels by proton (1H) NMR as a function of plasma glucose levels and fitting steady-state models to these data. Extraction of the kinetic parameters for cerebral glucose transport necessitated assuming a constant cerebral metabolic rate of glucose (CMRglc) obtained from other tracer studies, such as 13C NMR. Here we present new methodology to simultaneously obtain kinetic parameters for glucose transport and utilization in the human brain by fitting both dynamic and steady-state 1H NMR data with a reversible, non-steady-state Michaelis-Menten model. Dynamic data were obtained by measuring brain and plasma glucose time courses during glucose infusions to raise and maintain plasma concentration at ∼17 mmol/l for ∼2 h in five healthy volunteers. Steady-state brain vs. plasma glucose concentrations were taken from literature and the steady-state portions of data from the five volunteers. In addition to providing simultaneous measurements of glucose transport and utilization and obviating assumptions for constant CMRglc, this methodology does not necessitate infusions of expensive or radioactive tracers. Using this new methodology, we found that the maximum transport capacity for glucose through the blood-brain barrier was nearly twofold higher than maximum cerebral glucose utilization. The glucose transport and utilization parameters were consistent with previously published values for human brain. PMID:21791622
Influence of low oxygen tensions and sorption to sediment black carbon on biodegradation of pyrene.

PubMed

Ortega-Calvo, José-Julio; Gschwend, Philip M

2010-07-01

Sorption to sediment black carbon (BC) may limit the aerobic biodegradation of polycyclic aromatic hydrocarbons (PAHs) in resuspension events and intact sediment beds. We examined this hypothesis experimentally under conditions that were realistic in terms of oxygen concentrations and BC content. A new method, based on synchronous fluorescence observations of (14)C-pyrene, was developed for continuously measuring the uptake of dissolved pyrene by Mycobacterium gilvum VM552, a representative degrader of PAHs. The effect of oxygen and pyrene concentrations on pyrene uptake followed Michaelis-Menten kinetics, resulting in a dissolved oxygen half-saturation constant (K(om)) of 14.1 microM and a dissolved pyrene half-saturation constant (K(pm)) of 6 nM. The fluorescence of (14)C-pyrene in air-saturated suspensions of sediments and induced cells followed time courses that reflected simultaneous desorption and biodegradation of pyrene, ultimately causing a quasi-steady-state concentration of dissolved pyrene balancing desorptive inputs and biodegradation removals. The increasing concentrations of (14)CO(2) in these suspensions, as determined with liquid scintillation, evidenced the strong impact of sorption to BC-rich sediments on the biodegradation rate. Using the best-fit parameter values, we integrated oxygen and sorption effects and showed that oxygen tensions far below saturation levels in water are sufficient to enable significant decreases in the steady-state concentrations of aqueous-phase pyrene. These findings may be relevant for bioaccumulation scenarios that consider the effect of sediment resuspension events on exposure to water column and sediment pore water, as well as the direct uptake of PAHs from sediments.
Glyphosate sensitivity of 5-enol-pyruvylshikimate-3-phosphate synthase from Bacillus subtilis depends upon state of activation induced by monovalent cations.

PubMed

Fischer, R S; Rubin, J L; Gaines, C G; Jensen, R A

1987-07-01

The 5-enol-pyruvylshikimate-3-phosphate (EPSP) synthase from Bacillus subtilis was activated by monovalent cations, catalytic activity being negligible in the absence of monovalent cations. The order of cation effectiveness (NH4+ greater than K+ greater than Rb+ greater than Na+ = Cs+ = Li+) indicated that the extent of activation was directly related to the unhydrated cation radius. Ammonium salts, at physiological concentrations, were dramatically more effective than other cations. Activation by ammonium was instantaneous, was not influenced by the counter ion, and gave a hyperbolic saturation curve. Hill plots did not show detectable cooperativity in the binding of ammonium. Double-reciprocal plots indicated that ammonium increases the maximal velocity and decreases the apparent Michaelis constants of EPSP synthase with respect to both phosphoenol pyruvate (PEP) and shikimate 3-phosphate (S3P). A direct relationship between sensitivity to inhibition by glyphosate and the activation state of EPSP synthase was demonstrated. Hill plots indicated a single value for glyphosate binding throughout the range of ammonium activation. Double-reciprocal plots of substrate saturation data obtained with ammonium-activated enzyme in the presence of glyphosate showed glyphosate to behave as a competitive inhibitor with respect to PEP and as a mixed-type inhibitor relative to S3P. The increased glyphosate sensitivity of ammonium-activated EPSP synthase is attributed to a lowering of the inhibitor constant of glyphosate with respect to PEP. Erroneous underestimates of sensitivities of some bacterial EPSP synthases to inhibition by glyphosate may result from failure to recognize cation requirements of EPSP synthases.
Effects of Acute Lymphoblastic Leukemia on Ceruloplasmin Oxidase, Copper and Several Markers of Oxidative Damage, in Children.

PubMed

Mehdi, Wesen Adel; Yusof, Faridah; Mehde, Atheer Awad; Zainulabdeen, Jwan Abdulmohsin; Raus, Raha Ahmed; Abdulbari, Alaa Shawqi

2015-01-01

Acute leukaemia is characterized by fast growth of abnormal clones of haemopoietic precursor cells inside bone marrow leading to undue accumulation in the bone marrow. Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer. The study concerned 50 children diagnosed with ALL (mean age, 8.55±2.54) compared to 40 healthy controls (mean age, 8.00±1.85). The Hb, serum copper, ceruloplasmin oxidase, advanced oxidation protein products (AOPPs), total antioxidant activity (TAA) and protein were measured in all groups. One proteinous component was isolated by gel filtration chromatography from the precipitate produced by polyethylene glycol. Significantly higher levels of AOPP, copper and decrease in total antioxidant activity were noted in the cases. Statistical analysis also showed a significant increase (p<0.01) in the activity of serum ceruloplasmin oxidase in patients with ALL compared to normal subjects. The maximum velocity (Vmax) and Michaelis constant had values of 104.2 U/L and 11.7 mM, respectively. The ΔH* values for ceruloplasmin oxidase in ALL patients were positive, confirming the reaction to be endothermic. The results from this study showed a significant increase in AOPP, ceruloplasmine oxidase and decrease in total antioxidant activity .These parameters may play a role in development of DNA damage in childhood patients with acute lymphoblastic leukemia (ALL). The ΔS* and ΔG* values were negative, these refer that the reaction of ES formation is spontaneous, but needs energy in a so-called endergonic reaction. Also the negative ΔS* value of ceruloplasmin oxidase indicates that the complex [ES*] is further modulated through increasing structure arrangement.
Alternative Analysis of the Michaelis-Menten Equations

ERIC Educational Resources Information Center

Krogstad, Harald E.; Dawed, Mohammed Yiha; Tegegne, Tadele Tesfa

2011-01-01

Courses in mathematical modelling are always in need of simple, illustrative examples. The Michaelis-Menten reaction kinetics equations have been considered to be a basic example of scaling and singular perturbation. However, the leading order approximations do not easily show the expected behaviour, and this note proposes a different perturbation…
Fluorescent microplate assay method for high-throughput detection of lipase transesterification activity.

PubMed

Zheng, Jianyong; Wei, Wei; Lan, Xing; Zhang, Yinjun; Wang, Zhao

2018-05-15

This study describes a sensitive and fluorescent microplate assay method to detect lipase transesterification activity. Lipase-catalyzed transesterification between butyryl 4-methyl umbelliferone (Bu-4-Mu) and methanol in tert-butanol was selected as the model reaction. The release of 4-methylumbelliferone (4-Mu) in the reaction was determined by detecting the fluorescence intensity at λ ex 330 nm and λ em 390 nm. Several lipases were used to investigate the accuracy and efficiency of the proposed method. Apparent Michaelis constant (Km) was calculated for transesterification between Bu-4-Mu and methanol by the lipases. The main advantages of the assay method include high sensitivity, inexpensive reagents, and simple detection process. Copyright © 2018 Elsevier Inc. All rights reserved.
Phosphatidylethanolamine Synthesis by Castor Bean Endosperm 1

PubMed Central

Shin, Sungho; Moore, Thomas S.

1990-01-01

A base exchange reaction for synthesis of phosphatidylethanolamine by the endoplasmic reticulum of castor bean (Ricinus comminus L. var Hale) endosperm has been examined. The calculated Michaelis-Menten constant of the enzyme for ethanolamine was 5 micromolar and the optimal pH was 7.8 in the presence of 2 millimolar CaCl2. l-Serine, N-methylethanolamine and N,N-dimethylethanolamine all reduced ethanolamine incorporation, while d-serine and myo-inositol had little effect. These inhibitions of ethanolamine incorporation were found to be noncompetitive and ethanolamine also noncompetitively inhibited l-serine incorporation by exchange. The activity of the ethanolamine base exchange enzyme was affected by several detergents, with the best activity being obtained with the zwitterionic defjtergent 3-3-cholamidopropyl) dimethylammonio-2-hydroxyl-1-propanesulfonate. PMID:16667427
Origin of Cyanide in Cultures of a Psychrophilic Basidiomycete1

PubMed Central

Stevens, Dennis L.; Strobel, Gary A.

1968-01-01

An unidentified psychrophilic basidiomycete used valine and isoleucine as precursors to hydrocyanic acid (HCN). As probable intermediates in the pathway from valine and isoleucine two cyanogenic glucosides, linamarin and lotaustralin, were demonstrated in fungus cultures. The fungus contained two β-glucosidases and an oxynitrilase which, acting together, were capable of releasing cyanide from both linamarin and lotaustralin. The two β-glucosidases were purified and compared as to pH optimum, Michaelis constant, energy of activation, thermal stability, and substrate specificity. The products of methyl ethyl ketone cyanohydrin and acetone cyanohydrin dissociation by the oxynitrilase were demonstrated to be HCN together with methyl ethyl ketone and acetone, respectively. The oxynitrilase attacked aliphatic hydroxynitriles, but showed no activity on aromatic hydroxynitriles. Images PMID:5651322
Effect of antihypertensive agents - captopril and nifedipine - on the functional properties of rat heart mitochondria

PubMed Central

Kancirová, Ivana; Jašová, Magdaléna; Waczulíková, Iveta; Ravingerová, Táňa; Ziegelhöffer, Attila; Ferko, Miroslav

2016-01-01

Objective(s): Investigation of acute effect on cellular bioenergetics provides the opportunity to characterize the possible adverse effects of drugs more comprehensively. This study aimed to investigate the changes in biochemical and biophysical properties of heart mitochondria induced by captopril and nifedipine antihypertensive treatment. Materials and Methods: Male, 12-week-old Wistar rats in two experimental models (in vivo and in vitro) were used. In four groups, the effects of escalating doses of captopril, nifedipine and combination of captopril + nifedipine added to the incubation medium (in vitro) or administered per os to rat (in vivo) on mitochondrial ATP synthase activity and membrane fluidity were monitored. Results: In the in vitro model we observed a significant inhibitory effect of treatment on the ATP synthase activity (P<0.05) with nonsignificant differences in membrane fluidity. Decrease in the value of maximum reaction rate Vmax (P<0.05) without any change in the value of Michaelis-Menten constant Km, indicative of a noncompetitive inhibition, was presented. At the in vivo level, we did not demonstrate any significant changes in the ATP synthase activity and the membrane fluidity in rats receiving captopril, nifedipine, and combined therapy. Conclusion: In vitro kinetics study revealed that antihypertensive drugs (captopril and nifedipine) directly interact with mitochondrial ATP synthase. In vivo experiment did not prove any acute effect on myocardial bioenergetics and suggest that drugs do not enter cardiomyocyte and have no direct effect on mitochondria. PMID:27482342
A novel bifunctional endo-/exo-type cellulase from an anaerobic ruminal bacterium.

PubMed

Ko, Kyong-Cheol; Han, Yunjon; Choi, Jong Hyun; Kim, Geun-Joong; Lee, Seung-Goo; Song, Jae Jun

2011-03-01

An anaerobic microorganism termed AN-C16-KBRB was isolated from the bovine rumen and demonstrated cellulolytic activity on a NB agar plate containing azo-carboxymethyl cellulose. The 16S rRNA gene of the strain was 98% similar to that of Clostridiaceae bacterium SK082 (AB298754) as the highest homology. A novel celEdx16 gene encoding a bifunctional endo-/exocellulase (CelEdx16) was cloned by the shotgun method from AN-C16-KBRB, and the enzyme was characterized. The celEdx16 gene had an open reading frame of 1,104-base pairs, which encoded 367 amino acids to yield a protein of molecular mass 40.4 kDa. The amino acid sequence was 53% identical to that of an endoglucanase from Clostridium thermocellum. CelEdx16 was overexpressed in Escherichia coli and purified using Ni-NTA affinity chromatography. The specific endocellulase and exocellulase activities of CelEdx16 were 15.9 and 3.6 x 10⁻² U mg⁻¹, respectively. The Michaelis-Menten constant (K (m) values) and the maximal reaction velocities (V(max) values) of CelEdx16 were 47.1 μM and 9.6 x 10⁻³ μmole min⁻¹ when endocellulase activity was measured and 106.3 μM and 2.1 x 10⁻⁵ μmol min⁻¹ when exocellulase activity was assessed. CelEdx16 was optimally active at pH 5.0 and 40 °C.
Signal Amplification in Field Effect-Based Sandwich Enzyme-Linked Immunosensing by Tuned Buffer Concentration with Ionic Strength Adjuster.

PubMed

Kumar, Satyendra; Kumar, Narendra; Panda, Siddhartha

2016-04-01

Miniaturization of the sandwich enzyme-based immunosensor has several advantages but could result in lower signal strength due to lower enzyme loading. Hence, technologies for amplification of the signal are needed. Signal amplification in a field effect-based electrochemical immunosensor utilizing chip-based ELISA is presented in this work. First, the molarities of phosphate buffer saline (PBS) and concentrations of KCl as ionic strength adjuster were optimized to maximize the GOx glucose-based enzymatic reactions in a beaker for signal amplification measured by change in the voltage shift with an EIS device (using 20 μl of solution) and validated with a commercial pH meter (using 3 ml of solution). The PBS molarity of 100 μM with 25 mM KCl provided the maximum voltage shift. These optimized buffer conditions were further verified for GOx immobilized on silicon chips, and similar trends with decreased PBS molarity were obtained; however, the voltage shift values obtained on chip reaction were lower as compared to the reactions occurring in the beaker. The decreased voltage shift with immobilized enzyme on chip could be attributed to the increased Km (Michaelis-Menten constant) values in the immobilized GOx. Finally, a more than sixfold signal enhancement (from 8 to 47 mV) for the chip-based sandwich immunoassay was obtained by altering the PBS molarity from 10 to 100 μM with 25 mM KCl.
Concentration dependence of biotransformation in fish liver S9: Optimizing substrate concentrations to estimate hepatic clearance for bioaccumulation assessment.

PubMed

Lo, Justin C; Allard, Gayatri N; Otton, S Victoria; Campbell, David A; Gobas, Frank A P C

2015-12-01

In vitro bioassays to estimate biotransformation rate constants of contaminants in fish are currently being investigated to improve bioaccumulation assessments of hydrophobic contaminants. The present study investigates the relationship between chemical substrate concentration and in vitro biotransformation rate of 4 environmental contaminants (9-methylanthracene, pyrene, chrysene, and benzo[a]pyrene) in rainbow trout (Oncorhynchus mykiss) liver S9 fractions and methods to determine maximum first-order biotransformation rate constants. Substrate depletion experiments using a series of initial substrate concentrations showed that in vitro biotransformation rates exhibit strong concentration dependence, consistent with a Michaelis-Menten kinetic model. The results indicate that depletion rate constants measured at initial substrate concentrations of 1 μM (a current convention) could underestimate the in vitro biotransformation potential and may cause bioconcentration factors to be overestimated if in vitro biotransformation rates are used to assess bioconcentration factors in fish. Depletion rate constants measured using thin-film sorbent dosing experiments were not statistically different from the maximum depletion rate constants derived using a series of solvent delivery-based depletion experiments for 3 of the 4 test chemicals. Multiple solvent delivery-based depletion experiments at a range of initial concentrations are recommended for determining the concentration dependence of in vitro biotransformation rates in fish liver fractions, whereas a single sorbent phase dosing experiment may be able to provide reasonable approximations of maximum depletion rates of very hydrophobic substances. © 2015 SETAC.

Determining "small parameters" for quasi-steady state

NASA Astrophysics Data System (ADS)

Goeke, Alexandra; Walcher, Sebastian; Zerz, Eva

2015-08-01

For a parameter-dependent system of ordinary differential equations we present a systematic approach to the determination of parameter values near which singular perturbation scenarios (in the sense of Tikhonov and Fenichel) arise. We call these special values Tikhonov-Fenichel parameter values. The principal application we intend is to equations that describe chemical reactions, in the context of quasi-steady state (or partial equilibrium) settings. Such equations have rational (or even polynomial) right-hand side. We determine the structure of the set of Tikhonov-Fenichel parameter values as a semi-algebraic set, and present an algorithmic approach to their explicit determination, using Groebner bases. Examples and applications (which include the irreversible and reversible Michaelis-Menten systems) illustrate that the approach is rather easy to implement.
Reexamining Michaelis-Menten Enzyme Kinetics for Xanthine Oxidase

ERIC Educational Resources Information Center

Bassingthwaighte, James B.; Chinn, Tamara M.

2013-01-01

Abbreviated expressions for enzyme kinetic expressions, such as the Michaelis-Menten (M-M) equations, are based on the premise that enzyme concentrations are low compared with those of the substrate and product. When one does progress experiments, where the solute is consumed during conversion to form a series of products, the idealized conditions…
A Simple Classroom Teaching Technique to Help Students Understand Michaelis-Menten Kinetics

ERIC Educational Resources Information Center

Runge, Steven W.; Hill, Brent J. F.; Moran, William M.

2006-01-01

A new, simple classroom technique helps cell biology students understand principles of Michaelis-Menten enzyme kinetics. A student mimics the enzyme and the student's hand represents the enzyme's active site. The catalytic event is the transfer of marbles (substrate molecules) by hand from one plastic container to another. As predicted, increases…
New types of experimental data shape the use of enzyme kinetics for dynamic network modeling.

PubMed

Tummler, Katja; Lubitz, Timo; Schelker, Max; Klipp, Edda

2014-01-01

Since the publication of Leonor Michaelis and Maude Menten's paper on the reaction kinetics of the enzyme invertase in 1913, molecular biology has evolved tremendously. New measurement techniques allow in vivo characterization of the whole genome, proteome or transcriptome of cells, whereas the classical enzyme essay only allows determination of the two Michaelis-Menten parameters V and K(m). Nevertheless, Michaelis-Menten kinetics are still commonly used, not only in the in vitro context of enzyme characterization but also as a rate law for enzymatic reactions in larger biochemical reaction networks. In this review, we give an overview of the historical development of kinetic rate laws originating from Michaelis-Menten kinetics over the past 100 years. Furthermore, we briefly summarize the experimental techniques used for the characterization of enzymes, and discuss web resources that systematically store kinetic parameters and related information. Finally, describe the novel opportunities that arise from using these data in dynamic mathematical modeling. In this framework, traditional in vitro approaches may be combined with modern genome-scale measurements to foster thorough understanding of the underlying complex mechanisms. © 2013 FEBS.
Free energy landscape of the Michaelis complex of lactate dehydrogenase: A network analysis of atomistic simulations

NASA Astrophysics Data System (ADS)

Pan, Xiaoliang; Schwartz, Steven

2015-03-01

It has long been recognized that the structure of a protein is a hierarchy of conformations interconverting on multiple time scales. However, the conformational heterogeneity is rarely considered in the context of enzymatic catalysis in which the reactant is usually represented by a single conformation of the enzyme/substrate complex. Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate with concomitant interconversion of two forms of the cofactor nicotinamide adenine dinucleotide (NADH and NAD+). Recent experimental results suggest that multiple substates exist within the Michaelis complex of LDH, and they are catalytic competent at different reaction rates. In this study, millisecond-scale all-atom molecular dynamics simulations were performed on LDH to explore the free energy landscape of the Michaelis complex, and network analysis was used to characterize the distribution of the conformations. Our results provide a detailed view of the kinetic network the Michaelis complex and the structures of the substates at atomistic scale. It also shed some light on understanding the complete picture of the catalytic mechanism of LDH.
[Investigations on the physiology of the glands of carnivorous plants : IV. The kinetics of chloride secretion by the gland tissue of Nepenthes].

PubMed

Lüttge, U

1966-03-01

The transport of chloride in isolated tissue from Nepenthes pitchers was investigated using (36)Cl(-), an Aminco-Cotlove chloride-titrator for the determinations of Cl(-) concentrations, and KCN and AsO 4 (-) -as metabolic inhibitors.The tissue was brought in contact with different experimental solutions (=medium). The surface corresponding to the outside of the pitchers was cut with a razor blade to remove the cutinized epidermal layer. At this surface the Cl(-) uptake from the medium is a metabolic process which depends on the Cl(-)-concentration of the medium in a manner that corresponds to the MICHAELIS-MENTEN kinetics. The Michaelis-constant of this transport step was 3×10(-2)M. The Cl(-)-efflux into the medium, however, is a passive process.The opposite surface of the tissue slices (corresponding to the inside of the pitchers) carries the glands. The chloride secretion taking place here is also dependent on metabolism. In vitro it occurs even when a high gradient of chloride concentration has been set up between the medium and the solution which is in contact with the glands. In vivo the Cl(-)-concentration of the pitcher fluid and the amount of Cl(-) per gram of tissue water are almost equal.The rôle of chloride in the physiology of Nepenthes is still under investigation, A correlation between the chloride content of the pitcher fluid and its enzymatic activity (Casein-test), however, could already be demonstrated.
Preparation of poly(ethylene glycol) hydrogels with different network structures for the application of enzyme immobilization.

PubMed

Choi, Dongkil; Lee, Woojin; Park, Jinwon; Koh, Wongun

2008-01-01

In this study, poly(ethylene glycol) (PEG)-based hydrogels having different network structures were synthesized by UV-initiated photopolymerization and used for the enzyme immobilization. PEGs with different molecular weight were acrylated by derivatizing both ends with acryloyl chloride and photopolymerization of PEG-diacrylate (PEG-DA) yielded crosslinked hydrogel network within 5 seconds. Attachment of acrylate groups and gelation were confirmed by ATR/FT-IR and FT-Raman spectroscopy. Network structures of hydrogels could be easily controlled by changing the molecular weight (MW) of PEG-DA and characterized by calculating molecular weight between crosslinks and mesh size from the swelling measurement. Synthesis of hydrogels with higher MW of PEG produced less crosslinked hydrogels having higher water content, larger value of Mc and mesh size, which resulted in enhanced mass transfer but loss of mechanical properties. For the enzyme immobilization, glucose oxidase (GOX) was immobilized inside PEG hydrogels by means of physical entrapment and covalent immobilization. Encapsulated GOX were covalently bound to PEG backbone using acryloyl-PEG-N-hydroxysuccinimide and maintained their activity over a week period without leakage. Kinetic study indicated that immobilized enzyme inside hydrogel prepared from higher MW of PEG possessed lower apparent Km (Michaelis-Menten constant) and higher activity.
Modular rate laws for enzymatic reactions: thermodynamics, elasticities and implementation.

PubMed

Liebermeister, Wolfram; Uhlendorf, Jannis; Klipp, Edda

2010-06-15

Standard rate laws are a key requisite for systematically turning metabolic networks into kinetic models. They should provide simple, general and biochemically plausible formulae for reaction velocities and reaction elasticities. At the same time, they need to respect thermodynamic relations between the kinetic constants and the metabolic fluxes and concentrations. We present a family of reversible rate laws for reactions with arbitrary stoichiometries and various types of regulation, including mass-action, Michaelis-Menten and uni-uni reversible Hill kinetics as special cases. With a thermodynamically safe parameterization of these rate laws, parameter sets obtained by model fitting, sampling or optimization are guaranteed to lead to consistent chemical equilibrium states. A reformulation using saturation values yields simple formulae for rates and elasticities, which can be easily adjusted to the given stationary flux distributions. Furthermore, this formulation highlights the role of chemical potential differences as thermodynamic driving forces. We compare the modular rate laws to the thermodynamic-kinetic modelling formalism and discuss a simplified rate law in which the reaction rate directly depends on the reaction affinity. For automatic handling of modular rate laws, we propose a standard syntax and semantic annotations for the Systems Biology Markup Language. An online tool for inserting the rate laws into SBML models is freely available at www.semanticsbml.org. Supplementary data are available at Bioinformatics online.
Dual enzyme activities assay by quantitative electrospray ionization quadrupole-time-of-flight mass spectrometry.

PubMed

Cai, Tingting; Zhang, Li; Wang, Haoyang; Zhang, Jing; Wang, Rong; Zhang, Yurong; Guo, Yinlong

2012-01-01

A practical and rapid method based on electrospray ionization quadrupole-time of flight mass spectrometry (ESI-Q-ToF MS) was developed for detecting activities of both acetylcholinesterase IAChEI and glutathione S-transferase (GST). The simultaneous study of these two enzyme activities is significant for studying human bio-functions, especially for those who take in toxic compounds and have a risk of disease. Here, the enzyme activities were represented by the conversion of enzymatic substrates and determined by quantitatively analyzing enzymatic substrates. Different internal standards were used to quantify each enzymatic substrate and the good linearity of calibration curves demonstrated the feasibility of the internal standards. The Michaelis-Menten constants (Km) of both GST and AChE were measured by this method and were consistent with values previously reported. Furthermore, we applied this approach to detect GST and AChE activities of whole bloods from four deceased and healthy people. The variation in enzyme activity was in accord with information from gas chromatography mass spectrometry [GC/MS). The screening of AChE and GST provided reliable results and strong forensic evidence. This method offers an alternative choice for detecting enzyme activities and is anticipated to have wide applications in pharmaceutical research and prevention in toxic compounds.
Kinetics of Papain: An Introductory Biochemistry Laboratory Experiment

NASA Astrophysics Data System (ADS)

Cornely, Kathleen; Crespo, Eric; Earley, Michael; Kloter, Rachel; Levesque, Aime; Pickering, Mary

1999-05-01

Enzyme kinetics experiments are popular in the undergraduate laboratory. These experiments have pedagogic value because they reinforce the concepts of Michaelis-Menten kinetics covered in the lecture portion of the course and give students the experience of calculating kinetic constants from data they themselves have generated. In this experiment, we investigate the kinetics of the thiol protease papain. The source of the papain is commercially available papaya latex. A specific substrate, Na-benzoyl-arginine-p-nitroanilide (BAPNA), is used, which takes advantage of the fact that papain interacts with a phenylalanine residue two amino acids away from the peptide bond cleaved. Upon hydrolysis by papain, a bright yellow product is released, p-nitroaniline. This allows the reaction to be monitored spectrophotometrically by measuring the rate of formation of the p-nitroaniline product as a function of the increase in absorbance of the solution at the lmax of p-nitroaniline (400 nm) over time at various substrate concentrations. These data are used to plot a Lineweaver-Burk plot from which the vmax and KM are obtained. If time permits, students carry out additional investigations in which e of p-nitroaniline is measured, the enzyme solution protein concentration is measured, the enzyme purity is evaluated by SDS-PAGE, and a pH-rate profile is constructed from experimental data.
In situ monitoring of myenteric neuron activity using acetylcholinesterase-modified AlGaN/GaN solution-gate field-effect transistors.

PubMed

Müntze, Gesche Mareike; Pouokam, Ervice; Steidle, Julia; Schäfer, Wladimir; Sasse, Alexander; Röth, Kai; Diener, Martin; Eickhoff, Martin

2016-03-15

The response characteristics of acetylcholinesterase-modified AlGaN/GaN solution-gate field-effect transistors (AcFETs) are quantitatively analyzed by means of a kinetic model. The characterization shows that the covalent enzyme immobilization process yields reproducible AcFET characteristics with a Michaelis constant KM of (122 ± 4) μM for the immobilized enzyme layer. The increase of KM by a factor of 2.4 during the first four measurement cycles is attributed to partial denaturation of the enzyme. The AcFETs were used to record the release of acetylcholine (ACh) by neuronal tissue cultivated on the gate area upon stimulation by rising the extracellular K(+) concentration. The neuronal tissue constituted of isolated myenteric neurons from four to 12 days old Wistar rats, or sections from the muscularis propria containing the myenteric plexus from adult rats. For both cases the AcFET response was demonstrated to be related to the activity of the immobilized acetylcholinesterase using the reversible acetylcholinesterase blocker donepezil. A concentration response curve of this blocking agent revealed a half maximal inhibitory concentration of 40 nM which is comparable to values measured by complementary in vitro methods. Copyright © 2015 Elsevier B.V. All rights reserved.
Competition between isoprene emission and pigment synthesis during leaf development in aspen

PubMed Central

Rasulov, Bahtijor; Bichele, Irina; Laisk, Agu; Niinemets, Ülo

2014-01-01

In growing leaves, lack of isoprene synthase is considered responsible for delayed isoprene emission, but competition for dimethylallyl diphosphate (DMADP), the substrate for both isoprene synthesis and prenyltransferase reactions in photosynthetic pigment and phytohormone synthesis, can also play a role. We used a kinetic approach based on postillumination isoprene decay and modeling DMADP consumption to estimate in vivo kinetic characteristics of isoprene synthase and prenyltransferase reactions, and determine the share of DMADP use by different processes through leaf development in Populus tremula. Pigment synthesis rate was also estimated from pigment accumulation data, and distribution of DMADP use from isoprene emission changes due to alendronate, a selective inhibitor of prenyltransferases. Development of photosynthetic activity and pigment synthesis occurred with the greatest rate in 1-5 days old leaves when isoprene emission was absent. Isoprene emission commenced on days 5-6 and increased simultaneously with slowing down of pigment synthesis. In vivo Michaelis-Menten constant (Km) values obtained were 265 nmol m−2 (20 μM) for DMADP-consuming prenyltransferase reactions and 2560 nmol m−2 (190 μM) for isoprene synthase. Thus, despite decelerating pigment synthesis reactions in maturing leaves, isoprene emission in young leaves was limited by both isoprene synthase activity and competition for DMADP by prenyltransferase reactions. PMID:24033429
Purification and studies on characteristics of cholinesterases from Daphnia magna *

PubMed Central

Yang, Yan-xia; Niu, Li-zhi; Li, Shao-nan

2013-01-01

Due to their significant value in both economy and ecology, Daphnia had long been employed to investigate in vivo response of cholinesterase (ChE) in anticholinesterase exposures, whereas the type constitution and property of the enzyme remained unclear. A type of ChE was purified from Daphnia magna using a three-step procedure, i.e., Triton X-100 extraction, ammonium sulfate precipitation, and diethylaminoethyl (DEAE)-Sepharose™-Fast-Flow chromatography. According to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), molecular mass of the purified ChE was estimated to be 84 kDa. Based on substrate studies, the purified enzyme preferred butyrylthiocholine iodide (BTCh) [with maximum velocity (V max)/Michaelis constant (K m)=8.428 L/(min·mg protein)] to acetylthiocholine iodide (ATCh) [with V max/K m=5.346 L/(min·mg protein)] as its substrate. Activity of the purified enzyme was suppressed by high concentrations of either ATCh or BTCh. Inhibitor studies showed that the purified enzyme was more sensitive towards inhibition by tetraisopropylpyrophosphoramide (iso-OMPA) than by 1,5-bis(4-allyldimethylammoniumphenyl) pentan-3-one dibromide (BW284C51). Result of the study suggested that the purified ChE was more like a type of pseudocholinesterase, and it also suggested that Daphnia magna contained multiple types of ChE in their bodies. PMID:23549850
Methylmercury-cholinesterase interactions in rats.

PubMed Central

Hastings, F L; Lucier, G W; Klein, R

1975-01-01

The interaction of methylmercury hydroxide (MMH) and cholinesterases was studied in male and female rats. MMH administered subcutaneously in doses of 10 mg/kg for 2 days reduced the level of plasma cholinesterase (ButChE) by 68% in females and 47% in males while brain acetylcholinesterase (AChE) was unaffected. Normal females had higher but more variable ButChE levels than normal males. In a time-course experiment, a single dose of MMH (10 mg/kg) reduced ButChE levels when mercury levels reached 22 mug/ml in the blood. A 10% reduction in brain AChE was observed at 72 hours; however, mercury reached a concentration of only 2.0 mug/g in brain tissue. The determination of the Michaelis constant Km and maximum velocity value Vmax for butyrylcholine and ButChE in control and MMH-treated (1 mg/kg) animals indicated that MMH reduced Vmax only. Since no loss in ButChE activity occurred when MMH and control plasma were incubated in vitro, MMH is not a direct inhibitor of ButChE. Because only the inactive monomeric form of ButChE contains free sulfhydryl groups, it is postulated that MMH combines covalently with the sulfur, preventing formation of active enzyme. By analogy, it is believed this is also the case with AChE. PMID:1227853
Impact of capillary flow hydrodynamics on carrier-mediated transport of opioid derivatives at the blood-brain barrier, based on pH-dependent Michaelis-Menten and Crone-Renkin analyses.

PubMed

Yusof, Siti R; Abbott, N Joan; Avdeef, Alex

2017-08-30

Most studies of blood-brain barrier (BBB) permeability and transport are conducted at a single pH, but more detailed information can be revealed by using multiple pH values. A pH-dependent biophysical model was applied to the mechanistic analysis of published pH-dependent BBB luminal uptake data from three opioid derivatives in rat: pentazocine (Suzuki et al., 2002a, 2002b), naloxone (Suzuki et al., 2010a), and oxycodone (Okura et al., 2008). Two types of data were processed: in situ brain perfusion (ISBP) and brain uptake index (BUI). The published perfusion data were converted to apparent luminal permeability values, P app , and analyzed by the pCEL-X program (Yusof et al., 2014), using the pH-dependent Crone-Renkin equation (pH-CRE) to determine the impact of cerebrovascular flow on the Michaelis-Menten transport parameters (Avdeef and Sun, 2011). For oxycodone, the ISBP data had been measured at pH7.4 and 8.4. The present analysis indicates a 7-fold lower value of the cerebrovascular flow velocity, F pf , than that expected in the original study. From the pyrilamine-inhibited data, the flow-corrected passive intrinsic permeability value was determined to be P 0 =398×10 -6 cm·s -1 . The uptake data indicate that the neutral form of oxycodone is affected by a transporter at pH8.4. The extent of the cation uptake was less certain from the available data. For pentazocine, the brain uptake by the BUI method had been measured at pH5.5, 6.5, and 7.4, in a concentration range 0.1-40mM. Under similar conditions, ISBP data were also available. The pH-CRE determined values of F pf from both methods were nearly the same, and were smaller than the expected value in the original publication. The transport of the cationic pentazocine was not fully saturated at pH5.5 at 40mM. The transport of the neutral species at pH7.4 appeared to reach saturation at 40mM pentazocine concentration, but not at 12mM. In the case of naloxone, a pH-dependent Michaelis-Menten equation (pH-MME) analysis of the data indicated a smooth sigmoidal transition from a higher capacity uptake process affecting cationic naloxone (pH5.0-7.0) to a lower capacity uptake process affecting the neutral drug (pH8.0-8.5), with cross-over point near pH7.4. Evidently, measurements at multiple pH values can reveal important information about both cerebrovascular flow and BBB transport kinetics. Copyright © 2017 Elsevier B.V. All rights reserved.
Analysis of the activation of acetylcholinesterase by carbon nanoparticles using a monolithic immobilized enzyme microreactor: role of the water molecules in the active site gorge.

PubMed

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

2013-10-01

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

PubMed

Millington, W R; McCall, A L; Wurtman, R J

1978-10-01

Competition by deanol (dimethylaminoethanol) with choline for uptake from the bloodstream into the brain was demonstrated by simultaneous intracarotid administration of carbon 14-labeled choline with deanol (plus tritiated water and indium 113m, to calculate a brain uptake index) and by measuring the brain uptake of 14C-labeled choline mixed with sera from rats pretreated with deanol (300 or 500 mg/kg 8 or 30 minutes earlier). The inhibition constant for inhibition of choline uptake by deanol (159 micrograms) was actually lower than the Michaelis constant for choline itself (442 micrograms); hence, the affinity of the carrier mechanism for deanol is at least as great as it is for choline. Deanol administration also elevated blood choline levels; thus, the effect of the drug on brain choline (and acetylcholine) levels is the result of the increase it produces in blood choline and the suppression it causes in choline uptake. These findings may explain discrepant results from laboratories seeking increases in brain acetylcholine or clinical improvement in patients with tardive dyskinesia after deanol treatment.
DOE Office of Scientific and Technical Information (OSTI.GOV)

Adi, Y. A., E-mail: yudi.adi@math.uad.ac.id; Department of Mathematic Faculty of MIPA Universitas Gadjah Mada; Kusumo, F. A.

In this paper we consider a mathematical model of PI3K/AKT signaling pathways in phosphorylation AKT. PI3K/AKT pathway is an important mediator of cytokine signaling implicated in regulation of hematopoiesis. Constitutive activation of PI3K/AKT signaling pathway has been observed in Acute Meyloid Leukemia (AML) it caused by the mutation of Fms-like Tyrosine Kinase 3 in internal tandem duplication (FLT3-ITD), the most common molecular abnormality associated with AML. Depending upon its phosphorylation status, protein interaction, substrate availability, and localization, AKT can phosphorylate or inhibite numerous substrates in its downstream pathways that promote protein synthesis, survival, proliferation, and metabolism. Firstly, we present amore » mass action ordinary differential equation model describing AKT double phosphorylation (AKTpp) in a system with 11 equations. Finally, under the asumtion enzyme catalyst constant and steady state equilibrium, we reduce the system in 4 equation included Michaelis Menten constant. Simulation result suggested that a high concentration of PI3K and/or a low concentration of phospatase increased AKTpp activation. This result also indicates that PI3K is a potential target theraphy in AML.« less
A mathematical model of phosphorylation AKT in Acute Myeloid Leukemia

NASA Astrophysics Data System (ADS)

Adi, Y. A.; Kusumo, F. A.; Aryati, L.; Hardianti, M. S.

2016-04-01

In this paper we consider a mathematical model of PI3K/AKT signaling pathways in phosphorylation AKT. PI3K/AKT pathway is an important mediator of cytokine signaling implicated in regulation of hematopoiesis. Constitutive activation of PI3K/AKT signaling pathway has been observed in Acute Meyloid Leukemia (AML) it caused by the mutation of Fms-like Tyrosine Kinase 3 in internal tandem duplication (FLT3-ITD), the most common molecular abnormality associated with AML. Depending upon its phosphorylation status, protein interaction, substrate availability, and localization, AKT can phosphorylate or inhibite numerous substrates in its downstream pathways that promote protein synthesis, survival, proliferation, and metabolism. Firstly, we present a mass action ordinary differential equation model describing AKT double phosphorylation (AKTpp) in a system with 11 equations. Finally, under the asumtion enzyme catalyst constant and steady state equilibrium, we reduce the system in 4 equation included Michaelis Menten constant. Simulation result suggested that a high concentration of PI3K and/or a low concentration of phospatase increased AKTpp activation. This result also indicates that PI3K is a potential target theraphy in AML.
Lipase from liver of seabass (Lates calcarifer): Characteristics and the use for defatting of fish skin.

PubMed

Sae-Leaw, Thanasak; Benjakul, Soottawat

2018-02-01

Lipase from liver of seabass (Lates calcarifer), with a molecular weight of 60kDa, was purified to homogeneity using ammonium sulfate precipitation and a series of chromatographies, including diethylaminoethyl sepharose (DEAE) and Sephadex G-75 size exclusion columns. The optimal pH and temperature were 8.0 and 50°C, respectively. Purified lipase had Michaelis-Menten constant (K m ) and catalytic constant (k cat ) of 0.30mM and 2.16s -1 , respectively, when p-nitrophenyl palmitate (p-NPP) was used as the substrate. When seabass skin was treated with crude lipase from seabass liver at various levels (0.15 and 0.30units/g dry skin) for 1-3h at 30°C, the skin treated with lipase at 0.30 units/g dry skin for 3h had the highest lipid removal (84.57%) with lower lipid distribution in skin. Efficacy in defatting was higher than when isopropanol was used. Thus, lipase from liver of seabass could be used to remove fat in fish skin. Copyright © 2017 Elsevier Ltd. All rights reserved.

Purification and Characterization of the Crown Gall-specific Enzyme, Octopine Synthase 1

PubMed Central

Hack, Ethan; Kemp, John D.

1980-01-01

A single enzyme catalyzes the synthesis of all four N2-(1-carboxyethyl)-amino acid derivatives found in a crown gall tumor tissue induced by Agrobacterium tumefaciens (E. F. Sm. and Town.) Conn strain B6 on sunflower (Helianthus annuus L.). This enzyme, octopine synthase, has been purified by ammonium sulfate fractionation and chromatography on diethylaminoethylcellulose, blue agarose, and hydroxylapatite. The purified enzyme has all the N2-(1-carboxyethyl)-amino acid synthesizing activities found in crude preparations, and the relative activities with six amino acids remain nearly constant during purification. Although the maximum velocities (V) and Michaelis constants (Km) differ, the ratio V/Km is the same for all amino acid substrates. Thus an equimolar mixture of amino acids will give rise to an equimolar mixture of products. The kinetic properties of the enzyme are consistent with a partially ordered mechanism with arginine (NADPH, then arginine or pyruvate). Octopine synthase is a monomeric enzyme with a molecular weight of 39,000 by gel filtration and 38,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Images PMID:16661312
In vitro characterization of the inhibitory effects of ketoconazole on metabolic activities of cytochrome P-450 in canine hepatic microsomes.

PubMed

Kuroha, Masanori; Kuze, Yoji; Shimoda, Minoru; Kokue, Eiichi

2002-06-01

To evaluate the inhibitory potency of ketoconazole (KTZ) on the metabolic activities of isozymes of cytochrome P-450 (CYP) in dogs. 4 healthy 1-year-old male Beagles. Hepatic microsomes were harvested from 4 dogs after euthanasia. To investigate the effects of KTZ on CYP metabolic activities, 7-ethoxyresorufin, tolbutamide, bufuralol, and midazolam hydrochloride were used as specific substrates for CYP1A1/2, CYP2C21, CYP2D15, and CYP3A12, respectively. The concentrations of metabolites formed by CYP were measured by high-performance liquid chromatography, except for the resorufin concentrations that were measured by a fluorometric method. The reaction velocity-substrate concentration data were analyzed to obtain kinetic variables, including maximum reaction velocity, Michaelis-Menten constant, and inhibitory constant (Ki). KTZ competitively inhibited 7-ethoxyresorufin O-deethylation and midazolam 4-hydroxylation; it noncompetitively inhibited tolbutamide methylhydroxylation. Bufuralol 1'-hydroxylation was inhibited slightly by KTZ. The mean Ki values of KTZ were 10.6+/-6.0, 170+/-2.5, and 0.180+/-0.131 microM for 7-ethoxyresorufin O-deethylation, tolbutamide methylhydroxylation, and midazolam 4-hydroxylation, respectively. In dogs, KTZ at a therapeutic dose may change the pharmacokinetics of CYP3A12 substrates as a result of inhibition of their biotransformation. Furthermore, no influence of KTZ on the pharmacokinetics of CYP1A1/2, CYP2C21, and CYP2D15 substrates are likely. In clinical practice, adverse drug effects may develop when KTZ is administered concomitantly with a drug that is primarily metabolized by CYP3A12.
Effect of Exogenous and Endogenous Nitrate Concentration on Nitrate Utilization by Dwarf Bean 1

PubMed Central

Breteler, Hans; Nissen, Per

1982-01-01

The effect of the exogenous and endogenous NO3− concentration on net uptake, influx, and efflux of NO3− and on nitrate reductase activity (NRA) in roots was studied in Phaseolus vulgaris L. cv. Witte Krombek. After exposure to NO3−, an apparent induction period of about 6 hours occurred regardless of the exogenous NO3− level. A double reciprocal plot of the net uptake rate of induced plants versus exogenous NO3− concentration yielded four distinct phases, each with simple Michaelis-Menten kinetics, and separated by sharp breaks at about 45, 80, and 480 micromoles per cubic decimeter. Influx was estimated as the accumulation of 15N after 1 hour exposure to 15NO3−. The isotherms for influx and net uptake were similar and corresponded to those for alkali cations and Cl−. Efflux of NO3− was a constant proportion of net uptake during initial NO3− supply and increased with exogenous NO3− concentration. No efflux occurred to a NO3−-free medium. The net uptake rate was negatively correlated with the NO3− content of roots. Nitrate efflux, but not influx, was influenced by endogenous NO3−. Variations between experiments, e.g. in NO3− status, affected the values of Km and Vmax in the various concentration phases. The concentrations at which phase transitions occurred, however, were constant both for influx and net uptake. The findings corroborate the contention that separate sites are responsible for uptake and transitions between phases. Beyond 100 micromoles per cubic decimeter, root NRA was not affected by exogenous NO3− indicating that NO3− uptake was not coupled to root NRA, at least not at high concentrations. PMID:16662570
Inhibition of Fosfomycin Resistance Protein FosA by Phosphonoformate (Foscarnet) in Multidrug-Resistant Gram-Negative Pathogens

PubMed Central

Ito, Ryota; Tomich, Adam D.; McElheny, Christi L.; Mettus, Roberta T.; Sluis-Cremer, Nicolas

2017-01-01

ABSTRACT FosA proteins confer fosfomycin resistance to Gram-negative pathogens via glutathione-mediated modification of the antibiotic. In this study, we assessed whether inhibition of FosA by sodium phosphonoformate (PPF) (foscarnet), a clinically approved antiviral agent, would reverse fosfomycin resistance in representative Gram-negative pathogens. The inhibitory activity of PPF against purified recombinant FosA from Escherichia coli (FosA3), Klebsiella pneumoniae (FosAKP), Enterobacter cloacae (FosAEC), and Pseudomonas aeruginosa (FosAPA) was determined by steady-state kinetic measurements. The antibacterial activity of PPF against FosA in clinical strains of these species was evaluated by susceptibility testing and time-kill assays. PPF increased the Michaelis constant (Km) for fosfomycin in a dose-dependent manner, without affecting the maximum rate (Vmax) of the reaction, for all four FosA enzymes tested, indicating a competitive mechanism of inhibition. Inhibitory constant (Ki) values were 22.6, 35.8, 24.4, and 56.3 μM for FosAKP, FosAEC, FosAPA, and FosA3, respectively. Addition of clinically achievable concentrations of PPF (∼667 μM) reduced the fosfomycin MICs by ≥4-fold among 52% of the K. pneumoniae, E. cloacae, and P. aeruginosa clinical strains tested and led to a bacteriostatic or bactericidal effect in time-kill assays among representative strains. PPF inhibits FosA activity across Gram-negative species and can potentiate fosfomycin activity against the majority of strains with chromosomally encoded fosA. These data suggest that PPF may be repurposed as an adjuvant for fosfomycin to treat infections caused by some FosA-producing, multidrug-resistant, Gram-negative pathogens. PMID:28993329
Enzymatic and biochemical properties of a novel human serine dehydratase isoform.

PubMed

Ogawa, Hirofumi; Gomi, Tomoharu; Nishizawa, Mikio; Hayakawa, Yumiko; Endo, Shunro; Hayashi, Kyoko; Ochiai, Hiroshi; Takusagawa, Fusao; Pitot, Henry C; Mori, Hisashi; Sakurai, Hiroaki; Koizumi, Keiichi; Saiki, Ikuo; Oda, Hirofumi; Fujishita, Takashi; Miwa, Toshiro; Maruyama, Muneharu; Kobayashi, Masashi

2006-05-01

A cDNA clone similar to human serine dehydratase (SDH) is deposited in the GenBank/EMBL databases, but its structural and functional bases remain unknown. Despite the occurrence of mRNA, the expected protein level was found to be low in cultured cells. To learn about physicochemical properties of the protein, we expressed the cDNA in Escherichia coli, and compared the expressed protein with that of a hepatic SDH. The purified protein showed l-serine and l-threonine dehydratase activity, demonstrating to be an isoform of SDH. However, their Km and Vmax constants were different in a range of two-order. Removal of Pro128 from the hepatic SDH consisting of 328 residues, which is missing in the corresponding position of the isoform consisting of 329 residues, significantly changed the Michaelis constants and Kd value for pyridoxal 5'-phosphate, whereas addition of a proline residue to the isoform was without effect. These findings suggest the difference in the structures of the active sites of the two enzymes. Another striking feature was that the expressed level of the isoform in E. coli was 7-fold lower than that of the hepatic SDH. Substitution of Val for Leu287 in the isoform dramatically increased the protein level. The high yield of the mutated isoform was also confirmed by the in vitro transcription and translation experiment. The poor expression of the isoform could be explained by the more stable secondary structure of the mRNA than that of the hepatic SDH mRNA. The present findings may provide a clue as to why the protein level in cultured cells is low.
Simultaneous measurement of glucose transport and utilization in the human brain.

PubMed

Shestov, Alexander A; Emir, Uzay E; Kumar, Anjali; Henry, Pierre-Gilles; Seaquist, Elizabeth R; Öz, Gülin

2011-11-01

Glucose is the primary fuel for brain function, and determining the kinetics of cerebral glucose transport and utilization is critical for quantifying cerebral energy metabolism. The kinetic parameters of cerebral glucose transport, K(M)(t) and V(max)(t), in humans have so far been obtained by measuring steady-state brain glucose levels by proton ((1)H) NMR as a function of plasma glucose levels and fitting steady-state models to these data. Extraction of the kinetic parameters for cerebral glucose transport necessitated assuming a constant cerebral metabolic rate of glucose (CMR(glc)) obtained from other tracer studies, such as (13)C NMR. Here we present new methodology to simultaneously obtain kinetic parameters for glucose transport and utilization in the human brain by fitting both dynamic and steady-state (1)H NMR data with a reversible, non-steady-state Michaelis-Menten model. Dynamic data were obtained by measuring brain and plasma glucose time courses during glucose infusions to raise and maintain plasma concentration at ∼17 mmol/l for ∼2 h in five healthy volunteers. Steady-state brain vs. plasma glucose concentrations were taken from literature and the steady-state portions of data from the five volunteers. In addition to providing simultaneous measurements of glucose transport and utilization and obviating assumptions for constant CMR(glc), this methodology does not necessitate infusions of expensive or radioactive tracers. Using this new methodology, we found that the maximum transport capacity for glucose through the blood-brain barrier was nearly twofold higher than maximum cerebral glucose utilization. The glucose transport and utilization parameters were consistent with previously published values for human brain.
Intracellular pH recovery from alkalinization. Characterization of chloride and bicarbonate transport by the anion exchange system of human neutrophils

PubMed Central

1990-01-01

The nature of the intracellular pH-regulatory mechanism after imposition of an alkaline load was investigated in isolated human peripheral blood neutrophils. Cells were alkalinized by removal of a DMO prepulse. The major part of the recovery could be ascribed to a Cl- /HCO3- counter-transport system: specifically, a one-for-one exchange of external Cl- for internal HCO3-. This exchange mechanism was sensitive to competitive inhibition by the cinnamate derivative UK-5099 (Ki approximately 1 microM). The half-saturation constants for binding of HCO3- and Cl- to the external translocation site of the carrier were approximately 2.5 and approximately 5.0 mM. In addition, other halides and lyotropic anions could substitute for external Cl-. These ions interacted with the exchanger in a sequence of decreasing affinities: HCO3- greater than Cl approximately NO3- approximately Br greater than I- approximately SCN- greater than PAH-. Glucuronate and SO4(2-) lacked any appreciable affinity. This rank order is reminiscent of the selectivity sequence for the principal anion exchanger in resting cells. Cl- and HCO3- displayed competition kinetics at both the internal and external binding sites of the carrier. Finally, evidence compatible with the existence of an approximately fourfold asymmetry (Michaelis constants inside greater than outside) between inward- and outward-facing states is presented. These results imply that a Cl-/HCO3- exchange mechanism, which displays several properties in common with the classical inorganic anion exchanger of erythrocytes, is primarily responsible for restoring the pHi of human neutrophils to its normal resting value after alkalinization. PMID:2280252
Deltamethrin is metabolized by CYP6FU1, a cytochrome P450 associated with pyrethroid resistance, in Laodelphax striatellus.

PubMed

Elzaki, Mohammed Esmail Abdalla; Miah, Mohammad Asaduzzaman; Peng, Yingchuan; Zhang, Haomiao; Jiang, Ling; Wu, Min; Han, Zhaojun

2018-06-01

Cytochrome P450s (CYPs) are known to play a major role in metabolizing a wide range compounds. CYP6FU1 has been found to be over-expressed in a deltamethrin-resistant strain of Laodelphax striatellus. This study was conducted to express CYP6FU1 in Sf9 cells as a recombinant protein, to confirm its ability to degrade deltamethrin, chlorpyrifos, imidacloprid and traditional P450 probing substrates. Carbon monoxide difference spectrum analysis indicated that the intact CYP6FU1 protein was expressed in insect Sf9 cells. Catalytic activity tests with four traditional P450 probing substrates revealed that the expressed CYP6FU1 preferentially metabolized p-nitroanisole and ethoxyresorufin, but not ethoxycoumarin and luciferin-HEGE. The enzyme kinetic parameters were tested using p-nitroanisole. The michaelis constant (K m ) and catalytic constant (K cat ) values were 17.51 ± 4.29 µm and 0.218 ± 0.001 pmol min -1 mg -1 protein, respectively. Furthermore, CYP6FU1 activity for degradation of insecticides was tested by measuring substrate depletion and metabolite formation. The chromatogram analysis showed obvious nicotinamide-adenine dinucleotide phosphate (NADPH)-dependent depletion of deltamethrin, and formation of the unknown metabolite. Mass spectra and the molecular docking model showed that the metabolite was 4-hydroxy-deltamethrin. However, the recombinant CYP6FU1 could not metabolize imidacloprid and chlorpyrifos. These results confirmed that the over-expressed CYP6FU1 contributes to deltamethrin resistance in L. striatellus, and p-nitroanisole might be a potential diagnostic probe for deltamethrin metabolic resistance detection and monitoring. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.
Determination of Unbound Partition Coefficient and in Vitro-in Vivo Extrapolation for SLC13A Transporter-Mediated Uptake.

PubMed

Riccardi, Keith; Li, Zhenhong; Brown, Janice A; Gorgoglione, Matthew F; Niosi, Mark; Gosset, James; Huard, Kim; Erion, Derek M; Di, Li

2016-10-01

Unbound partition coefficient (Kpuu) is important to an understanding of the asymmetric free drug distribution of a compound between cells and medium in vitro, as well as between tissue and plasma in vivo, especially for transporter-mediated processes. Kpuu was determined for a set of compounds from the SLC13A family that are inhibitors and substrates of transporters in hepatocytes and transporter-transfected cell lines. Enantioselectivity was observed, with (R)-enantiomers achieving much higher Kpuu (>4) than the (S)-enantiomers (<1) in human hepatocytes and SLC13A5-transfected human embryonic 293 cells. The intracellular free drug concentration correlated directly with in vitro pharmacological activity rather than the nominal concentration in the assay because of the high Kpuu mediated by SLC13A5 transporter uptake. Delivery of the diacid PF-06649298 directly or via hydrolysis of the ethyl ester prodrug PF-06757303 resulted in quite different Kpuu values in human hepatocytes (Kpuu of 3 for diacid versus 59 for prodrug), which was successfully modeled on the basis of passive diffusion, active uptake, and conversion rate from ester to diacid using a compartmental model. Kpuu values changed with drug concentrations; lower values were observed at higher concentrations possibly owing to a saturation of transporters. Michaelis-Menten constant (Km) of SLC13A5 was estimated to be 24 μM for PF-06649298 in human hepatocytes. In vitro Kpuu obtained from rat suspension hepatocytes supplemented with 4% fatty acid free bovine serum albumin showed good correlation with in vivo Kpuu of liver-to-plasma, illustrating the potential of this approach to predict in vivo Kpuu from in vitro systems. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.
Luminal glucose concentrations in the gut under normal conditions.

PubMed

Ferraris, R P; Yasharpour, S; Lloyd, K C; Mirzayan, R; Diamond, J M

1990-11-01

Luminal glucose (Glc) concentrations in the small intestine (SI) are widely assumed to be 50-500 mM. These values have posed problems for interpreting SI luminal osmolality and absorptive capacity, Glc transporter Michaelis-Menten constants (Km), and the physiological role of active Glc transport and its regulation. Hence we measured luminal contents, osmolality, and Glc, Na+, and K+ concentrations in normally feeding rats, rabbits, and dogs. Measured Glc concentrations were compatible with the portion of measured osmolality not accounted for by Na+ and K+ salts, amino acids, and peptides. Mean SI luminal osmolalities were less than or equal to 100 mosmol/kg hypertonic. For animals on the most nearly physiological diets, SI Glc concentrations averaged 0.4-24 mM and ranged with time and SI region from 0.2 to a maximum of 48 mM. The older published very high values are artifacts of direct infusion of concentrated Glc solutions into the gut, nonspecific Glc assays, and failure to test for quantitative recovery or to centrifuge samples in the cold. By storing food after meals and releasing it between meals, rat stomach greatly damps diurnal fluctuations in quantity and osmolality of food reaching the SI and hence also damps fluctuations in absorption rates. These new values for luminal Glc have five important physiological implications: the problem of accounting for apparently very hypertonic SI contents in the face of high osmotic water permeability disappears; the effective Km of the SI Glc transporter is now comparable to prevailing Glc concentrations; the SI no longer appears to have enormous excess absorptive capacity for Glc; regulation of Glc transport by dietary intake now makes functional sense; and the claim that high luminal Glc concentrations permit solvent drag to become the major mode of Glc absorption under normal conditions is undermined.
Response to a temperature modulation as a signature of chemical mechanisms.

PubMed

Berthoumieux, H; Jullien, L; Lemarchand, A

2007-11-01

We consider n reactive species involved in unimolecular reactions and submitted to a temperature modulation of small amplitude. We determine the conditions on the rate constants for which the deviations from the equilibrium concentrations of each species can be optimized and find the analytical expression of the frequency associated with an extremum of concentration shift in the case n=3. We prove that the frequency dependence of the displacement of equilibrium gives access to the number n of species involved in the mechanism. We apply the results to the case of the transformation of a reactant into a product through a possible reactive intermediate and find the order relation obeyed by the activation energies of the different barriers. The results typically apply to enzymatic catalysis with kinetics of Michaelis-Menten type.
Action mechanism of tyrosinase on meta- and para-hydroxylated monophenols.

PubMed

Fenoll, L G; Rodríguez-López, J N; Varón, R; García-Ruiz, P A; García-Cánovas, F; Tudela, J

2000-04-01

The relationship between the structure and activity of meta- and para-hydroxylated monophenols was studied during their tyrosinase-catalysed hydroxylation and the rate-limiting steps of the reaction mechanism were identified. The para-hydroxylated substrates permit us to study the effect of a substituent (R) in the carbon-1 position (C-1) of the benzene ring on the nucleophilic attack step, while the meta group permits a similar study of the effect on the electrophilic attack step. Substrates with a -OCH3 group on C-1, as p-hydroxyanisol (4HA) and m-hydroxyanisol (3HA), or with a -CH2OH group, as p-hydroxybenzylalcohol (4HBA) and m-hydroxybenzylalcohol (3HBA), were used because the effect of the substituent (R) size was assumed to be similar. However, the electron-donating effect of the -OCH3 group means that the carbon-4 position (C-4) is favoured for nucleophilic attack (para-hydroxylated substrates) or for electrophilic attack (meta-hydroxylated substrates). The electron-attracting effect of the -CH2OH group has the opposite effect, hindering nucleophilic (para) or electrophilic (meta) attack of C-4. The experimental data point to differences between the maximum steady-state rate (V(M)Max) of the different substrates, the value of this parameter depends on the nucleophilic and electrophilic attack. However, differences are greatest in the Michaelis constants (K(M)m), with the meta-hydroxylated substrates having very large values. The catalytic efficiency k(M)cat/K(M)m is much greater for thepara-hydroxylated substrates although it varies greatly between one substrate and the other. However, it varies much less in the meta-hydroxylated substrates since this parameter describes the power of the nucleophilic attack, which is weaker in the meta OH. The large increase in the K(M)m of the meta-hydroxylated substrates might suggest that the phenolic OH takes part in substrate binding. Since this is a weaker nucleophil than the para-hydroxylated substrates, the binding constant decreases, leading to an increase in K(M)m. The catalytic efficiency of tyrosinase on a monophenol (para or meta) is directly related to the nucleophilic power of the oxygen of the phenolic OH. The oxidation step is not limiting since if this were the case, the para and meta substrates would have the same V(M)max. The small difference between the absolute values of V(M)max suggests that the rate constants of the nucleophilic and electrophilic attacks are on the same order of magnitude.
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
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. Copyright © 2014 Elsevier Ltd. All rights reserved.
Concentration-Dependent Interactions of the Organophosphates Chlorpyrifos Oxon and Methyl Paraoxon with Human Recombinant Acetylcholinesterase

PubMed Central

Kaushik, R.; Rosenfeld, Clint A.; Sultatos, L.G.

2007-01-01

For many decades it has been thought that oxygen analogs (oxons) of organophosphorus insecticides phosphylate the catalytic site of acetylcholinesterase by a mechanism that follows simple Michaelis-Menten kinetics. More recently, the interactions of at least some oxons have been shown to be far more complex, and likely involve binding of oxons to a second site on acetylcholinesterase that modulates the inhibitory capacity of other oxon molecules at the catalytic site. The current study has investigated the interactions of chlorpyrifos oxon and methyl paraoxon with human recombinant acetylcholinesterase. Both chlorpyrifos oxon and methyl paraoxon were found to have ki’s that change as a function of oxon concentration. Furthermore, 10 nM chlorpyrifos oxon resulted in a transient increase in acetylthiocholine hydrolysis, followed by inhibition. Moreover, in the presence of 100 nM chlorpyrifos oxon, acetylthiocholine was found to influence both the Kd (binding affinity) and k2 (phosphorylation constant) of this oxon. Collectively, these results demonstrate that the interactions of chlorpyrifos oxon and methyl paraoxon with acetylcholinesterase cannot be described by simple Michaelis-Menten kinetics, but instead support the hypothesis that these oxons bind to a secondary site on acetylcholinesterase, leading to activation/inhibition of the catalytic site, depending on the nature of the substrate and inhibitor. Additionally, these data raise questions regarding the adequacy of estimating risk of low levels of insecticide exposure from direct extrapolation of insecticide dose-response curves since the capacity of individual oxon molecules at low oxon levels could be greater than individual oxon molecules in vivo associated with the dose response curve. PMID:17467020
Structural Characterization of Early Michaelis Complexes in the Reaction Catalyzed by (+)-Limonene Synthase from Citrus sinensis Using Fluorinated Substrate Analogues.

PubMed

Kumar, Ramasamy P; Morehouse, Benjamin R; Matos, Jason O; Malik, Karan; Lin, Hongkun; Krauss, Isaac J; Oprian, Daniel D

2017-03-28

The stereochemical course of monoterpene synthase reactions is thought to be determined early in the reaction sequence by selective binding of distinct conformations of the geranyl diphosphate (GPP) substrate. We explore here formation of early Michaelis complexes of the (+)-limonene synthase [(+)-LS] from Citrus sinensis using monofluorinated substrate analogues 2-fluoro-GPP (FGPP) and 2-fluoroneryl diphosphate (FNPP). Both are competitive inhibitors for (+)-LS with K I values of 2.4 ± 0.5 and 39.5 ± 5.2 μM, respectively. The K I values are similar to the K M for the respective nonfluorinated substrates, indicating that fluorine does not significantly perturb binding of the ligand to the enzyme. FGPP and FNPP are also substrates, but with dramatically reduced rates (k cat values of 0.00054 ± 0.00005 and 0.00024 ± 0.00002 s -1 , respectively). These data are consistent with a stepwise mechanism for (+)-LS involving ionization of the allylic GPP substrate to generate a resonance-stabilized carbenium ion in the rate-limiting step. Crystals of apo-(+)-LS were soaked with FGPP and FNPP to obtain X-ray structures at 2.4 and 2.2 Å resolution, respectively. The fluorinated analogues are found anchored in the active site through extensive interactions involving the diphosphate, three metal ions, and three active-site Asp residues. Electron density for the carbon chains extends deep into a hydrophobic pocket, while the enzyme remains mostly in the open conformation observed for the apoprotein. While FNPP was found in multiple conformations, FGPP, importantly, was in a single, relatively well-defined, left-handed screw conformation, consistent with predictions for the mechanism of stereoselectivity in the monoterpene synthases.
The logistic growth of duckweed (Lemna minor) and kinetics of ammonium uptake.

PubMed

Zhang, Kun; Chen, You-Peng; Zhang, Ting-Ting; Zhao, Yun; Shen, Yu; Huang, Lei; Gao, Xu; Guo, Jin-Song

2014-01-01

Mathematical models have been developed to describe nitrogen uptake and duckweed growth experimentally to study the kinetics of ammonium uptake under various concentrations. The kinetics of duckweed ammonium uptake was investigated using the modified depletion method after plants were grown for two weeks at different ammonium concentrations (0.5-14 mg/L) in the culture medium. The maximum uptake rate and Michaelis-Menten constant for ammonium were estimated as 0.082 mg/(g fresh weight x h) and 1.877 mg/L, respectively. Duckweed growth was assessed when supplied at different total nitrogen (TN) concentrations (1-5 mg/L) in the culture medium. The results showed that the intrinsic growth rate was from 0.22 to 0.26 d(-1), and TN concentrations had no significant influence on the duckweed growth rate.
PURIFICATION AND ACTIVITY OF PROTEINASE OF STREPTOCOCCUS FAECALIS VAR. LIQUEFACIENS

PubMed Central

Shugart, Lee R.; Beck, Raymond W.

1964-01-01

Shugart, Lee R. (University of Tennessee, Knoxville) and Raymond W. Beck. Purification and activity of proteinase of Streptococcus faecalis var. liquefaciens. J. Bacteriol. 88:586–590. 1964.—A proteolytic enzyme from Streptococcus faecalis var. liquefaciens was purified 480-fold by ammonium sulfate fractionation and treatment with calcium phosphate gel. Approximately 20% of the original enzyme activity was recovered in the purified fraction. Optimal enzyme activity was found to be at pH 7.6 and 35 C. The enzyme is apparently more susceptible to heat denaturation when complexed with substrate than when heated in the absence of substrate. Michaelis-Menten constants were found to be 0.655% for hemoglobin and 0.133% for casein. Apparent energies of activation on these substrates were calculated to be 9,060 and 12,020 cal, respectively. PMID:14208492
Biosensor reveals multiple sources for mitochondrial NAD⁺.

PubMed

Cambronne, Xiaolu A; Stewart, Melissa L; Kim, DongHo; Jones-Brunette, Amber M; Morgan, Rory K; Farrens, David L; Cohen, Michael S; Goodman, Richard H

2016-06-17

Nicotinamide adenine dinucleotide (NAD(+)) is an essential substrate for sirtuins and poly(adenosine diphosphate-ribose) polymerases (PARPs), which are NAD(+)-consuming enzymes localized in the nucleus, cytosol, and mitochondria. Fluctuations in NAD(+) concentrations within these subcellular compartments are thought to regulate the activity of NAD(+)-consuming enzymes; however, the challenge in measuring compartmentalized NAD(+) in cells has precluded direct evidence for this type of regulation. We describe the development of a genetically encoded fluorescent biosensor for directly monitoring free NAD(+) concentrations in subcellular compartments. We found that the concentrations of free NAD(+) in the nucleus, cytoplasm, and mitochondria approximate the Michaelis constants for sirtuins and PARPs in their respective compartments. Systematic depletion of enzymes that catalyze the final step of NAD(+) biosynthesis revealed cell-specific mechanisms for maintaining mitochondrial NAD(+) concentrations. Copyright © 2016, American Association for the Advancement of Science.
Mannitol and Mannitol Dehydrogenases in Conidia of Aspergillus oryzae

PubMed Central

Horikoshi, Koki; Iida, Shigeji; Ikeda, Yonosuke

1965-01-01

Horikoshi, Koki (The Institute of Physical and Chemical Research, Tokyo, Japan), Shigeji Iida, and Yonosuke Ikeda. Mannitol and mannitol dehydrogenases in conidia of Aspergillus oryzae. J. Bacteriol. 89:326–330. 1965.—A sugar alcohol was isolated from the conidia of Aspergillus oryzae and identified as d-mannitol. Two types of d-mannitol dehydrogenases, nicotinamide adenine dinucleotide phosphate-linked and nicotinamide adenine dinucleotide-linked, were found in the conidia. Substrate specificities, pH optima, Michaelis-Menton constants, and the effects of inhibitors were studied. d-Mannitol was converted to fructose by the dehydrogenases. Synthesis of d-mannitol dehydrogenases was not observed during germination; the content of d-mannitol decreased at an early stage of germination. It was assumed, therefore, that d-mannitol might be used as the source of endogenous respiration and provide energy for the germination. PMID:14255698

Construction of uric acid biosensor based on biomimetic titanate nanotubes.

PubMed

Tao, Haisheng; Wang, Xuebin; Wang, Xizhang; Hu, Yemin; Ma, Yanwen; Lu, Yinong; Hu, Zheng

2010-02-01

A uric acid biosensor has been fabricated through the immobilization of uricase on glassy carbon electrode modified by biomimetic titanate nanotubes of high specific surface area synthesized by hydrothermal decomposition. The so-constructed biosensor presents a high affinity to uric acid with a small apparent Michaelis-Menten constant of only 0.66 mM. The biosensor exhibits fairly good electrochemical properties such as the high sensitivity of 184.3 microAcm(-2)mM(-1), the fast response of less than 2 s, as well as the wide linear range from 1 microM to 5 mM. These performances indicate that titanate nanotubes could provide a favorable microenvironment for uricase immobilization, stabilize its biological activity, and function as an efficient electron conducting tunnel to facilitate the electron transfer. This suggests an important potential of titanate nanotubes in uric acid biosensors.
Species Differences in the Oxidative Desulfurization of a Thiouracil-Based Irreversible Myeloperoxidase Inactivator by Flavin-Containing Monooxygenase Enzymes.

PubMed

Eng, Heather; Sharma, Raman; Wolford, Angela; Di, Li; Ruggeri, Roger B; Buckbinder, Leonard; Conn, Edward L; Dalvie, Deepak K; Kalgutkar, Amit S

2016-08-01

N1-Substituted-6-arylthiouracils, represented by compound 1 [6-(2,4-dimethoxyphenyl)-1-(2-hydroxyethyl)-2-thioxo-2,3-dihydropyrimidin-4(1H)-one], are a novel class of selective irreversible inhibitors of human myeloperoxidase. The present account is a summary of our in vitro studies on the facile oxidative desulfurization in compound 1 to a cyclic ether metabolite M1 [5-(2,4-dimethoxyphenyl)-2,3-dihydro-7H-oxazolo[3,2-a]pyrimidin-7-one] in NADPH-supplemented rats (t1/2 [half-life = mean ± S.D.] = 8.6 ± 0.4 minutes) and dog liver microsomes (t1/2 = 11.2 ± 0.4 minutes), but not in human liver microsomes (t1/2 > 120 minutes). The in vitro metabolic instability also manifested in moderate-to-high plasma clearances of the parent compound in rats and dogs with significant concentrations of M1 detected in circulation. Mild heat deactivation of liver microsomes or coincubation with the flavin-containing monooxygenase (FMO) inhibitor imipramine significantly diminished M1 formation. In contrast, oxidative metabolism of compound 1 to M1 was not inhibited by the pan cytochrome P450 inactivator 1-aminobenzotriazole. Incubations with recombinant FMO isoforms (FMO1, FMO3, and FMO5) revealed that FMO1 principally catalyzed the conversion of compound 1 to M1. FMO1 is not expressed in adult human liver, which rationalizes the species difference in oxidative desulfurization. Oxidation by FMO1 followed Michaelis-Menten kinetics with Michaelis-Menten constant, maximum rate of oxidative desulfurization, and intrinsic clearance values of 209 μM, 20.4 nmol/min/mg protein, and 82.7 μl/min/mg protein, respectively. Addition of excess glutathione essentially eliminated the conversion of compound 1 to M1 in NADPH-supplemented rat and dog liver microsomes, which suggests that the initial FMO1-mediated S-oxygenation of compound 1 yields a sulfenic acid intermediate capable of redox cycling to the parent compound in a glutathione-dependent fashion or undergoing further oxidation to a more electrophilic sulfinic acid species that is trapped intramolecularly by the pendant alcohol motif in compound 1. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.
Drug oxygenation activities mediated by liver microsomal flavin-containing monooxygenases 1 and 3 in humans, monkeys, rats, and minipigs.

PubMed

Yamazaki, Miho; Shimizu, Makiko; Uno, Yasuhiro; Yamazaki, Hiroshi

2014-07-15

Liver microsomal flavin-containing monooxygenases (FMO, EC 1.14.13.8) 1 and 3 were functionally characterized in terms of expression levels and molecular catalytic capacities in human, cynomolgus monkey, rat, and minipig livers. Liver microsomal FMO3 in humans and monkeys and FMO1 and FMO3 in rats and minipigs could be determined immunochemically with commercially available anti-human FMO3 peptide antibodies or rat FMO1 peptide antibodies. With respect to FMO-dependent N-oxygenation of benzydamine and tozasertib and S-oxygenation of methimazole and sulindac sulfide activities, rat and minipig liver microsomes had high maximum velocity values (Vmax) and high catalytic efficiency (Vmax/Km, Michaelis constant) compared with those for human or monkey liver microsomes. Apparent Km values for recombinantly expressed rat FMO3-mediated N- and S-oxygenations were approximately 10-100-fold those of rat FMO1, although these enzymes had similar Vmax values. The mean catalytic efficiencies (Vmax/Km, 1.4 and 0.4 min(-1)μM(-1), respectively) of recombinant human and monkey FMO3 were higher than those of FMO1, whereas Vmax/Km values for rat and minipig FMO3 were low compared with those of FMO1. Minipig liver microsomal FMO1 efficiently catalyzed N- and S-oxygenation reactions; in addition, the minipig liver microsomal FMO1 concentration was higher than the levels in rats, humans, and monkeys. These results suggest that liver microsomal FMO1 could contribute to the relatively high FMO-mediated drug N- and S-oxygenation activities in rat and minipig liver microsomes and that lower expression of FMO1 in human and monkey livers could be a determinant factor for species differences in liver drug N- and S-oxygenation activities between experimental animals and humans. Copyright © 2014 Elsevier Inc. All rights reserved.
Production and characterization of alpha-amylase from mango kernel by Fusarium solani NAIMCC-F-02956 using submerged fermentation.

PubMed

Kumar, Devendra; Yadav, Kaushlesh K; Muthukumar, M; Garg, Neelima

2013-11-01

Microbial production of enzymes using low valued agro industrial wastes is gaining importance globally. Mango is one of the major fruit processed into a variety of products. During processing 40-50% of solid waste is generated in form of peel and stones. After decortications of mango stone, kernel is obtained which is a rich source of starch (upto 60%). It was utilized as a substrate for alpha-amylase production using Fusarium soloni. Maximum alpha-amylase production (0.889 U g(-1)) was recorded using a substrate concentration of 5% (w/v), pH-4 and temperature 30 degrees C on 9th day of incubation. Supplementation of production medium with micronutrients viz., Ca2+, Fe2+ or Mg2+ improved the enzyme production while, Zn2+, B3+ or Mn2+ ions exhibited inhibitory effect. The extracellular protein was precipitated by ammonium sulphate up to 70% saturation, dialyzed and purified (27.84 fold) by gel-exclusion (Sephadex G-75) chromatography. Protein profiling on 12% SDS-PAGE revealed three bands corresponding to 26, 27 and 30 kDa molecular sizes. The optimum amylase activity was achieved at pH 5.0 at 40 degrees C. The Michaelis constant (KM), Vmax and activation energy (-Ea) were found to be 3.7 mg ml(-1), 0.24 U mg(-1) and 42.39 kJ mole(-1), respectively.
A novel enzymatic glucose sensor based on Pt nanoparticles-decorated hollow carbon spheres-modified glassy carbon electrode

NASA Astrophysics Data System (ADS)

Luhana, Charles; Bo, Xiang-Jie; Ju, Jian; Guo, Li-Ping

2012-10-01

A new glucose biosensor was developed based on hollow carbon spheres decorated with platinum nanoparticles (Pt/HCSs)-modified glassy carbon electrode immobilized with glucose oxidase (GOx) with the help of Nafion. The Pt nanoparticles were well dispersed on the HCSs with an average size of 2.29 nm. The detection of glucose was achieved via electrochemical detection of the enzymatically liberated H2O2 at +0.5 V versus Ag/AgCl at physiologic pH of 7.4. The Pt/HCSs-modified electrode exhibited excellent electrocatalytic activities toward both the oxidation and reduction of H2O2. The glucose biosensor showed good electrocatalytic performance in terms of high sensitivity (4.1 μA mM-1), low detection limit (1.8 μM), fast response time <3 s, and wide linear range (0.04-8.62 mM). The apparent Michaelis-Menten constant ( K m) and the maximum current density ( i max) values for the biosensor were 10.94 mM and 887 μA cm-2 respectively. Furthermore, this biosensor showed an acceptable reproducibility and high stability. The interfering signals from ascorbic acid and uric acid at concentration levels normally found in human blood were not much compared with the response to glucose. Blood serum samples were also tested with this biosensor and a good recovery was achieved for the two spiked serum samples.
Biocompatibility selenium nanoparticles with an intrinsic oxidase-like activity

NASA Astrophysics Data System (ADS)

Guo, Leilei; Huang, Kaixun; Liu, Hongmei

2016-03-01

Selenium nanoparticles (SeNPs) are considered to be the new selenium supplement forms with high biological activity and low toxicity; however, the molecular mechanism by which SeNPs exert the biological function is unclear. Here, we reported that biocompatibility SeNPs possessed intrinsic oxidase-like activity. Using Na2SeO3 as a precursor and glutathione as a reductant, biocompatibility SeNPs were synthesized by the wet chemical reduction method in the presence of bovine serum albumin (BSA). The results of structure characterization revealed that synthesized SeNPs were amorphous red elementary selenium with spherical morphology, and ranged in size from 25 to 70 nm size with a narrow distribution (41.4 ± 6.7 nm). The oxidase-like activity of the as-synthesized SeNPs was tested with 3,3',5,5'-tetramethylbenzidine (TMB) as a substrate. The results indicated that SeNPs could catalyze the oxidization of TMB by dissolved oxygen. These SeNPs showed an optimum catalytic activity at pH 4 and 30 °C, and the oxidase-like activity was higher as the concentration of SeNPs increased and the size of SeNPs decreased. The Michaelis constant ( K m) values and maximal reaction velocity ( V max) of the SeNPs for TMB oxidation were 0.0083 mol/L and 3.042 μmol/L min, respectively.
Comparison of Free and Immobilized L-asparaginase Synthesized by Gamma-Irradiated Penicillium cyclopium.

PubMed

El-Refai, Heba A; Shafei, Mona S; Mostafa, Hanan; El-Refai, Abdel-Monem H; Araby, Eman M; El-Beih, Fawkia M; Easa, Saadia M; Gomaa, Sanaa K

2016-01-01

Gamma irradiation is used on Penicillium cyclopium in order to obtain mutant cells of high L-asparaginase productivity. Using gamma irradiation dose of 4 KGy, P. cyclopium cells yielded L-asparaginase with extracellular enzyme activity of 210.8 ± 3 U/ml, and specific activity of 752.5 ± 1.5 U/mg protein, which are 1.75 and 1.53 times, respectively, the activity of the wild strain. The enzyme was partially purified by 40-60% acetone precipitation. L-asparaginase was immobilized onto Amberlite IR-120 by ionic binding. Both free and immobilized enzymes exhibited maximum activity at pH 8 and 40 degrees C. The immobilization process improved the enzyme thermal stability significantly. The immobilized enzyme remained 100% active at temperatures up to 60 degrees C, while the free asparaginase was less tolerant to high temperatures. The immobilized enzyme was more stable at pH 9.0 for 50 min, retaining 70% of its relative activity. The maximum reaction rate (V(max)) and Michaelis-Menten constant (K(m)) of the free form were significantly changed after immobilization. The K(m) value for immobilized L-asparaginase was about 1.3 times higher than that of free enzyme. The ions K+, Ba2+ and Na+ showed stimulatory effect on enzyme activity with percentages of 110%, 109% and 106% respectively.
Purification and characterization of polyphenol oxidase from rape flower.

PubMed

Sun, Han-Ju; Wang, Jing; Tao, Xue-Ming; Shi, Juan; Huang, Mei-Ying; Chen, Zhe

2012-01-25

The purification and partial enzymology characteristics of polyphenol oxidase (PPO) from rape flower were studied. After preliminary treatments, the crude enzyme solution was in turn purified with ammonium sulfate, dialysis, and Sephadex G-75 gel chromatography. The optimal conditions and stability of PPO were examined at different pH values and temperatures. Subsequently, PPO was also characterized by substrate (catechol) concentrations, inhibitors, kinetic parameters, and molecular weight. Results showed that the optimal pH for PPO activity was 5.5 in the presence of catechol and that PPO was relatively stable at pH 3.5-5.5. PPO was moderately stable at temperatures from 60 to 70 °C, whereas it was easily denatured at 80-90 °C. Ethylenediaminetetraacetic acid, sodium chloride, and calcium chloride had little inhibitive effects on PPO, whereas citric acid, sodium sulfite, and ascorbic acid had strongly inhibitive effects. The Michaelis-Menten constant (K(m)) and maximal reaction velocity (V(max)) of PPO were 0.767 mol/L and 0.519 Ab/min/mL of the crude PPO solution, respectively. PPO was finally purified to homogeneity with a purification factor of 4.41-fold and a recovery of 12.41%. Its molecular weight was 60.4 kDa, indicating that the PPO is a dimer. The data obtained in this research may help to prevent the enzymatic browning of rape flower during its storage and processing.
Competition between isoprene emission and pigment synthesis during leaf development in aspen.

PubMed

Rasulov, Bahtijor; Bichele, Irina; Laisk, Agu; Niinemets, Ülo

2014-03-01

In growing leaves, lack of isoprene synthase (IspS) is considered responsible for delayed isoprene emission, but competition for dimethylallyl diphosphate (DMADP), the substrate for both isoprene synthesis and prenyltransferase reactions in photosynthetic pigment and phytohormone synthesis, can also play a role. We used a kinetic approach based on post-illumination isoprene decay and modelling DMADP consumption to estimate in vivo kinetic characteristics of IspS and prenyltransferase reactions, and to determine the share of DMADP use by different processes through leaf development in Populus tremula. Pigment synthesis rate was also estimated from pigment accumulation data and distribution of DMADP use from isoprene emission changes due to alendronate, a selective inhibitor of prenyltransferases. Development of photosynthetic activity and pigment synthesis occurred with the greatest rate in 1- to 5-day-old leaves when isoprene emission was absent. Isoprene emission commenced on days 5 and 6 and increased simultaneously with slowing down of pigment synthesis. In vivo Michaelis-Menten constant (Km ) values obtained were 265 nmol m(-2) (20 μm) for DMADP-consuming prenyltransferase reactions and 2560 nmol m(-2) (190 μm) for IspS. Thus, despite decelerating pigment synthesis reactions in maturing leaves, isoprene emission in young leaves was limited by both IspS activity and competition for DMADP by prenyltransferase reactions. © 2013 John Wiley & Sons Ltd.
Kinetics of butyrate, acetate, and hydrogen metabolism in a thermophilic, anaerobic, butyrate-degrading triculture.

PubMed

Ahring, B K; Westermann, P

1987-02-01

Kinetics of butyrate, acetate, and hydrogen metabolism were determined with butyrate-limited, chemostat-grown tricultures of a thermophilic butyrate-utilizing bacterium together with Methanobacterium thermoautotrophicum and the TAM organism, a thermophilic acetate-utilizing methanogenic rod. Kinetic parameters were determined from progress curves fitted to the integrated form of the Michaelis-Menten equation. The apparent half-saturation constants, K(m), for butyrate, acetate, and dissolved hydrogen were 76 muM, 0.4 mM, and 8.5 muM, respectively. Butyrate and hydrogen were metabolized to a concentration of less than 1 muM, whereas acetate uptake usually ceased at a concentration of 25 to 75 muM, indicating a threshold level for acetate uptake. No significant differences in K(m) values for butyrate degradation were found between chemostat- and batch-grown tricultures, although the maximum growth rate was somewhat higher in the batch cultures in which the medium was supplemented with yeast extract. Acetate utilization was found to be the rate-limiting reaction for complete degradation of butyrate to methane and carbon dioxide in continuous culture. Increasing the dilution rate resulted in a gradual accumulation of acetate. The results explain the low concentrations of butyrate and hydrogen normally found during anaerobic digestion and the observation that acetate is the first volatile fatty acid to accumulate upon a decrease in retention time or increase in organic loading of a digestor.
A Facile synthesis of superparamagnetic Fe3O4 nanofibers with superior peroxidase-like catalytic activity for sensitive colorimetric detection of L-cysteine

NASA Astrophysics Data System (ADS)

Chen, Sihui; Chi, Maoqiang; Zhu, Yun; Gao, Mu; Wang, Ce; Lu, Xiaofeng

2018-05-01

Superaramagnetic Fe3O4 nanomaterials are good candidates as enzyme mimics due to their excellent catalytic activity, high stability and facile synthesis. However, the morphology of Fe3O4 nanomaterials has much influence on their enzyme-like catalytic activity. In this work, we have developed a simple polymer-assisted thermochemical reduction approach to prepare Fe3O4 nanofibers for peroxidase-like catalytic applications. The as-prepared Fe3O4 nanofibers show a higher catalytic activity than commercial Fe3O4 nanoparticles. The steady-state kinetic assay result shows that the Michaelis-Menten constant value of the as-obtained Fe3O4 nanofibers is similar to that of horseradish peroxidase (HRP), indicating their superior affinity to the 3,3‧,5,5‧-tetramethylbenzidine (TMB) and H2O2 substrate. Based on the outstanding catalytic activity, a sensing platform for the detection of L-cysteine has been performed and the limit of detection is as low as 0.028 μM. In addition, an excellent selectivity toward L-cysteine over other types of amino acids, glucose and metal ions has been achieved as well. This work offers an original means for the fabrication of superparamagnetic Fe3O4 nanofibers and demonstrates their delightful potential applications in the fields of biosensing, environmental monitoring, and medical diagnostics.
Single transporter for sulfate, selenate, and selenite in Escherichia coli K-12.

PubMed Central

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

1985-01-01

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

PubMed

Apostoł, I; Kuciel, R; Wasylewska, E; Ostrowski, W S

1985-01-01

A new spectrophotometric method for following dephosphorylation of phosphotyrosine has been described. The absorption spectra of phosphotyrosine and tyrosine were plotted over the pH range from 3 to 9. The change in absorbance accompanying the conversion of phosphotyrosine to tyrosine was the greatest at 286 nm. The difference absorption coefficients were calculated for several pH values. Dephosphorylation of phosphotyrosine by acid phosphatases from human prostate gland, from wheat germ and potatoes obeys the Michaelis-Menten equation, whereas alkaline phosphatases calf intestine and E. coli are inhibited by excess of substrate.
Macromolecular crowding effect upon in vitro enzyme kinetics: mixed activation-diffusion control of the oxidation of NADH by pyruvate catalyzed by lactate dehydrogenase.

PubMed

Balcells, Cristina; Pastor, Isabel; Vilaseca, Eudald; Madurga, Sergio; Cascante, Marta; Mas, Francesc

2014-04-17

Enzyme kinetics studies have been usually designed as dilute solution experiments, which differ substantially from in vivo conditions. However, cell cytosol is crowded with a high concentration of molecules having different shapes and sizes. The consequences of such crowding in enzymatic reactions remain unclear. The aim of the present study is to understand the effect of macromolecular crowding produced by dextran of different sizes and at diverse concentrations in the well-known reaction of oxidation of NADH by pyruvate catalyzed by L-lactate dehydrogenase (LDH). Our results indicate that the reaction rate is determined by both the occupied volume and the relative size of dextran obstacles with respect to the enzyme present in the reaction. Moreover, we analyzed the influence of macromolecular crowding on the Michaelis-Menten constants, vmax and Km. The obtained results show that only high concentrations and large sizes of dextran reduce both constants suggesting a mixed activation-diffusion control of this enzymatic reaction due to the dextran crowding action. From our knowledge, this is the first experimental study that depicts mixed activation-diffusion control in an enzymatic reaction due to the effect of crowding.
Small intestinal sulphoxidation of albendazole.

PubMed

Villaverde, C; Alvarez, A I; Redondo, P; Voces, J; Del Estal, J L; Prieto, J G

1995-05-01

1. The in vitro sulphoxidation of Albendazole (ABZ) by rat intestinal microsomes has been examined. The results revealed intestinal sulphoxidation of ABZ by intestinal microsomes in a NADPH-dependent enzymatic system. The kinetic constants for sulphoxidase activity were Vmax = 46 pmol/min/mg protein and Michaelis constant Km = 6.8 microM. 2. The possible effect of inducers (Arochlor 1254 and ABZ pretreatment) and inhibitors (erythromycin, methimazole, carbon monoxide and fenbendazole), was also studied. In rat pretreated with Arochlor 1254, Vmax was 52 pmol/min/mg protein, whereas oral administration of ABZ increased the intestinal sulphoxidation of the drug, Vmax being 103 pmol/min/mg protein. 3. Erythromycin did not change the enzymatic bioconversion of ABZ, but methimazole and carbon monoxide inhibited the enzyme activity by approximately 60 and 30% respectively. Fenbendazole (a structural analogue of ABZ) was a competitive inhibitor of the sulphoxidation process, characterized by a Ki or 69 microM. 4. These data demonstrate that the intestinal enzymes contributing to the initial sulphoxidation of ABZ may be similar to the hepatic enzymes involved in the biotransformation process by the P450 and FMO systems, a conclusion that needs to be further established.
A novel nitrite biosensor based on gold dendrites with egg white as template.

PubMed

He, Yaping; Zhang, Dawei; Dong, Sheying; Zheng, Jianbin

2012-01-01

Gold dendrites (AuD) were synthesized with egg white as the soft template and a novel nitrite (NO(2)(-)) biosensor was fabricated by assembly of a myoglobin (Mb)-L-cysteamine (Cys)-AuD biological hybrid. The results of Fourier transform infrared spectra and UV-visible spectra indicated that Mb retained its original structure in the resulting Mb-Cys-AuD. Electrochemical investigation of the biosensor showed a pair of well-defined, quasi-reversible redox peaks with E(pa) = -0.314 V and E(pc) = -0.344 V (vs. SCE) in 0.1 M, pH 7.0 sodium phosphate buffered saline at the scan rate of 200 mV/s. The transfer rate constant (k(s)) was 1.49 s(-1). The Mb-Cys-AuD showed a good electrochemical catalytic response for the reduction of NO(2)(-), with the linear range from 0.5 to 400 µM and the detection limit of 0.3 µM (S/N = 3). The apparent Michaelis-Menten constant (K(M)(app)) was estimated to be 0.2 mM. Therefore, the assembled bio-hybrid as a novel matrix opened up a further possibility for study on the design of enzymatic biosensors with potential applications.
Absolute Bioavailability and Pharmacokinetics of Linezolid in Hospitalized Patients Given Enteral Feedings

PubMed Central

Beringer, Paul; Nguyen, Megan; Hoem, Nils; Louie, Stan; Gill, Mark; Gurevitch, Michael; Wong-Beringer, Annie

2005-01-01

Linezolid is a new antimicrobial agent effective against drug-resistant gram-positive pathogens which are common causes of infections in hospitalized patients. Many such patients rely on the intravenous or enteral route for nutrition and drug administration. Therefore, the bioavailability of linezolid administered enterally in the presence of enteral feedings in hospitalized patients was examined. Eighteen subjects were assessed in a randomized single-dose crossover study; 12 received continuous enteral feedings, while 6 did not (controls). Both groups received linezolid 600 mg intravenously and orally (control) or enterally, with the alternate route of administration separated by a 24-h washout period. Pharmacokinetic parameters derived from noncompartmental and compartmental analysis incorporating linear and nonlinear elimination pathways were compared between groups: F, Ka, Vs, K23, K32, Vmax, Km, and K20 (bioavailability, absorption rate constant, volume of central compartment normalized to body weight, intercompartmental rate constants, maximum velocity, Michaelis-Menten constant, and elimination rate constant, respectively). Pharmacokinetic (PK) data were available from 17 patients. The linezolid oral suspension was rapidly and completely absorbed by either the oral or enteral route of administration. Bioavailability was unaltered in the presence of enteral feedings. PK estimates remain similar regardless of the model applied. At the therapeutic dose used, only slight nonlinearity in elimination was observed. A linezolid oral suspension may be administered via the enteral route to hospitalized patients without compromise in its excellent bioavailability and rapid rate of absorption. Compartmental pharmacokinetic analysis offers a more flexible study application, since bioavailability (F) can be estimated directly with intermixed intravenous/oral doses without a need for a washout period. PMID:16127039
Zero-order metoprolol pharmacokinetics after therapeutic doses: severe toxicity and cardiogenic shock.

PubMed

Isbister, Geoffrey K; Ang, Karyn; Gorman, Kieron; Cooper, Joyce; Mostafa, Ahmed; Roberts, Michael S

2016-11-01

Acute beta-blocker overdose can cause severe cardiac dysfunction. Chronic toxicity is rare but potentially severe. We report therapeutic dosing of metoprolol resulting in unusual pharmacokinetics and toxicity, given high-dose insulin therapy for treatment. A 90-year-old female presented with hypotension, tachycardia and severe cardiac dysfunction after commencing a rapidly increasing metoprolol dose of 250 mg split daily. She was admitted to intensive care and given high-dose insulin therapy (10 U/kg/h), noradrenaline, adrenaline and dobutamine for severe cardiac dysfunction (cardiac index, 0.76 L/min/m 2 ). She developed acute renal failure, ischaemic hepatitis and disseminated intravascular coagulopathy. Inotropes and high-dose insulin were weaned over four days with complete recovery. Metoprolol was quantified with liquid chromatography-tandem mass spectrometry and concentration-time data were analysed using MONOLIX ® vs 4.3 ( www.lixoft.com ). Admission metoprolol concentration was 2.39 μg/mL (therapeutic reference range: 0.035-0.5 μg/mL). Data best fitted a one compartmental model with Michaelis-Menten kinetics and zero order elimination at high concentrations. Final parameter estimates were V, 63.4 L, maximum rate [V m ], 9.57 mg h -1 , Michaelis constant [K m ], 1.97 mg L -1 . Predicted elimination half-life decreased from 20 h over time until there was first order elimination with a half-life 9 h. The time course of cardiac dysfunction was longer than acute overdose but consistent with prolonged zero order elimination of metoprolol, suggesting the patient was a poor CYP2D6 metaboliser. High-dose insulin euglycaemia appeared to be effective in combination with vasoconstrictors/inotropes.
In vitro metabolic stability of moisture-sensitive rabeprazole in human liver microsomes and its modulation by pharmaceutical excipients.

PubMed

Ren, Shan; Park, Mi-Jin; Kim, Aera; Lee, Beom-Jin

2008-03-01

A reliable method to assess in vitro metabolic stability of rabeprazole and its modulation by Generally Recognized As Safe (GRAS)-listed pharmaceutical excipients was established in human liver microsomes. The metabolic stability of rabeprazole decreased as a function of incubation time, resulting in the formation of thioether rabeprazole via nonenzymatic degradation and enzymatic metabolism. Buffer type was also a determining factor for the degree of both nonenzymatic degradation and enzymatic metabolism. The net extent of enzymatic drug metabolism, obtained by calculating the difference in drug degradation between a microsome-present reaction system and a microsome-free solution, was about 9.20 +/- 0.67% in phosphate buffer and 2.27 +/- 1.76% in Tris buffer, respectively. Rabeprazole exhibited first-order kinetics in microsome-free solution but showed non-linear kinetics in the microsome-present reaction system. The maximal velocity, Vmax, in phosphate buffer was 5.07 microg mL(-1) h(-1) and the Michaelis-Menten constant, Km, was 10.39 microg mL(-1) by computer-fitting to the classical Michaelis-Menten equation for pattern of time-dependent change in the substrate concentration. The intact drug and its thioether form were well resolved and successfully identified by HPLC chromatography and liquid chromatography mass spectroscopy (LC/MS). The metabolic stability of rabeprazole was also modulated by the presence of pharmaceutical excipients. Among the five pharmaceutical excipients tested, poloxamer 188 and Gelucire 44/14 had potentially inhibitory effects on rabeprazole metabolism in human liver microsomes (p < 0.05). A greater understanding of metabolic stability and its modulation by pharmaceutical excipients would be useful for optimizing the bioavailability of rabeprazole at the early formulation stages.
Concentration-dependent interactions of the organophosphates chlorpyrifos oxon and methyl paraoxon with human recombinant acetylcholinesterase

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

Kaushik, R.; Rosenfeld, Clint A.; Sultatos, L.G.

2007-06-01

For many decades it has been thought that oxygen analogs (oxons) of organophosphorus insecticides phosphorylate the catalytic site of acetylcholinesterase by a mechanism that follows simple Michaelis-Menten kinetics. More recently, the interactions of at least some oxons have been shown to be far more complex and likely involve binding of oxons to a second site on acetylcholinesterase that modulates the inhibitory capacity of other oxon molecules at the catalytic site. The current study has investigated the interactions of chlorpyrifos oxon and methyl paraoxon with human recombinant acetylcholinesterase. Both chlorpyrifos oxon and methyl paraoxon were found to have k {sub i}'smore » that change as a function of oxon concentration. Furthermore, 10 nM chlorpyrifos oxon resulted in a transient increase in acetylthiocholine hydrolysis, followed by inhibition. Moreover, in the presence of 100 nM chlorpyrifos oxon, acetylthiocholine was found to influence both the K {sub d} (binding affinity) and k {sub 2} (phosphorylation constant) of this oxon. Collectively, these results demonstrate that the interactions of chlorpyrifos oxon and methyl paraoxon with acetylcholinesterase cannot be described by simple Michaelis-Menten kinetics but instead support the hypothesis that these oxons bind to a secondary site on acetylcholinesterase, leading to activation/inhibition of the catalytic site, depending on the nature of the substrate and inhibitor. Additionally, these data raise questions regarding the adequacy of estimating risk of low levels of insecticide exposure from direct extrapolation of insecticide dose-response curves since the capacity of individual oxon molecules at low oxon levels could be greater than individual oxon molecules in vivo associated with the dose-response curve.« less

Adaptive response due to changes in gene regulation: a study with Drosophila.

PubMed Central

McDonald, J F; Chambers, G K; David, J; Ayala, F J

1977-01-01

In spite of the critical role of the process of adaptation in evolution, there are few detailed studies of the genotypic and molecular basis of the process. Drosophila melanogaster flies selected for increased tolerance to ethanol exhibited higher levels of alcohol dehydrogenase (alcohol:NAD+ oxidoreductase; EC 1.1.1.1) activity than unselected controls. A series of tests (electrophoresis, product inhibition, temperature stability, pH optima, substrate specificity, and Michaelis constants) gave no evidence of structural differences in the enzyme of the selected and the control flies. However, quantitative immunological assays showed that the selected flies contained significantly higher amounts of alcohol dehydrogenase. Adaptation of the selected flies to higher alcohol tolerance has most likely taken place by changes not in the structural gene locus coding for the enzyme, but by regulatory changes affecting the amount of gene product. Images PMID:412190
Transgalactosylation and hydrolytic activities of commercial preparations of β-galactosidase for the synthesis of prebiotic carbohydrates.

PubMed

Guerrero, Cecilia; Vera, Carlos; Conejeros, Raúl; Illanes, Andrés

2015-03-01

β-Galactosidases exhibit both hydrolytic and transgalactosylation activities; the former has been used traditionally for the production of delactosed milk and dairies, while the latter is being increasingly used for the synthesis of lactose-derived oligosaccharides: balance between both activities was highly dependent on the enzyme origin: β-galactosidases from Aspegillus oryzae and Bacillus circulans exhibited high transgalactosylation activity, while those from one from Kluyveromyces exhibited high hydrolytic activity but quite low transgalactosylation activity. Also the affinity for the donors (lactose or lactulose) and the acceptors (lactose, lactulose or fructose) of transgalactosylated galactose was dependent on the enzyme origin, as reflected by the Michaelis constants obtained in the synthesis of galacto-oligosaccharides, fructosyl-galacto-oligosaccharides and lactulose. Finally, the balance between transgalactosylation and hydrolytic activities of β-galactosidases could be tuned by changing the concentration of galactose donor. Copyright © 2014 Elsevier Inc. All rights reserved.
Insights into ligand binding to a Glutathione S-transferase from mango: structure, thermodynamics and kinetics

PubMed Central

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

2017-01-01

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

PubMed Central

Allen, J D; Thoma, J A

1976-01-01

We have developed a depolymerase computer model that uses a minimization routine. The model is designed so that, given experimental bond-cleavage frequencies for oligomeric substrates and experimental Michaelis parameters as a function of substrate chain length, the optimum subsite map is generated. The minimized sum of the weighted-squared residuals of the experimental and calculated data is used as a criterion of the goodness-of-fit for the optimized subsite map. The application of the minimization procedure to subsite mapping is explored through the use of simulated data. A procedure is developed whereby the minimization model can be used to determine the number of subsites in the enzymic binding region and to locate the position of the catalytic amino acids among these subsites. The degree of propagation of experimental variance into the subsite-binding energies is estimated. The question of whether hydrolytic rate coefficients are constant or a function of the number of filled subsites is examined. PMID:999629
Evidence of enzymatic catalysis of oxygen reduction on stainless steels under marine biofilm.

PubMed

Faimali, Marco; Benedetti, Alessandro; Pavanello, Giovanni; Chelossi, Elisabetta; Wrubl, Federico; Mollica, Alfonso

2011-04-01

Cathodic current trends on stainless steel samples with different surface percentages covered by biofilm and potentiostatically polarized in natural seawater were studied under oxygen concentration changes, temperature increases, and additions of enzymic inhibitors to the solution. The results showed that on each surface fraction covered by biofilm the oxygen reduction kinetics resembled a reaction catalyzed by an immobilised enzyme with high oxygen affinity (apparent Michaelis-Menten dissociation constant close to K(O(2))(M) ≈ 10 μM) and low activation energy (W ≈ 20 KJ mole(-1)). The proposed enzyme rapidly degraded when the temperature was increased above the ambient (half-life time of ∼1 day at 25°C, and of a few minutes at 50°C). Furthermore, when reversible enzymic inhibitors (eg sodium azide and cyanide) were added, the cathodic current induced by biofilm growth was inhibited.
Spectral Quasi-Equilibrium Manifold for Chemical Kinetics.

PubMed

Kooshkbaghi, Mahdi; Frouzakis, Christos E; Boulouchos, Konstantinos; Karlin, Iliya V

2016-05-26

The Spectral Quasi-Equilibrium Manifold (SQEM) method is a model reduction technique for chemical kinetics based on entropy maximization under constraints built by the slowest eigenvectors at equilibrium. The method is revisited here and discussed and validated through the Michaelis-Menten kinetic scheme, and the quality of the reduction is related to the temporal evolution and the gap between eigenvalues. SQEM is then applied to detailed reaction mechanisms for the homogeneous combustion of hydrogen, syngas, and methane mixtures with air in adiabatic constant pressure reactors. The system states computed using SQEM are compared with those obtained by direct integration of the detailed mechanism, and good agreement between the reduced and the detailed descriptions is demonstrated. The SQEM reduced model of hydrogen/air combustion is also compared with another similar technique, the Rate-Controlled Constrained-Equilibrium (RCCE). For the same number of representative variables, SQEM is found to provide a more accurate description.
Preparation and characterization of tannase immobilized onto carboxyl-functionalized superparamagnetic ferroferric oxide nanoparticles.

PubMed

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

2016-04-01

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

NASA Astrophysics Data System (ADS)

Wang, Min; Bao, Wen-Jing; Wang, Jiong; Wang, Kang; Xu, Jing-Juan; Chen, Hong-Yuan; Xia, Xing-Hua

2014-10-01

3D hierarchical layer double hydroxides (LDHs) have attracted extensive interest due to their unique electronic and catalytic properties. Unfortunately, the existing preparation methods require high temperature or toxic organic compounds, which limits the applications of the 3D hierarchical LDHs in biocatalysis and biomedicine. Herein, we present a green strategy to synthesize ``Desert Rose Stone''-like Mg-Al-CO3 LDH nanoflowers in situ deposited on aluminum substrates via a coprecipitation method using atmospheric carbon dioxide. Using this method, we construct a novel ``Desert Rose Stone''-like nanobiocatalytic system by using HRP as the model enzyme. Compared with the free HRP, the HRP/Mg-Al-LDH nanobiocatalytic system exhibits higher catalytic activity and stability. A smaller apparent Michaelis-Menten constant (0.16 mM) of this system suggests that the encapsulated HRP shows higher affinity towards H2O2.
Properties of thymidylate synthetase from Ehrlich ascites carcinoma cells. Effect of Mg2/ and MgATP2-.

PubMed

Jastreboff, M; Kedzierska, B; Rode, W

1982-01-15

Ehrlich ascites carcinoma thymidylate synthetase was purified to electrophoretic homogeneity by affinity chromatography on 10-formyl-5,8-dideazofolate-ethyl-Sepharose. Electrophoretic analysis of the formation of the enzyme-5-fluorodeoxyuridylate-5,10-methylenetetrahydrofolate complexes showed the presence of two binding sites for 5-fluorodeoxyuridylate on the enzyme molecule. Molecular weight of the native enzyme was found to be 78,5000, whereas that of its monomer was 38, 500. The apparent Michaelis constants for dUMP and (+/-)-L-5,10-methylenetetrahydrofolate were 1.3 +/- 0.4 and 32.2 +/- 0.7 micrometers respectively. Phosphate acted as a weak inhibitor, competitive toward dUMP. The enzyme reaction exhibited a temperature-dependent change of activation energy, reflected in the binding affinity of dUMP, with a transitional temperature of 35.8 degrees. Both Mg2+ and MgATP2- were strong activators of the enzyme, MgATP2- being more effective.
Energy dissipation drives the gradient signal amplification through an incoherent type-1 feed-forward loop

NASA Astrophysics Data System (ADS)

Lan, Ganhui

2015-09-01

We present here the analytical relation between the gain of eukaryotic gradient sensing network and the associated thermodynamic cost. By analyzing a general incoherent type-1 feed-forward loop, we derive the gain function (G ) through the reaction network and explicitly show that G depends on the nonequilibrium factor (0 ≤γ ≤1 with γ =0 and 1 representing irreversible and equilibrium reaction systems, respectively), the Michaelis constant (KM), and the turnover ratio (rcat) of the participating enzymes. We further find the maximum possible gain is intrinsically determined by KM/Gmax=(1 /KM+2 ) /4 . Our model also indicates that the dissipated energy (measured by -lnγ ), from the intracellular energy-bearing bioparticles (e.g., ATP), is used to generate a force field Fγ∝(1 -√{γ }) that reshapes and disables the effective potential around the zero gain region, which leads to the ultrasensitive response to external chemical gradients.
Inhibition of recombinant Pneumocystis carinii dihydropteroate synthetase by sulfa drugs.

PubMed Central

Hong, Y L; Hossler, P A; Calhoun, D H; Meshnick, S R

1995-01-01

Forty-four sulfa drugs were screened against crude preparations of recombinant Pneumocystis carinii dihydropteroate synthetase. The apparent Michaelis-Menten constants (Km) for p-aminobenzoic acid and 7,8-dihydro-6-hydroxymethylpterin pyrophosphate were 0.34 +/- 0.02 and 2.50 +/- 0.71 microM, respectively. Several sulfa drugs, including sulfathiazole, sulfachlorpyridazine, sulfamethoxypyridazine, and sulfathiourea, inhibited dihydropteroate synthetase approximately as well as sulfamethoxazole, as determined by the concentrations which cause 50% inhibition and/or by Ki. For all sulfones and sulfonamides tested, unsubstituted p-amino groups were necessary for activity, and sulfonamides containing an N1-heterocyclic substituent were found to be the most effective inhibitors. Folate biosynthesis in isolated intact P. carinii was approximately equally sensitive to inhibition by sulfamethoxazole, sulfachlorpyridazine, sulfamethoxypyridazine, sulfisoxazole, and sulfathiazole. Two of these drugs, sulfamethoxypyridazine and sulfisoxazole, are known to be less toxic than sulfamethoxazole and should be further evaluated for the treatment of P. carinii pneumonia. PMID:7486915
Ceramic membrane microfilter as an immobilized enzyme reactor.

PubMed

Harrington, T J; Gainer, J L; Kirwan, D J

1992-10-01

This study investigated the use of a ceramic microfilter as an immobilized enzyme reactor. In this type of reactor, the substrate solution permeates the ceramic membrane and reacts with an enzyme that has been immobilized within its porous interior. The objective of this study was to examine the effect of permeation rate on the observed kinetic parameters for the immobilized enzyme in order to assess possible mass transfer influences or shear effects. Kinetic parameters were found to be independent of flow rate for immobilized penicillinase and lactate dehydrogenase. Therefore, neither mass transfer nor shear effects were observed for enzymes immobilized within the ceramic membrane. Both the residence time and the conversion in the microfilter reactor could be controlled simply by regulating the transmembrane pressure drop. This study suggests that a ceramic microfilter reactor can be a desirable alternative to a packed bed of porous particles, especially when an immobilized enzyme has high activity and a low Michaelis constant.
Parallel synthesis of libraries of anodic and cathodic functionalized electrodeposition paints as immobilization matrix for amperometric biosensors.

PubMed

Ngounou, Bertrand; Aliyev, Elchin H; Guschin, Dmitrii A; Sultanov, Yusif M; Efendiev, Ayaz A; Schuhmann, Wolfgang

2007-09-01

The integration of flexible anchoring groups bearing imidazolyl or pyridyl substituents into the structure of electrodeposition paints (EDP) is the basis for the parallel synthesis of a library containing 107 members of different cathodic and anodic EDPs with a high variation in polymer properties. The obtained EDPs were used as immobilization matrix for biosensor fabrication using glucose oxidase as a model enzyme. Amperometric glucose sensors based on the different EDPs showed a wide variation in their sensor characteristics with respect to the apparent Michaelis-Menten constant (KM(app)) representing the linear measuring range and the maximum current (Imax(app)). Based on these results first assumptions concerning the impact of different side chains in the EDP on the expected biosensor properties could be obtained allowing for an improved rational optimization of EDPs used as immobilization matrix in amperometric biosensors.
Conformational heterogeneity within the Michaelis complex of lactate dehydrogenase†

PubMed Central

Deng, Hua; Vu, Dung V.; Clinch, Keith; Desamero, Ruel; Dyer, R. Brian; Callender, Robert

2011-01-01

A series of isotope edited IR measurements, both static as well as temperature jump relaxation spectroscopy, are performed on lactate dehydrogenase (LDH) to determine the ensemble of structures available to its Michaelis complex. There clearly has been a substantial reduction in the number of states available to the pyruvate substrate (as modeled by the substrate mimic, oxamate) and NADH when bound to protein compared to dissolved in solution, as determined by the bandwidths and positions of the critical C2=O band of bound substrate mimic and the C4-H stretch of NADH reduced nicotinamide group. Moreover, it is found that a strong ionic bond (characterized by a signature IR band discovered in this study) is formed between the carboxyl group of bound pyruvate with (presumably) Arg171, forming a strong ‘anchor’ within the protein matrix. However, conformational heterogeneity within the Michaelis complex is found that has an impact on both catalytic efficiency and thermodynamics of the enzyme. PMID:21568287
Protein kinase A catalytic subunit primed for action: Time-lapse crystallography of Michaelis complex formation

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

Das, Amit; Gerlits, Oksana O.; Parks, Jerry M.

The catalytic subunit of the cyclic AMP-dependent protein kinase A (PKAc) catalyzes the transfer of the γ-phosphate of bound Mg 2ATP to a serine or threonine residue of a protein substrate. Here, time-lapse X-ray crystallography was used to capture a series of complexes of PKAc with an oligopeptide substrate and unreacted Mg 2ATP, including the Michaelis complex, that reveal important geometric rearrangements in and near the active site preceding the phosphoryl transfer reaction. Contrary to the prevailing view, Mg 2+ binds first to the M1 site as a complex with ATP and is followed by Mg 2+ binding to themore » M2 site. Furthermore, the target serine hydroxyl of the peptide substrate rotates away from the active site toward the bulk solvent, which breaks the hydrogen bond with D166. In conclusion, the serine hydroxyl of the substrate rotates back toward D166 to form the Michaelis complex with the active site primed for phosphoryl transfer.« less
Protein kinase A catalytic subunit primed for action: Time-lapse crystallography of Michaelis complex formation

DOE PAGES

Das, Amit; Gerlits, Oksana O.; Parks, Jerry M.; ...

2015-11-12

The catalytic subunit of the cyclic AMP-dependent protein kinase A (PKAc) catalyzes the transfer of the γ-phosphate of bound Mg 2ATP to a serine or threonine residue of a protein substrate. Here, time-lapse X-ray crystallography was used to capture a series of complexes of PKAc with an oligopeptide substrate and unreacted Mg 2ATP, including the Michaelis complex, that reveal important geometric rearrangements in and near the active site preceding the phosphoryl transfer reaction. Contrary to the prevailing view, Mg 2+ binds first to the M1 site as a complex with ATP and is followed by Mg 2+ binding to themore » M2 site. Furthermore, the target serine hydroxyl of the peptide substrate rotates away from the active site toward the bulk solvent, which breaks the hydrogen bond with D166. In conclusion, the serine hydroxyl of the substrate rotates back toward D166 to form the Michaelis complex with the active site primed for phosphoryl transfer.« less
Mechanistic kinetic modeling generates system-independent P-glycoprotein mediated transport elementary rate constants for inhibition and, in combination with 3D SIM microscopy, elucidates the importance of microvilli morphology on P-glycoprotein mediated efflux activity.

PubMed

Ellens, Harma; Meng, Zhou; Le Marchand, Sylvain J; Bentz, Joe

2018-06-01

In vitro transporter kinetics are typically analyzed by steady-state Michaelis-Menten approximations. However, no clear evidence exists that these approximations, applied to multiple transporters in biological membranes, yield system-independent mechanistic parameters needed for reliable in vivo hypothesis generation and testing. Areas covered: The classical mass action model has been developed for P-glycoprotein (P-gp) mediated transport across confluent polarized cell monolayers. Numerical integration of the mass action equations for transport using a stable global optimization program yields fitted elementary rate constants that are system-independent. The efflux active P-gp was defined by the rate at which P-gp delivers drugs to the apical chamber, since as much as 90% of drugs effluxed by P-gp partition back into nearby microvilli prior to reaching the apical chamber. The efflux active P-gp concentration was 10-fold smaller than the total expressed P-gp for Caco-2 cells, due to their microvilli membrane morphology. The mechanistic insights from this analysis are readily extrapolated to P-gp mediated transport in vivo. Expert opinion: In vitro system-independent elementary rate constants for transporters are essential for the generation and validation of robust mechanistic PBPK models. Our modeling approach and programs have broad application potential. They can be used for any drug transporter with minor adaptations.
Cloning, Expression, and Purification of Choline Dehydrogenase from the Moderate Halophile Halomonas elongata

PubMed Central

Gadda, Giovanni; McAllister-Wilkins, Elien Elizabeth

2003-01-01

Choline dehydrogenase (EC 1.1.99.1) catalyzes the four-electron oxidation of choline to glycine-betaine via a betaine-aldehyde intermediate. Such a reaction is of considerable interest for biotechnological applications in that transgenic plants engineered with bacterial glycine-betaine-synthesizing enzymes have been shown to have enhanced tolerance towards various environmental stresses, such as hypersalinity, freezing, and high temperatures. To date, choline dehydrogenase has been poorly characterized in its biochemical and kinetic properties, mainly because its purification has been hampered by instability of the enzyme in vitro. In the present report, we cloned and expressed in Escherichia coli the betA gene from the moderate halophile Halomonas elongata which codes for a hypothetical choline dehydrogenase. The recombinant enzyme was purified to more than 70% homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by treatment with 30 to 50% saturation of ammonium sulfate followed by column chromatography using DEAE-Sepharose. The purified enzyme showed similar substrate specificities with either choline or betaine-aldehyde as the substrate, as indicated by the apparent V/K values (where V is the maximal velocity and K is the Michaelis constant) of 0.9 and 0.6 μmol of O2 min−1 mg−1 mM−1 at pH 7 and 25°C, respectively. With 1 mM phenazine methosulfate as the primary electron acceptor, the apparent Vmax values for choline and betaine-aldehyde were 10.9 and 5.7 μmol of O2 min−1 mg−1, respectively. These Vmax values decreased four- to sevenfold when molecular oxygen was used as the electron acceptor. Altogether, the kinetic data are consistent with the conclusion that H. elongata betA codes for a choline dehydrogenase that can also act as an oxidase when electron acceptors other than molecular oxygen are not available. PMID:12676692
Conformation change of tRNA/sub Glu/ in the complex with glutamyl-tRNA synthetase is required for the specific binding of L-glutamate

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

Hara-Yokoyama, M.; Yokoyama, S.; Miyazawa, T.

1986-11-04

The binding of Thermus thermophilus glutamyl-tRNA synthetase (GluRS) with T. thermophilus tRNA/sup Glu/, Escherichia coli tRNA/sup Glu/, and amino acids was studied by fluorescence measurements. In the absence of tRNA/sup Glu/, GluRS binds with D-glutamate as well as L-glutamate. However, in the presence of E.coli tRNA/sup Glu/, GluRS binds specifically with L-glutamate. The KCl effects on the Michaelis constants (K/sub m/) for tRNA/sup Glu/, L-glutamate, and ATP were studied for the aminoacylation of the homologous tRNA/sup Glu/ and heterologous tRNA/sup Glu/ species. As the KCl concentration is raised from 0 to 100 mM, the K/sub m/ value for L-glutamate inmore » the heterologous system is remarkably increased whereas the K/sub m/ value for L-glutamate in the homologous system is only slightly increased. The circular dichroism analyses were made mainly of the bands due to the 2-thiouridine derivatives of tRNA/sup Glu/ in the complex. The conformation change of T. thermophilus tRNA/sup Glu/ upon complex formation with GluRS is not affected by addition of KCl. In contrast, the heterologous tRNA/sup Glu/GluRS complex is in equilibrium of two forms that depends on KCl concentration. The predominant form at low KCl concentration is closely related to the small K/sub m/ value for L-glutamate. In this form of the complex, the conformation of tRNA/sup Glu/ is appreciably different from that of free molecule. Accordingly, such a conformation change of tRNA/sup Glu/ in the complex with GluRS is required for the specific binding of L-glutamate as the substrate.« less
Mechanism of acute silver toxicity in Daphnia magna.

PubMed

Bianchini, Adalto; Wood, Chris M

2003-06-01

Daphnids (Daphnia magna) were exposed to AgNO3 at 0.303 +/- 0.017 microg silver/L (46.9% as Ag+), in the absence of food, in moderately hard synthetic water under static conditions for up to 48 h. Results from accumulation experiments demonstrated that silver body burden was inversely related to body mass. Daphnids exposed to silver exhibited ionoregulatory disturbance, which was characterized by decreases in whole-body sodium concentration. This ionoregulatory disturbance was explained, at least in part, by a competitive inhibition of the whole-body sodium uptake (six- to sevenfold increase in the Michaelis constant with no change in maximal velocity), which was complete by 1 h of exposure, and resulted in approximately 40% inhibition of sodium influx from the water. A rapidly developing inhibition of whole-body Na+,K(+)-dependent adenosine triphosphatase (Na+,K(+)-ATPase) activity, significant by 2 h and complete at 90% blockade by 12 h, also was observed during exposure to AgNO3. Therefore, these findings clearly demonstrate that the key mechanism involved in acute Ag+ toxicity in D. magna, the most sensitive freshwater organism tested to date, resembles that described for freshwater fish--that is, inhibition of active sodium uptake by blockade of Na+,K(+)-ATPase. Furthermore, the results showed that Na+,K(+)-ATPase inhibition was directly related to silver accumulation in the whole body of D. magna. However, the nature of the sodium uptake inhibition (competitive vs noncompetitive in fish) and the fact that whole-body chloride concentration was not disturbed in daphnids was different from fish. With regard to the biotic ligand model (BLM) for silver, our results yielded a log K value of about 8.9. However, the current version of the BLM uses a rainbow trout log K value (7.3) but achieves the correct sensitivity of the model for daphnids by reducing the saturation of toxic sites needed to cause toxicity. An alternative way may be to use the log K value derived from the present results.

Nitrous oxide production kinetics during nitrate reduction in river sediments.

PubMed

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

2010-03-01

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

DTIC Science & Technology

1989-03-31

Jv we make use of the numerical algorithm developed by Stodden and Micha 17, extending it to the equations of motion in curvilinear coordinates. To be...in the field of the average potential V(Q). The set of equations (4.13’) have been recently derived by Stodden and Michat 5 in a more tedious.way by...B. Bloom, J. Chem. Phys. 83, 5703 (1985) 5 P. K. Swamninathan, C. D. Stodden , and D. A. Micha, J. Chem. Phys., in press (1989). 6 R. A. Marcus, Chem
Real-time monitoring of glucose-6-phosphate dehydrogenase activity using liquid droplet arrays and its application to human plasma samples.

PubMed

Jung, Se-Hui; Ji, Su-Hyun; Han, Eun-Taek; Park, Won Sun; Hong, Seok-Ho; Kim, Young-Myeong; Ha, Kwon-Soo

2016-05-15

Glucose-6-phosphate dehydrogenase (G6PD) regulates nicotinamide adenine dinucleotide phosphate (NADPH) levels and is related to the pathogenesis of various diseases, including G6PD deficiency, type 2 diabetes, aldosterone-induced endothelial dysfunction, and cancer. Therefore, a highly sensitive array-based assay for determining quantitative G6PD activity is required. Here, we developed an on-chip G6PD activity assay using liquid droplet fluorescence arrays. Quantitative G6PD activity was determined by calculating reduced resorufin concentrations in liquid droplets. The limit of detection (LOD) of this assay was 0.162 mU/ml (2.89 pM), which is much more sensitive than previous assays. We used our activity assay to determine kinetic parameters, including Michaelis-Menten constants (Km) and maximum rates of enzymatic reaction (Vmax) for NADP(+) and G6P, and half-maximal inhibitory concentrations (IC50). We successfully applied this new assay to determine G6PD activity in human plasma from normal healthy individuals (n=30) and patients with inflammation (n=30). The inflammatory group showed much higher G6PD activities than did the normal group (p<0.001), with a high area under the curve value of 0.939. Therefore, this new activity assay has the potential to be used for diagnosis of G6PD-associated diseases and utilizing kinetic studies. Copyright © 2016 Elsevier B.V. All rights reserved.
Characterization of Active Anthocyanin Degradation in the Petals of Rosa chinensis and Brunfelsia calycina Reveals the Effect of Gallated Catechins on Pigment Maintenance

PubMed Central

Luo, Honghui; Deng, Shuangfan; Fu, Wei; Zhang, Xin; Zhang, Xuelian; Zhang, Zhaoqi; Pang, Xuequn

2017-01-01

Anthocyanin degradation decreases ornamental or nutritional values of horticultural products. To investigate factors that may influence colour change in flower development, anthocyanin degradation was compared between the flowers of Brunfelsia calycina and Rosa chinensis, which show rapid and slow degradation, respectively. In-gel activity assays, high performance liquid chromatography (HPLC) analysis of tannins, enzyme kinetics measurement and immune-detection of anthocyanin degradation related-perioxidases (PODs) were carried out for the comparison. Rose petals possessed significantly lower anthocyanin degradation-related POD activities than Brunfelsia petals, which may be related to the high tannin contents. Epicatechin gallate (ECG) and gallocatechin gallate (GCG) were detected in rose as 161.3 ± 12.34 and 273.56 ± 41.23 μg/g FW (Fresh Weight) respectively, while not detected in Brunfelsia. ECG and GCG inhibited the activities of the Brunfelsia POD with half maximal inhibitory concentrations (IC50s) as 21.5 and 29.7 μM respectively, and increased the colour intensities of the anthocyanins. Catechin and epicatechin did not inhibit the POD activity, while serving as POD substrates, with Km (the Michaelis constant) as 0.48 and 1.23 mM. Similar protein levels of the anthocyanin degradation-related 40-kDa PODs were detected in Brunfelsia and rose. In summary, high amount of tannins, particularly ECG and GCG, in red rose petals may inhibit the degradation-related enzymes, leading to the maintenance of anthocyanins in vivo. PMID:28346355
Determination of the inhibitory effect of green tea extract on glucose-6-phosphate dehydrogenase based on multilayer capillary enzyme microreactor.

PubMed

Camara, Mohamed Amara; Tian, Miaomiao; Liu, Xiaoxia; Liu, Xin; Wang, Yujia; Yang, Jiqing; Yang, Li

2016-08-01

Natural herbal medicines are an important source of enzyme inhibitors for the discovery of new drugs. A number of natural extracts such as green tea have been used in prevention and treatment of diseases due to their low-cost, low toxicity and good performance. The present study reports an online assay of the activity and inhibition of the green tea extract of the Glucose 6-phosphate dehydrogenase (G6PDH) enzyme using multilayer capillary electrophoresis based immobilized enzyme microreactors (CE-IMERs). The multilayer CE-IMERs were produced with layer-by-layer electrostatic assembly, which can easily enhance the enzyme loading capacity of the microreactor. The activity of the G6PDH enzyme was determined and the enzyme inhibition by the inhibitors from green tea extract was investigated using online assay of the multilayer CE-IMERs. The Michaelis constant (Km ) of the enzyme, the IC50 and Ki values of the inhibitors were achieved and found to agree with those obtained using offline assays. The results show a competitive inhibition of green tea extract on the G6PDH enzyme. The present study provides an efficient and easy-to-operate approach for determining G6PDH enzyme reaction and the inhibition of green tea extract, which may be beneficial in research and the development of natural herbal medicines. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.
Aryl acylamidase activity of human serum albumin with o-nitrotrifluoroacetanilide as the substrate.

PubMed

Masson, Patrick; Froment, Marie-Thérèse; Darvesh, Sultan; Schopfer, Lawrence M; Lockridge, Oksana

2007-08-01

Albumin is generally regarded as an inert protein with no enzyme activity. However, albumin has esterase activity as well as aryl acylamidase activity. A new acetanilide substrate, o-nitrotrifluoroacetanilide (o-NTFNAC), which is more reactive than the classical o-nitroacetanilide, made it possible to determine the catalytic parameters for hydrolysis by fatty-acid free human serum albumin. Owing to the low enzymatic activity of albumin, kinetic studies were performed at high albumin concentration (0.075 mM). The albumin behavior with this substrate was Michaelis-Menten like. Kinetic analysis was performed according to the formalism used for catalysis at high enzyme concentration. This approach provided values for the turnover and dissociation constant of the albumin-substrate complex: k(cat) = 0.13 +/- 0.02 min(-1) and Ks = 0.67 +/- 0.04 mM. MALDI-TOF experiments showed that unlike the ester substrate p-nitrophenyl acetate, o-NTFNAC does not form a stable adduct (acetylated enzyme). Kinetic analysis and MALDI-TOF experiments demonstrated that hydrolysis of o-NTFNAC by albumin is fully rate-limited by the acylation step (k(cat) = k2). Though the aryl acylamidase activity of albumin is low (k(cat)/Ks = 195 M(-1)min(-1)), because of its high concentration in human plasma (0.6-1 mM), albumin may participate in hydrolysis of aryl acylamides through second-order kinetics. This suggests that albumin may have a role in the metabolism of endogenous and exogenous aromatic amides, including drugs and xenobiotics.
Characterization of Active Anthocyanin Degradation in the Petals of Rosa chinensis and Brunfelsia calycina Reveals the Effect of Gallated Catechins on Pigment Maintenance.

PubMed

Luo, Honghui; Deng, Shuangfan; Fu, Wei; Zhang, Xin; Zhang, Xuelian; Zhang, Zhaoqi; Pang, Xuequn

2017-03-25

Anthocyanin degradation decreases ornamental or nutritional values of horticultural products. To investigate factors that may influence colour change in flower development, anthocyanin degradation was compared between the flowers of Brunfelsia calycina and Rosa chinensis , which show rapid and slow degradation, respectively. In-gel activity assays, high performance liquid chromatography (HPLC) analysis of tannins, enzyme kinetics measurement and immune-detection of anthocyanin degradation related-perioxidases (PODs) were carried out for the comparison. Rose petals possessed significantly lower anthocyanin degradation-related POD activities than Brunfelsia petals, which may be related to the high tannin contents. Epicatechin gallate (ECG) and gallocatechin gallate (GCG) were detected in rose as 161.3 ± 12.34 and 273.56 ± 41.23 μg/g FW (Fresh Weight) respectively, while not detected in Brunfelsia . ECG and GCG inhibited the activities of the Brunfelsia POD with half maximal inhibitory concentrations (IC50s) as 21.5 and 29.7 μM respectively, and increased the colour intensities of the anthocyanins. Catechin and epicatechin did not inhibit the POD activity, while serving as POD substrates, with K m (the Michaelis constant) as 0.48 and 1.23 mM. Similar protein levels of the anthocyanin degradation-related 40-kDa PODs were detected in Brunfelsia and rose. In summary, high amount of tannins, particularly ECG and GCG, in red rose petals may inhibit the degradation-related enzymes, leading to the maintenance of anthocyanins in vivo.
Purification and characterization of an extracellular trypsin-like protease of Fusarium oxysporum var. lini.

PubMed

Barata, Ricardo Andrade; Andrade, Milton Hercules Guerra; Rodrigues, Roberta Dias; Castro, Ieso Miranda

2002-01-01

An alkaline serineprotease, capable of hydrolyzing Nalpha-benzoyl- dl arginine p-nitroanilide, was secreted by Fusarium oxysporum var. lini grown in the presence of gelatin as the sole nitrogen and carbon source. The protease was purified 65-fold to electrophoretic homogenity from the culture supernatant in a three-step procedure comprising QSepharose chromatography, affinity chromatography, and FPLC on a MonoQ column. SDS-PAGE analysis of the purified protein indicated an estimated molecular mass of 41 kDa. The protease had optimum activity at a reaction temperature of 45 degrees C and showed a rapid decrease of activity at 48 degrees C. The optimum pH was around 8.0. Characterization of the protease showed that Ca2+ and Mg2+ cations increased the activity, which was not inhibited by EDTA or 1,10-phenanthroline. The enzyme activity on Nalpha-benzoyl-DL arginine p-nitroanilide was inhibited by 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, p-aminobenzamidine dihydrochloride, aprotinin, 3-4 dichloroisocoumarin, and N-tosyl-L-lysine chloromethyl ketone. The enzyme is also inhibited by substrate concentrations higher than 2.5 x 10(-4)M. The protease had a Michaelis-Menten constant of 0.16 mM and a V(max) of 0.60 mumol released product.min(-1).mg(-1) enzyme when assayed in a non-inhibiting substrate concentration. The activity on Nalpha-benzoyl- dl arginine p-nitroanilide was competitively inhibited by p-aminobenzamidine dihydrochoride. A K(i) value of 0.04 mM was obtained.
Hepatic and intestinal glucuronidation of mono(2-ethylhexyl) phthalate, an active metabolite of di(2-ethylhexyl) phthalate, in humans, dogs, rats, and mice: an in vitro analysis using microsomal fractions.

PubMed

Hanioka, Nobumitsu; Isobe, Takashi; Kinashi, Yu; Tanaka-Kagawa, Toshiko; Jinno, Hideto

2016-07-01

Mono(2-ethylhexyl) phthalate (MEHP) is an active metabolite of di(2-ethylhexyl) phthalate (DEHP) and has endocrine-disrupting effects. MEHP is metabolized into glucuronide by UDP-glucuronosyltransferase (UGT) enzymes in mammals. In the present study, the hepatic and intestinal glucuronidation of MEHP in humans, dogs, rats, and mice was examined in an in vitro system using microsomal fractions. The kinetics of MEHP glucuronidation by liver microsomes followed the Michaelis-Menten model for humans and dogs, and the biphasic model for rats and mice. The K m and V max values of human liver microsomes were 110 µM and 5.8 nmol/min/mg protein, respectively. The kinetics of intestinal microsomes followed the biphasic model for humans, dogs, and mice, and the Michaelis-Menten model for rats. The K m and V max values of human intestinal microsomes were 5.6 µM and 0.40 nmol/min/mg protein, respectively, for the high-affinity phase, and 430 µM and 0.70 nmol/min/mg protein, respectively, for the low-affinity phase. The relative levels of V max estimated by Eadie-Hofstee plots were dogs (2.0) > mice (1.4) > rats (1.0) ≈ humans (1.0) for liver microsomes, and mice (8.5) > dogs (4.1) > rats (3.1) > humans (1.0) for intestinal microsomes. The percentages of the V max values of intestinal microsomes to liver microsomes were mice (120 %) > rats (57 %) > dogs (39 %) > humans (19 %). These results suggest that the metabolic abilities of UGT enzymes expressed in the liver and intestine toward MEHP markedly differed among species, and imply that these species differences are strongly associated with the toxicity of DEHP.
Enzymatic hydrolysis of p-nitroacetanilide: mechanistic studies of the aryl acylamidase from Pseudomonas fluorescens.

PubMed

Stein, Ross L

2002-01-22

Aryl acylamidase (EC 3.1.5.13; AAA) catalyzes the hydrolysis of p-nitroacetanilide (PNAA) via the standard three-step mechanism of serine hydrolases: binding of substrate (K(s)), acylation of active-site serine (k(acyl)), and hydrolytic deacylation (k(deacyl)). Key mechanistic findings that emerged from this study include that (1) AAA requires a deprotonated base with a pK(a) of 8.3 for expression of full activity toward PNAA. Limiting values of kinetic parameters at high pH are k(c) = 7 s(-1), K(m) = 20 microM, and k(c)/K(m) = 340 000 M(-1) s(-1). (2) At pH 10, where all the isotope effects were conducted, k(c) is equally rate-limited by k(acyl) and k(deacyl). (3) The following isotope effects were determined: (D)()2(O)(k(c)/K(m)) = 1.7 +/- 0.2, (D)()2(O)k(c) = 3.5 +/- 0.3, and (beta)(D)(k(c)/K(m)) = 0.83 +/- 0.04, (beta)(D)k(c) = 0.96 +/- 0.01. These values, together with proton inventories for k(c)/K(m) and k(c), suggest the following mechanism: (i) The initial binding of substrate to enzyme to form the Michaelis complex is accompanied by solvation changes that generate solvent deuterium isotope effects originating from hydrogen ion fractionation at multiple sites on the enzyme surface. (ii) From within the Michaelis complex, the active site serine attacks the carbonyl carbon of PNAA with general-base catalysis to form a substantially tetrahedral transition state enroute to the acyl-enzyme. (iii) Finally, deacylation occurs through a process involving a rate-limiting solvent isotope effect, generating conformational change of the acyl-enzyme that positions the carbonyl bond in a polarizing environment that is optimal for attack by water.
At the centennial of Michaelis and Menten, competing Michaelis-Menten steps explain effect of GLP-1 on blood-brain transfer and metabolism of glucose.

PubMed

Gejl, Michael; Rungby, Jørgen; Brock, Birgitte; Gjedde, Albert

2014-08-01

Glucagon-like peptide-1 (GLP-1) is a potent insulinotropic incretin hormone with both pancreatic and extrapancreatic effects. Studies of GLP-1 reveal significant effects in regions of brain tissue that regulate appetite and satiety. GLP-1 mimetics are used for the treatment of type 2 diabetes mellitus. GLP-1 interacts with peripheral functions in which the autonomic nervous system plays an important role, and emerging pre-clinical findings indicate a potential neuroprotective role of the peptide, for example in models of stroke and in neurodegenerative disorders. A century ago, Leonor Michaelis and Maud Menten described the steady-state enzyme kinetics that still apply to the multiple receptors, transporters and enzymes that define the biochemical reactions of the brain, including the glucose-dependent impact of GLP-1 on blood-brain glucose transfer and metabolism. This MiniReview examines the potential of GLP-1 as a molecule of interest for the understanding of brain energy metabolism and with reference to the impact on brain metabolism related to appetite and satiety regulation, stroke and neurodegenerative disorders. These effects can be understood only by reference to the original formulation of the Michaelis-Menten equation as applied to a chain of kinetically controlled steps. Indeed, the effects of GLP-1 receptor activation on blood-brain glucose transfer and brain metabolism of glucose depend on the glucose concentration and relative affinities of the steps both in vitro and in vivo, as in the pancreas. © 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).
Kinetic Analysis of Phospholipase C from Catharanthus roseus Transformed Roots Using Different Assays1

PubMed Central

Hernández-Sotomayor, S.M. Teresa; De Los Santos-Briones, César; Muñoz-Sánchez, J. Armando; Loyola-Vargas, Victor M.

1999-01-01

The properties of phospholipase C (PLC) partially purified from Catharanthus roseus transformed roots were analyzed using substrate lipids dispersed in phospholipid vesicles, phospholipid-detergent mixed micelles, and phospholipid monolayers spread at an air-water interface. Using [33P]phosphatidylinositol 4,5-bisphosphate (PIP2) of high specific radioactivity, PLC activity was monitored directly by measuring the loss of radioactivity from monolayers as a result of the release of inositol phosphate and its subsequent dissolution on quenching in the subphase. PLC activity was markedly affected by the surface pressure of the monolayer, with reduced activity at extremes of initial pressure. The optimum surface pressure for PIP2 hydrolysis was 20 mN/m. Depletion of PLC from solution by incubation with sucrose-loaded PIP2 vesicles followed by ultracentrifugation demonstrated stable attachment of PLC to the vesicles. A mixed micellar system was established to assay PLC activity using deoxycholate. Kinetic analyses were performed to determine whether PLC activity was dependent on both bulk PIP2 and PIP2 surface concentrations in the micelles. The interfacial Michaelis constant was calculated to be 0.0518 mol fraction, and the equilibrium dissociation constant of PLC for the lipid was 45.5 μm. These findings will add to our understanding of the mechanisms of regulation of plant PLC. PMID:10444091
Mechanisms of Enhanced Catalysis in Enzyme-DNA Nanostructures Revealed through Molecular Simulations and Experimental Analysis.

PubMed

Gao, Yingning; Roberts, Christopher C; Toop, Aaron; Chang, Chia-En A; Wheeldon, Ian

2016-08-03

Understanding and controlling the molecular interactions between enzyme substrates and DNA nanostructures has important implications in the advancement of enzyme-DNA technologies as solutions in biocatalysis. Such hybrid nanostructures can be used to create enzyme systems with enhanced catalysis by controlling the local chemical and physical environments and the spatial organization of enzymes. Here we have used molecular simulations with corresponding experiments to describe a mechanism of enhanced catalysis due to locally increased substrate concentrations. With a series of DNA nanostructures conjugated to horseradish peroxidase, we show that binding interactions between substrates and the DNA structures can increase local substrate concentrations. Increased local substrate concentrations in HRP(DNA) nanostructures resulted in 2.9- and 2.4-fold decreases in the apparent Michaelis constants of tetramethylbenzidine and 4-aminophenol, substrates of HRP with tunable binding interactions to DNA nanostructures with dissociation constants in the micromolar range. Molecular simulations and kinetic analysis also revealed that increased local substrate concentrations enhanced the rates of substrate association. Identification of the mechanism of increased local concentration of substrates in close proximity to enzymes and their active sites adds to our understanding of nanostructured biocatalysis from which we can develop guidelines for enhancing catalysis in rationally designed systems. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Direct electrochemistry of glucose oxidase and glucose biosensing on a hydroxyl fullerenes modified glassy carbon electrode.

PubMed

Gao, Yun-Fei; Yang, Tian; Yang, Xiao-Lu; Zhang, Yu-Shuai; Xiao, Bao-Lin; Hong, Jun; Sheibani, Nader; Ghourchian, Hedayatollah; Hong, Tao; Moosavi-Movahedi, Ali Akbar

2014-10-15

Direct electrochemistry of glucose oxidase (GOD) was achieved when GOD-hydroxyl fullerenes (HFs) nano-complex was immobilized on a glassy carbon (GC) electrode and protected with a chitosan (Chit) membrane. The ultraviolet-visible absorption spectrometry (UV-vis), transmission electron microscopy (TEM), and circular dichroism spectropolarimeter (CD) methods were utilized for additional characterization of the GOD, GOD-HFs and Chit/GOD-HFs. Chit/HFs may preserve the secondary structure and catalytic properties of GOD. The cyclic voltammograms (CVs) of the modified GC electrode showed a pair of well-defined quasi-reversible redox peaks with the formal potential (E°') of 353 ± 2 mV versus Ag/AgCl at a scan rate of 0.05 V/s. The heterogeneous electron transfer constant (ks) was calculated to be 2.7 ± 0.2s(-1). The modified electrode response to glucose was linear in the concentrations ranging from 0.05 to 1.0mM, with a detection limit of 5 ± 1 μM. The apparent Michaelis-Menten constant (Km(app)) was 694 ± 8 μM. Thus, the modified electrode could be applied as a third generation biosensor for glucose with high sensitivity, selectivity and low detection limit. Copyright © 2014 Elsevier B.V. All rights reserved.
Direct electron transfer of glucose oxidase and biosensing for glucose based on PDDA-capped gold nanoparticle modified graphene/multi-walled carbon nanotubes electrode.

PubMed

Yu, Yanyan; Chen, Zuanguang; He, Sijing; Zhang, Beibei; Li, Xinchun; Yao, Meicun

2014-02-15

In this work, poly (diallyldimethylammonium chloride) (PDDA)-capped gold nanoparticles (AuNPs) functionalized graphene (G)/multi-walled carbon nanotubes (MWCNTs) nanocomposites were fabricated. Based on the electrostatic attraction, the G/MWCNTs hybrid material can be decorated with AuNPs uniformly and densely. The new hierarchical nanostructure can provide a larger surface area and a more favorable microenvironment for electron transfer. The AuNPs/G/MWCNTs nanocomposite was used as a novel immobilization platform for glucose oxidase (GOD). Direct electron transfer (DET) was achieved between GOD and the electrode. Field emission scanning electron microscopy (FESEM), UV-vis spectroscopy and cyclic voltammetry (CV) were used to characterize the electrochemical biosensor. The glucose biosensor fabricated based on GOD electrode modified with AuNPs/G/MWCNTs demonstrated satisfactory analytical performance with high sensitivity (29.72mAM(-1)cm(-2)) and low limit of detection (4.8 µM). The heterogeneous electron transfer rate constant (ΚS) and the apparent Michaelis-Menten constant (Km) of GOD were calculated to be 11.18s(-1) and 2.09 mM, respectively. With satisfactory selectivity, reproducibility, and stability, the nanostructure we proposed offered an alternative for electrode fabricating and glucose biosensing. © 2013 Elsevier B.V. All rights reserved.
Solution of non-steady-state substrate concentration in the action of biosensor response at mixed enzyme kinetics

NASA Astrophysics Data System (ADS)

Senthamarai, R.; Jana Ranjani, R.

2018-04-01

In this paper, a mathematical model of an amperometric biosensor at mixed enzyme kinetics and diffusion limitation in the case of substrate inhibition has been developed. The model is based on time dependent reaction diffusion equation containing a non -linear term related to non -Michaelis - Menten kinetics of the enzymatic reaction. Solution for the concentration of the substrate has been derived for all values of parameters using the homotopy perturbation method. All the approximate analytic expressions of substrate concentration are compared with simulation results using Scilab/Matlab program. Finally, we have given a satisfactory agreement between them.
Evaluation of rate law approximations in bottom-up kinetic models of metabolism.

PubMed

Du, Bin; Zielinski, Daniel C; Kavvas, Erol S; Dräger, Andreas; Tan, Justin; Zhang, Zhen; Ruggiero, Kayla E; Arzumanyan, Garri A; Palsson, Bernhard O

2016-06-06

The mechanistic description of enzyme kinetics in a dynamic model of metabolism requires specifying the numerical values of a large number of kinetic parameters. The parameterization challenge is often addressed through the use of simplifying approximations to form reaction rate laws with reduced numbers of parameters. Whether such simplified models can reproduce dynamic characteristics of the full system is an important question. In this work, we compared the local transient response properties of dynamic models constructed using rate laws with varying levels of approximation. These approximate rate laws were: 1) a Michaelis-Menten rate law with measured enzyme parameters, 2) a Michaelis-Menten rate law with approximated parameters, using the convenience kinetics convention, 3) a thermodynamic rate law resulting from a metabolite saturation assumption, and 4) a pure chemical reaction mass action rate law that removes the role of the enzyme from the reaction kinetics. We utilized in vivo data for the human red blood cell to compare the effect of rate law choices against the backdrop of physiological flux and concentration differences. We found that the Michaelis-Menten rate law with measured enzyme parameters yields an excellent approximation of the full system dynamics, while other assumptions cause greater discrepancies in system dynamic behavior. However, iteratively replacing mechanistic rate laws with approximations resulted in a model that retains a high correlation with the true model behavior. Investigating this consistency, we determined that the order of magnitude differences among fluxes and concentrations in the network were greatly influential on the network dynamics. We further identified reaction features such as thermodynamic reversibility, high substrate concentration, and lack of allosteric regulation, which make certain reactions more suitable for rate law approximations. Overall, our work generally supports the use of approximate rate laws when building large scale kinetic models, due to the key role that physiologically meaningful flux and concentration ranges play in determining network dynamics. However, we also showed that detailed mechanistic models show a clear benefit in prediction accuracy when data is available. The work here should help to provide guidance to future kinetic modeling efforts on the choice of rate law and parameterization approaches.
Hydroxylation of p-substituted phenols by tyrosinase: Further insight into the mechanism of tyrosinase activity

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

Munoz-Munoz, Jose Luis; Berna, Jose; Garcia-Molina, Maria del Mar

2012-07-27

Highlights: Black-Right-Pointing-Pointer The action the copper complexes and tyrosinase on phenols is equivalent. Black-Right-Pointing-Pointer Isotope effect showed that nucleophilic attack to copper atom may be the slower step. Black-Right-Pointing-Pointer The value of {rho} (Hammett constant) supports an electrophilic aromatic substitution. Black-Right-Pointing-Pointer Data obtained in steady state pH 7 conditions support the mechanism of Scheme 1SM. -- Abstract: A study of the monophenolase activity of tyrosinase by measuring the steady state rate with a group of p-substituted monophenols provides the following kinetic information: k{sub cat}{sup m} and the Michaelis constant, K{sub M}{sup m}. Analysis of these data taking into account chemicalmore » shifts of the carbon atom supporting the hydroxyl group ({delta}) and {sigma}{sub p}{sup +}, enables a mechanism to be proposed for the transformation of monophenols into o-diphenols, in which the first step is a nucleophilic attack on the copper atom on the form E{sub ox} (attack of the oxygen of the hydroxyl group of C-1 on the copper atom) followed by an electrophilic attack (attack of the hydroperoxide group on the ortho position with respect to the hydroxyl group of the benzene ring, electrophilic aromatic substitution with a reaction constant {rho} of -1.75). These steps show the same dependency on the electronic effect of the substituent groups in C-4. Furthermore, a study of a solvent deuterium isotope effect on the oxidation of monophenols by tyrosinase points to an appreciable isotopic effect. In a proton inventory study with a series of p-substituted phenols, the representation of k{sub cat}{sup f{sub n}}/k{sub cat}{sup f{sub 0}} against n (atom fractions of deuterium), where k{sub cat}{sup f{sub n}} is the catalytic constant for a molar fraction of deuterium (n) and k{sub cat}{sup f{sub 0}} is the corresponding kinetic parameter in a water solution, was linear for all substrates. These results indicate that only one of the proton transfer processes from the hydroxyl groups involved the catalytic cycle is responsible for the isotope effects. We suggest that this step is the proton transfer from the hydroxyl group of C-1 to the peroxide of the oxytyrosinase form (E{sub ox}). After the nucleophilic attack, the incorporation of the oxygen in the benzene ring occurs by means of an electrophilic aromatic substitution mechanism in which there is no isotopic effect.« less
An alternative explicit model expression equivalent to the integrated michaelis-menten equation and its application to nonlinear saturation pharmacokinetics.

PubMed

Goličnik, Marko

2011-06-01

Many pharmacodynamic processes can be described by the nonlinear saturation kinetics that are most frequently based on the hyperbolic Michaelis-Menten equation. Thus, various time-dependent solutions for drugs obeying such kinetics can be expressed in terms of the Lambert W(x)-omega function. However, unfortunately, computer programs that can perform the calculations for W(x) are not widely available. To avoid this problem, the replacement of the integrated Michaelis-Menten equation with an empiric integrated 1--exp alternative model equation was proposed recently by Keller et al. (Ther Drug Monit. 2009;31:783-785), although, as shown here, it was not necessary. Simulated concentrations of model drugs obeying Michaelis-Menten elimination kinetics were generated by two approaches: 1) calculation of time-course data based on an approximation equation W2*(x) performed using Microsoft Excel; and 2) calculation of reference time-course data based on an exact W(x) function built in to the Wolfram Mathematica. I show here that the W2*(x) function approximates the actual W(x) accurately. W2*(x) is expressed in terms of elementary mathematical functions and, consequently, it can be easily implemented using any of the widely available software. Hence, with the example of a hypothetical drug, I demonstrate here that an equation based on this approximation is far better, because it is nearly equivalent to the original solution, whereas the same characteristics cannot be fully confirmed for the 1--exp model equation. The W2*(x) equation proposed here might have an important role as a useful shortcut in optional software to estimate kinetic parameters from experimental data for drugs, and it might represent an easy and universal analytical tool for simulating and designing dosing regimens.
Improving the enzymolysis efficiency of potato protein by simultaneous dual-frequency energy-gathered ultrasound pretreatment: Thermodynamics and kinetics.

PubMed

Cheng, Yu; Liu, Yun; Wu, Juan; Ofori Donkor, Prince; Li, Ting; Ma, Haile

2017-07-01

The thermodynamics and kinetics of traditional and simultaneous dual frequency energy-gathered ultrasound (SDFU) assisted enzymolysis of potato protein were investigated to get the knowledge of the mechanisms on the SDFU's promoting efficiency during enzymolysis. The concentration of potato protein hydrolysate and parameters of thermodynamic and kinetic during traditional and SDFU assisted enzymolysis were determined. The results showed that potato protein hydrolysate concentration of SDFU assisted enzymolysis was higher than traditional enzymolysis at the hydrolysis time of 60min (p<0.05) whereas not significantly different at 120min (p>0.05). In some cases, SDFU assisted enzymolysis took less hydrolysis time than traditional enzymolysis when the similar conversion rates of potato protein were obtained. The thermodynamic papameters including the energy of activation (E a ), enthalpy of activation (△H), entropy of activation (△S) were reduced by ultrasound pretreatment while Gibbs free energy of activation (△G) increased little (1.6%). Also, kinetic papameters including Michaelis constant (K M ) and catalytic rate constant (k cat ) decreased by ultrasound pretreatment. On the contrary, reaction rate constants (k) of SDFU assisted enzymolysis were higher than that of traditional enzymolysis (p<0.05). It was indicated that the efficiency of SDFU assisted enzymolysis was higher than traditional enzymolysis in a limited time. The higher efficiency of SDFU assisted enzymolysis was related with the decrease of E a and K M by lowering the energy barrier between ground and active state and increasing affinity between substrate and enzyme. Copyright © 2017 Elsevier B.V. All rights reserved.

Spectrofluorimetric assay method for glutathione and glutathione transferase using monobromobimane.

PubMed

Yakubu, S I; Yakasai, I A; Musa, A

2011-06-01

The primary role of glutathione transferase is to defend an organism from toxicities through catalyzing the reaction of glutathione (GSH) with potentially toxic compounds or metabolites to their chemically and biologically inert conjugates. The objective of the study was to develop a simple and sensitive spectrofluorimetric assay method for glutathione transferase using monobromobimane (MBB), a non fluorescent compound with electrophilic site. MBB slowly reacted with glutathione to form fluorescent glutathione conjugate and that the reaction was catalysed by glutathione transferase. Both non-enzymatic and enzymatic reaction products of MBB, in presence of GSH in phosphate buffer (pH 6.5), were measured by following increase of fluorescence at wavelength of 475nm. For validation of the assay method, the kinetic parameters such as the apparent Michaelis-Mente constants and maximum rates of conjugate formation as well as the specific activity of rat hepatic glutathione transferase were determined. The method was found to be sensitive, thus, applied to measure glutathione contents of crude preparation of rat hepatic cytosol fraction.
( sup 3 H)Dopamine uptake by platelet storage granules in schizophrenia

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

Rabey, J.M.; Graff, E.; Oberman, Z.

1992-01-01

({sup 3}H)Dopamine (DA) uptake by platelet storage granules was determined in 26 schizophrenic male patients, paranoid type (14 acute stage; 12 in remission) and 20 age-matched, normal controls. maximum velocity (Vmax) of DA uptake was significantly higher in acute patients, than patients in remission or controls (p>0.05). The apparent Michaelis constant (kM) of DA uptake in acute patients was also significantly different from chronic patients a substantial diminution of DA uptake, while haloperidol produced a substantial diminution of DA uptake, while haloperidol (10{sup {minus}4}, 10{sup {minus}5} M) did not affect the assay. Considering that a DA disequilibrium in schizophrenia maymore » be expressed not only in the brain, but also in the periphery and that an increased amount of DA accumulated in the vesicles, implies that an increased quantity of catecholamine is available for release, our findings suggest additional evidence for the role of DA overactivity in the pathophysiology of this disorder.« less
Inhibition effect of graphene oxide on the catalytic activity of acetylcholinesterase enzyme.

PubMed

Wang, Yong; Gu, Yao; Ni, Yongnian; Kokot, Serge

2015-11-01

Variations in the enzyme activity of acetylcholinesterase (AChE) in the presence of the nano-material, graphene oxide (GO), were investigated with the use of molecular spectroscopy UV-visible and fluorescence methods. From these studies, important kinetic parameters of the enzyme were extracted; these were the maximum reaction rate, Vm , and the Michaelis constant, Km . A comparison of these parameters indicated that GO inhibited the catalytic activity of the AChE because of the presence of the AChE-GO complex. The formation of this complex was confirmed with the use of fluorescence data, which was resolved with the use of the MCR-ALS chemometrics method. Furthermore, it was found that the resonance light-scattering (RLS) intensity of AChE changed in the presence of GO. On this basis, it was demonstrated that the relationship between AChE and GO was linear and such models were used for quantitative analyses of GO. Copyright © 2015 John Wiley & Sons, Ltd.
Insights into ligand binding to a glutathione S-transferase from mango: Structure, thermodynamics and kinetics

DOE PAGES

Valenzuela-Chavira, Ignacio; Contreras-Vergara, Carmen A.; Arvizu-Flores, Aldo A.; ...

2017-01-17

We studied a mango glutathione S-transferase (GST) ( Mangifera indica) bound to glutathione (GSH) and S-hexyl glutathione (GSX). This GST Tau class (MiGSTU) had a molecular mass of 25.5 kDa. MiGSTU Michaelis-Menten kinetic constants were determined for their substrates obtaining a K m, V max and k cat for CDNB of 0.792 mM, 80.58 mM min -1 and 68.49 s -1 respectively and 0.693 mM, 105.32 mM min -1 and 89.57 s -1, for reduced GSH respectively. MiGSTU had a micromolar affinity towards GSH (5.2 mM) or GSX (7.8 mM). As a result, the crystal structure of the MiGSTU inmore » apo or bound to GSH or GSX generated a model that explains the thermodynamic signatures of binding and showed the importance of enthalpic-entropic compensation in ligand binding to Tau-class GST enzymes.« less
A fibrinolytic, alkaline and thermostable metalloprotease from the newly isolated Serratia sp RSPB11.

PubMed

Lakshmi Bhargavi, P; Prakasham, R S

2013-10-01

This study shows the purification and characterization of metalloprotease (serralysin) with fibrin and fibrinogenolytic property, from the newly isolated Serratia marcescens RSPB11. This protein macro molecule was more stable over a wide range of pH (6-10) and the temperatures up to 60 °C. It showed optimum enzyme activity at pH 9.0 and at a temperature of 37 °C. Inhibitory analysis revealed that this enzyme is metalloprotease and its enzyme activity could be regained by the addition of Co(2+), Cu(2+), Fe(2+), Mg(2+)and Zn(2+) ions after chelation of ions with EDTA. This enzyme showed the Michaelis-Menten's constant Km (1.261 mg/ml) for its substrate, casein and the observed maximum attainable velocity was Vmax (24,842 U/min). The purified enzyme showed an apparent molecular mass of approximately 50 kDa in SDS-PAGE. The results also suggested that this serralysin is having potential application thrombolytic therapy. Copyright © 2013 Elsevier B.V. All rights reserved.
An amperometric new methylene blue N-mediating sensor for hydrogen peroxide based on regenerated silk fibroin as an immobilization matrix for peroxidase.

PubMed

Qian, J; Liu, Y; Liu, H; Yu, T; Deng, J

1996-05-01

A simple and effective procedure was described for the immobilization of peroxidase in regenerated silk fibroin membrane prepared from waste silk. The membranes of regenerated silk fibroin with or without peroxidase, before or after the ethanol treatment, were characterized by ir spectra. An amperometric H202 sensor, based on the immobilized peroxidase in regenerated silk fibroin membrane, in the use of new methylene blue N as an electron transfer mediator, was fabricated. The characteristics of the sensor with respect to linearity, response time, effect of pH and temperature, stability, and reproducibility were investigated. Dependences of Michaelis-Menten constant KMapp on the concentration of the mediator, and the applied potential were also studied and the results were presented. The sensor was highly sensitive to H2O2 with a detection limit of 1.0 x 10(-7)M and with response time of less than 40 s.
Metabolism of acetylcholine in the nervous system of Aplysia californica. I. Source of choline and its uptake by intact nervous tissue

PubMed Central

1975-01-01

Although acetylcholine is a major neurotransmitter in Aplysia, labeling studies with methionine and serine showed that little choline was synthesized by nervous tissue and indicated that the choline required for the synthesis of acetylcholine must be derived exogenously. Aanglia in the central nervous system (abdominal, cerebral, and pleuropedals) all took up about 0.5 nmol of choline per hour at 9 muM, the concentration of choline we found in hemolymph. This rate was more than two orders of magnitude greater than that of synthesis from the labeled precursors. Ganglia accumulated choline by a process which has two kinetic components, one with a Michaelis constant between 2-8 muM. The other component was not saturated at 420 muM. Presumably the process with the high affinity functions to supply choline for synthesis of transmitter, since the efficiency of conversion to acetylcholine was maximal in the range of external concentrations found in hemolymph. PMID:1117282
Characterization and in vitro sensitivity of cholinesterases of gilthead seabream (Sparus aurata) to organophosphate pesticides.

PubMed

Albendín, G; Arellano, J M; Mánuel-Vez, M P; Sarasquete, C; Arufe, M I

2017-04-01

The characterization of cholinesterase activity in brain and muscle of gilthead seabream was carried out using four specific substrates and three selective inhibitors. In addition, K m and V max were calculated from the Michaelis-Menten equation for ASCh and BSCh substrates. Finally, the in vitro sensitivity of brain and muscle cholinesterases to three organophosphates (OPs) was also investigated by estimating inhibition kinetics. The results indicate that AChE is the enzyme present in the brain, whereas in muscle, a typical AChE form is present along with an atypical form of BChE. Very low ChE activity was found in plasma with all substrates used. The inhibitory potency of the studied OPs on brain and muscle AChEs based on bimolecular inhibition constants (k i ) was: omethoate < dichlorvos < azinphosmethyl-oxon. Furthermore, muscle BChE was found to be several orders of magnitude (from 2 to 4) more sensitive than brain and muscle AChE inhibition by dichlorvos and omethoate.
Measurement and Modeling of Respiration Rate of Tomato (Cultivar Roma) for Modified Atmosphere Storage.

PubMed

Kandasamy, Palani; Moitra, Ranabir; Mukherjee, Souti

2015-01-01

Experiments were conducted to determine the respiration rate of tomato at 10, 20 and 30 °C using closed respiration system. Oxygen depletion and carbon dioxide accumulation in the system containing tomato was monitored. Respiration rate was found to decrease with increasing CO2 and decreasing O2 concentration. Michaelis-Menten type model based on enzyme kinetics was evaluated using experimental data generated for predicting the respiration rate. The model parameters that obtained from the respiration rate at different O2 and CO2 concentration levels were used to fit the model against the storage temperatures. The fitting was fair (R2 = 0.923 to 0.970) when the respiration rate was expressed as O2 concentation. Since inhibition constant for CO2 concentration tended towards negetive, the model was modified as a function of O2 concentration only. The modified model was fitted to the experimental data and showed good agreement (R2 = 0.998) with experimentally estimated respiration rate.
Electroosmotic perfusion of tissue: sampling the extracellular space and quantitative assessment of membrane-bound enzyme activity in organotypic hippocampal slice cultures.

PubMed

Ou, Yangguang; Wu, Juanfang; Sandberg, Mats; Weber, Stephen G

2014-10-01

This review covers recent advances in sampling fluid from the extracellular space of brain tissue by electroosmosis (EO). Two techniques, EO sampling with a single fused-silica capillary and EO push-pull perfusion, have been developed. These tools were used to investigate the function of membrane-bound enzymes with outward-facing active sites, or ectoenzymes, in modulating the activity of the neuropeptides leu-enkephalin and galanin in organotypic-hippocampal-slice cultures (OHSCs). In addition, the approach was used to determine the endogenous concentration of a thiol, cysteamine, in OHSCs. We have also investigated the degradation of coenzyme A in the extracellular space. The approach provides information on ectoenzyme activity, including Michaelis constants, in tissue, which, as far as we are aware, has not been done before. On the basis of computational evidence, EO push-pull perfusion can distinguish ectoenzyme activity with a ~100 μm spatial resolution, which is important for studies of enzyme kinetics in adjacent regions of the rat hippocampus.
Dependence of nitrite oxidation on nitrite and oxygen in low-oxygen seawater

NASA Astrophysics Data System (ADS)

Sun, Xin; Ji, Qixing; Jayakumar, Amal; Ward, Bess B.

2017-08-01

Nitrite oxidation is an essential step in transformations of fixed nitrogen. The physiology of nitrite oxidizing bacteria (NOB) implies that the rates of nitrite oxidation should be controlled by concentration of their substrate, nitrite, and the terminal electron acceptor, oxygen. The sensitivities of nitrite oxidation to oxygen and nitrite concentrations were investigated using 15N tracer incubations in the Eastern Tropical North Pacific. Nitrite stimulated nitrite oxidation under low in situ nitrite conditions, following Michaelis-Menten kinetics, indicating that nitrite was the limiting substrate. The nitrite half-saturation constant (Ks = 0.254 ± 0.161 μM) was 1-3 orders of magnitude lower than in cultivated NOB, indicating higher affinity of marine NOB for nitrite. The highest rates of nitrite oxidation were measured in the oxygen depleted zone (ODZ), and were partially inhibited by additions of oxygen. This oxygen sensitivity suggests that ODZ specialist NOB, adapted to low-oxygen conditions, are responsible for apparently anaerobic nitrite oxidation.
Insights into ligand binding to a glutathione S-transferase from mango: Structure, thermodynamics and kinetics

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

Valenzuela-Chavira, Ignacio; Contreras-Vergara, Carmen A.; Arvizu-Flores, Aldo A.

We studied a mango glutathione S-transferase (GST) ( Mangifera indica) bound to glutathione (GSH) and S-hexyl glutathione (GSX). This GST Tau class (MiGSTU) had a molecular mass of 25.5 kDa. MiGSTU Michaelis-Menten kinetic constants were determined for their substrates obtaining a K m, V max and k cat for CDNB of 0.792 mM, 80.58 mM min -1 and 68.49 s -1 respectively and 0.693 mM, 105.32 mM min -1 and 89.57 s -1, for reduced GSH respectively. MiGSTU had a micromolar affinity towards GSH (5.2 mM) or GSX (7.8 mM). As a result, the crystal structure of the MiGSTU inmore » apo or bound to GSH or GSX generated a model that explains the thermodynamic signatures of binding and showed the importance of enthalpic-entropic compensation in ligand binding to Tau-class GST enzymes.« less
The construction of glucose biosensor based on platinum nanoclusters-multiwalled carbon nanotubes nanocomposites.

PubMed

Wang, Cheng Yan; Tan, Xing Rong; Chen, Shi Hong; Hu, Fang Xin; Zhong, Hua An; Zhang, Yu

2012-02-01

One-step synthesis method was proposed to obtain the nanocomposites of platinum nanoclusters and multiwalled carbon nanotubes (PtNCs-MWNTs), which were used as a novel immobilization matrix for the enzyme to fabricate glucose biosensor. The fabrication process of the biosensor was characterized by cyclic voltammetry, electrochemical impedance spectroscopy, atomic force microscopy and scanning electron microscope. Due to the favorable characteristic of PtNCs-MWNTs nanocomposites, the biosensor exhibited good characteristics, such as wide linear range (3.0 μM-12.1 mM), low detection limit (1.0 μM), high sensitivity (12.8 μA mM⁻¹), rapid response time (within 6 s). The apparent Michaelis-Menten constant (K(app)(m)) is 2.1 mM. The performance of the resulting biosensor is more prominent than that of most of the reported glucose biosensors. Furthermore, it was demonstrated that this biosensor can be used for the assay of glucose in human serum samples.
Chloroplast Phosphofructokinase

PubMed Central

Kelly, Grahame J.; Latzko, Erwin

1977-01-01

Chloroplast phosphofructokinase from spinach (Spinacia oleracea L.) was purified approximately 40-fold by a combination of fractionations with ammonium sulfate and acetone followed by chromatography on DEAE-Sephadex A-50. Positive cooperative kinetics was observed for the interaction between the enzyme and the substrate fructose 6-phosphate. The optimum pH shifted from 7.7 toward 7.0 as the fructose 6-phosphate concentration was taken below 0.5 mm. The second substrate was MgATP2− (Michaelis constant 30 μm). Free ATP inhibited the enzyme. Chloroplast phosphofructokinase was sensitive to inhibition by low concentration of phosphoenolpyruvate and glycolate 2-phosphate (especially at higher pH); these compounds inhibited in a positively cooperative fashion. Inhibitions by glycerate 2-phosphate (and probably glycerate 3-phosphate), citrate, and inorganic phosphate were also recorded; however, inorganic phosphate effectively relieved the inhibitions by phosphoenolpyruvate and glycolate 2-phosphate. These regulatory properties are considered to complement those of ADP-glucose pyrophosphorylase and fructosebisphosphatase in the regulation of chloroplast starch metabolism. PMID:16660079
Evaluation of free and immobilized Aspergillus niger NRC1ami pectinase applicable in industrial processes.

PubMed

Esawy, Mona A; Gamal, Amira A; Kamel, Zeinat; Ismail, Abdel-Mohsen S; Abdel-Fattah, Ahmed F

2013-02-15

The Aspergillus niger NRC1ami pectinase was evaluated according to its hydrolysis efficiency of dry untreated orange peels (UOP), HCl-treated orange peels and NaOH-treated orange peels (HOP and NOP). Pectinase was entrapped in polyvinyl alcohol (PVA) sponge and the optimum pH and temperature of the free and immobilized enzymes were shifted from 4, 40 °C to 6, 50 °C respectively. The study of pH stability of free and immobilized pectinase showed that the immobilization process protected the enzyme strongly from severe alkaline pHs. The immobilization process improved the enzyme thermal stability to great instant. The unique feature of the immobilization process is its ability to solve the orange juice haze problem completely. Immobilized enzyme was reused 12 times in orange juice clarification with 9% activity loss from the original activity. Maximum reaction rate (V(max)) and Michaelis-Menten constant (K(m)) of the partially purified form were significantly changed after immobilization. Copyright © 2012 Elsevier Ltd. All rights reserved.
A Century of Enzyme Kinetic Analysis, 1913 to 2013

PubMed Central

Johnson, Kenneth A.

2013-01-01

This review traces the history and logical progression of methods for quantitative analysis of enzyme kinetics from the 1913 Michaelis and Menten paper to the application of modern computational methods today. Following a brief review of methods for fitting steady state kinetic data, modern methods are highlighted for fitting full progress curve kinetics based upon numerical integration of rate equations, including a re-analysis of the original Michaelis-Menten full time course kinetic data. Finally, several illustrations of modern transient state kinetic methods of analysis are shown which enable the elucidation of reactions occurring at the active sites of enzymes in order to relate structure and function. PMID:23850893
A Generalized Michaelis-Menten Equation in Protein Synthesis: Effects of Mis-Charged Cognate tRNA and Mis-Reading of Codon.

PubMed

Dutta, Annwesha; Chowdhury, Debashish

2017-05-01

The sequence of amino acid monomers in the primary structure of a protein is decided by the corresponding sequence of codons (triplets of nucleic acid monomers) on the template messenger RNA (mRNA). The polymerization of a protein, by incorporation of the successive amino acid monomers, is carried out by a molecular machine called ribosome. We develop a stochastic kinetic model that captures the possibilities of mis-reading of mRNA codon and prior mis-charging of a tRNA. By a combination of analytical and numerical methods, we obtain the distribution of the times taken for incorporation of the successive amino acids in the growing protein in this mathematical model. The corresponding exact analytical expression for the average rate of elongation of a nascent protein is a 'biologically motivated' generalization of the Michaelis-Menten formula for the average rate of enzymatic reactions. This generalized Michaelis-Menten-like formula (and the exact analytical expressions for a few other quantities) that we report here display the interplay of four different branched pathways corresponding to selection of four different types of tRNA.
The role of CYP2D6 in primary and secondary oxidative metabolism of dextromethorphan: in vitro studies using human liver microsomes.

PubMed Central

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

1994-01-01

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

PubMed

Liang, Huiyun; Bourdon, Allen K; Chen, Liao Y; Phelix, Clyde F; Perry, George

2018-06-11

Fourteen glucose transporters (GLUTs) play essential roles in human physiology by facilitating glucose diffusion across the cell membrane. Due to its central role in the energy metabolism of the central nervous system, GLUT3 has been thoroughly investigated. However, the Gibbs free-energy gradient (what drives the facilitated diffusion of glucose) has not been mapped out along the transport path. Some fundamental questions remain. Here we present a molecular dynamics study of GLUT3 embedded in a lipid bilayer to quantify the free-energy profile along the entire transport path of attracting a β-d-glucose from the interstitium to the inside of GLUT3 and, from there, releasing it to the cytoplasm by Arrhenius thermal activation. From the free-energy profile, we elucidate the unique Michaelis-Menten characteristics of GLUT3, low K M and high V MAX , specifically suitable for neurons' high and constant demand of energy from their low-glucose environments. We compute GLUT3's binding free energy for β-d-glucose to be -4.6 kcal/mol in agreement with the experimental value of -4.4 kcal/mol ( K M = 1.4 mM). We also compute the hydration energy of β-d-glucose, -18.0 kcal/mol vs the experimental data, -17.8 kcal/mol. In this, we establish a dynamics-based connection from GLUT3's crystal structure to its cellular thermodynamics with quantitative accuracy. We predict equal Arrhenius barriers for glucose uptake and efflux through GLUT3 to be tested in future experiments.
Aspartic acid 413 is important for the normal allosteric functioning of ADP-glucose pyrophosphorylase

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

Greene, T.W.; Woodbury, R.L.; Okita, T.W.

1996-11-01

As part of a structure-function analysis of the higher-plant ADP-glucose pyrophosphorylase (AGP), we used a random mutagenesis approach in combination with a novel bacterial complementation system to isolate over 100 mutants that were defective in glycogen production. One mutant of the large subunit M27 was identified by its capacity to only partially complement a mutation in the structural gene for the bacterial AGP (glg C), as determined by its light-staining phenotype when cells were exposed to I{sub 2} vapors. Enzyme-linked immunosorbent assay and enzymatic pyrophosphorylysis assays of M27 cell extracts showed that the level of expression and AGP activity wasmore » comparable to those of cells that expressed the wildtype recombinant enzyme. Kinetic analysis indicated that the M27 AGP displays normal Michaelis constant values for the substrates glucose-1-phosphate and ATP but requires 6- to 10-fold greater levels of 3-phosphoglycerate (3-PGA) than the wild-type recombinant enzyme for maximum activation. DNA sequence analysis showed that M27 contains a single point mutation that resulted in the replacement of aspartic acid 413 to alanine. Substitution of a lysine residue at this site almost completely abolished activation by 3-PGA. Aspartic acid 413 is adjacent to a lysine residue that was previously identified by chemical modification studies to be important in the binding of 3-PGA. The kinetic properties of M27 corroborate the importance of this region in the allosteric regulation of a higher-plant AGP. 28 refs., 3 figs., 1 tab.« less

6-Mercaptopurine transport by equilibrative nucleoside transporters in conditionally immortalized rat syncytiotrophoblast cell lines TR-TBTs.

PubMed

Lee, Na-young; Sai, Yoshimichi; Nakashima, Emi; Ohtsuki, Sumio; Kang, Young-sook

2011-09-01

Recently, more women were provided with 6-mercaptopurine (6-MP) during pregnancy. Therefore, we attempted to clarify the transport mechanisms of 6-MP through blood-placenta barrier using rat conditionally immortalized syncytiotrophoblast cell lines (TR-TBTs). The uptake of 6-MP was time- and ATP dependent, but sodium independent in TR-TBTs. 6-MP was eliminated over 50% from the cells within 30 min. The uptake of 6-MP was saturable with Michaelis-Menten constant values of 198 μM and 250 μM in TR-TBT 18d-1 and TR-TBT 18d-2, respectively. 6-Thioguanine, azathioprine, and hypoxantine, structural analogues of 6-MP, strongly inhibited [(14) C]6-MP uptake. Equilibrative nucleoside transporter (ENT) inhibitors, adenosine and uridine, significantly inhibited [(14) C]6-MP uptake. However, several organic anions and cations had no effect on [(14) C]6-MP uptake in TR-TBTs. These results suggest that sodium-independent transporters, ENTs, may be involved in 6-MP uptake at the placenta. In addition, multidrug resistance protein (MRP) inhibitors, methotrexate, probenecid, cefmetazole, and sulfinpyrazone, significantly increased the accumulation of [(14) C]6-MP in the cells. It is indicated that 6-MP may be eliminated across the blood-placental barrier via MRPs. TR-TBTs expressed mRNA of ENT1, ENT2, MRP4, and MRP5. These findings are important for the therapy of acute lymphoblastic leukemia and autoimmune diseases of pregnant women, and should be useful data in elucidating teratogenicity of 6-MP during pregnancy. Copyright © 2011 Wiley-Liss, Inc.
Enzymatic glycosylation, inhibitor design, and synthesis and formation of glyco-self assembled monolayers for simulation of recognition.

PubMed

Scheppokat, Angela M; Gerber, Agnes; Schroven, Andreas; Meinke, Sebastian; Kopitzki, Sebastian; Beketow, Eugen; Thimm, Julian; Thiem, Joachim

2010-01-01

Glycosyltransferases from the albumen gland of Helix pomatia could be used in tandem mode for the chemoenzymatic synthesis of beta,1-3/beta,1-6-linked oligogalactans. By employing recombinant trans-sialidase of Trypanosoma cruzi (TcTS) the formation of a range of modified Galbeta,1-3GalNAc derivatives could be terminally alpha,2-3 sialylated. Biacore studies indicated the binding of these modified trisaccharides to myelin-associated glycoprotein (MAG). Using an eight-step synthetic route N-acyl-modified sialyl donor structures could be obtained. TcTS was used to transfer these structures to an isolactoside, and Michaelis constants of the donors indicated the kind and size of modifications allowed at the 5-nitrogen site. A number of sialic acid C-glycosides could be obtained via the C-allyl sialoside and subsequent metathesis. Biacore measurements showed derivatives substituted with aromatic residues to give K(D) values in the mM range. Benzaldehyde-functionalized glycosides of mono and disaccharides were synthesized by metathesis and could be used for the formation of novel glyco-self assembled monolayers (glyco-SAMs) employing various tether structures and attached to gold surfaces. Initial experiments were performed with concanavalin A and manno-SAMs. By atomic force microscopic measurements of tethered glycosides attached to gold-coated tips and surfaces weak forces in the nN range could be detected. Structure activity correlation of forces suggested rationales for complex interactions of various glycosides including minor stereochemical variations. Copyright (c) 2009 Elsevier GmbH. All rights reserved.
A human lung mast cell chymotrypsin-like enzyme. Identification and partial characterization.

PubMed

Wintroub, B U; Kaempfer, C E; Schechter, N M; Proud, D

1986-01-01

We have used a high performance liquid chromatography assay, which detects chymotryptic cleavage of the phe8-his9 bond of angiotensin I to yield angiotensin II, in order to examine human lung mast cells for the presence of chymotryptic activity. Mast cells, purified from human lung by enzymatic dispersion, countercurrent elutriation, and Percoll gradient centrifugation, were lysed or challenged with goat anti-human IgE. In multiple experiments angiotensin II-converting activity was detected in lysates of 10-99% pure mast cell preparations. Regression analysis of net percent release values of histamine and the angiotensin I-converting activity from dose-response experiments demonstrated a correlation between the two parameters, indicating that the chymotrypsin-like enzyme is a constituent of the mast cell secretory granule. The chymotryptic activity was completely inhibited by 10(-3) M phenylmethylsulfonylfluoride but not by 10(-3) M Captopril, and the pH optimum of activity was 7.5-9.5. Gel filtration of released material separated the activity from tryptase and demonstrated an approximate molecular weight of 30-35,000. The mast cell enzyme, like a human skin chymotrypsin-like proteinase, can be distinguished from leukocyte cathepsin G by lack of susceptibility to inhibition by bovine pancreatic trypsin inhibitor. Thus, an enzyme with limited chymotryptic specificity is present in human lung mast cells. The Michaelis constant of the enzyme for angiotensin I of 6.0 X 10(-5) M is similar to that of endothelial cell angiotensin-converting enzyme and is consistent with a reaction of physiologic importance.
Highly efficient enzymatic acetylation of flavonoids: Development of solvent-free process and kinetic evaluation

DOE PAGES

Milivojevic, Ana; Corovic, Marija; Carevic, Milica; ...

2017-09-23

Solubility and stability of flavonoid glycosides, valuable natural constituents of cosmetics and pharmaceuticals, could be improved by lipase-catalyzed acylation. Focus of this study was on development of eco-friendly process for the production of flavonoid acetates. By using phloridzin as model compound and triacetin as acetyl donor and solvent, 100% conversion and high productivity (23.32 g l –1 day –1) were accomplished. Complete conversions of two other glycosylated flavonoids, naringin and esculin, in solvent-free system were achieved, as well. Comprehensive kinetic mechanism based on two consecutive mono-substrate reactions was established where first one represents formation of flavonoid monoacetate and within secondmore » reaction diacetate is being produced from monoacetate. Both steps were regarded as reversible Michaelis-Menten reactions without inhibition. Apparent kinetic parameters for two consecutive reactions (V m constants for substrates and products and K m constants for forward and reverse reactions) were estimated for three examined acetyl acceptors and excellent fitting of experimental data (R 2 > 0.97) was achieved. Obtained results showed that derived kinetic model could be applicable for solvent-free esterifications of different flavonoid glycosides. As a result, it was valid for entire transesterification course (72 h of reaction) which, combined with complete conversions and green character of synthesis, represents firm basis for further process development.« less
Temperature Responses of C4 Photosynthesis: Biochemical Analysis of Rubisco, Phosphoenolpyruvate Carboxylase, and Carbonic Anhydrase in Setaria viridis.

PubMed

Boyd, Ryan A; Gandin, Anthony; Cousins, Asaph B

2015-11-01

The photosynthetic assimilation of CO2 in C4 plants is potentially limited by the enzymatic rates of Rubisco, phosphoenolpyruvate carboxylase (PEPc), and carbonic anhydrase (CA). Therefore, the activity and kinetic properties of these enzymes are needed to accurately parameterize C4 biochemical models of leaf CO2 exchange in response to changes in CO2 availability and temperature. There are currently no published temperature responses of both Rubisco carboxylation and oxygenation kinetics from a C4 plant, nor are there known measurements of the temperature dependency of the PEPc Michaelis-Menten constant for its substrate HCO3 (-), and there is little information on the temperature response of plant CA activity. Here, we used membrane inlet mass spectrometry to measure the temperature responses of Rubisco carboxylation and oxygenation kinetics, PEPc carboxylation kinetics, and the activity and first-order rate constant for the CA hydration reaction from 10°C to 40°C using crude leaf extracts from the C4 plant Setaria viridis. The temperature dependencies of Rubisco, PEPc, and CA kinetic parameters are provided. These findings describe a new method for the investigation of PEPc kinetics, suggest an HCO3 (-) limitation imposed by CA, and show similarities between the Rubisco temperature responses of previously measured C3 species and the C4 plant S. viridis. © 2015 American Society of Plant Biologists. All Rights Reserved.
[Biodegradation characteristics of o-chlorophenol with photosynthetic bacteria PSB-1D].

PubMed

Hu, Xiao-min; Dong, Yi-hu; Li, Liang; Lu, Juan; He, Ying-dian; Gao, Yang

2010-07-01

A strain of photosynthetic bacteria named PSB-1D with degradation of o-chlorophenol (2-CP) was isolated and screened from the shallow substrate sludge in downstream side of the sewage outfall of an insecticide factory. The PSB-1D is identified preliminarily as Rhodopseudomonas sp. according to its colony and cell morphological properties, physiological biochemical characteristics and absorption spectrum analysis of living cells. The experiments results of relationship between PSB-1D growth and o-chlorophenol degradation showed that the degradation rate of o-chlorophenol was up to 57.26% after 7 days cultural time. The main environmental factors including way of illumination and oxygen, initial pH, cultural temperature, illumination intensity had distinctly influenced on the o-chlorophenol degradation with PSB-1D. The results showed that the optimum conditions were as following: an anaerobic light, pH 7.0, temperature 30 degrees C, illumination intensity 4000 lx,initial o-chlorophenol concentration 50 mg/L. Under that cultural condition, the degradation rate of o-chlorophenol could reach to 62.08%. The degradation kinetic data fitted the Andrews model well. In addition, the biodegradation process of o-chlorophenol can be well described by enzymatic reaction of high concentration inhibition, with the maximum substrate utilization rate 0.309 d(-1), Michaelis-Menten constant 2.733 mg/L, inhibitory constant 230.15 mg/L respectively.
Temperature-Dependent Kinetic Model for Nitrogen-Limited Wine Fermentations▿

PubMed Central

Coleman, Matthew C.; Fish, Russell; Block, David E.

2007-01-01

A physical and mathematical model for wine fermentation kinetics was adapted to include the influence of temperature, perhaps the most critical factor influencing fermentation kinetics. The model was based on flask-scale white wine fermentations at different temperatures (11 to 35°C) and different initial concentrations of sugar (265 to 300 g/liter) and nitrogen (70 to 350 mg N/liter). The results show that fermentation temperature and inadequate levels of nitrogen will cause stuck or sluggish fermentations. Model parameters representing cell growth rate, sugar utilization rate, and the inactivation rate of cells in the presence of ethanol are highly temperature dependent. All other variables (yield coefficient of cell mass to utilized nitrogen, yield coefficient of ethanol to utilized sugar, Monod constant for nitrogen-limited growth, and Michaelis-Menten-type constant for sugar transport) were determined to vary insignificantly with temperature. The resulting mathematical model accurately predicts the observed wine fermentation kinetics with respect to different temperatures and different initial conditions, including data from fermentations not used for model development. This is the first wine fermentation model that accurately predicts a transition from sluggish to normal to stuck fermentations as temperature increases from 11 to 35°C. Furthermore, this comprehensive model provides insight into combined effects of time, temperature, and ethanol concentration on yeast (Saccharomyces cerevisiae) activity and physiology. PMID:17616615
Highly efficient enzymatic acetylation of flavonoids: Development of solvent-free process and kinetic evaluation

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

Milivojevic, Ana; Corovic, Marija; Carevic, Milica

Solubility and stability of flavonoid glycosides, valuable natural constituents of cosmetics and pharmaceuticals, could be improved by lipase-catalyzed acylation. Focus of this study was on development of eco-friendly process for the production of flavonoid acetates. By using phloridzin as model compound and triacetin as acetyl donor and solvent, 100% conversion and high productivity (23.32 g l –1 day –1) were accomplished. Complete conversions of two other glycosylated flavonoids, naringin and esculin, in solvent-free system were achieved, as well. Comprehensive kinetic mechanism based on two consecutive mono-substrate reactions was established where first one represents formation of flavonoid monoacetate and within secondmore » reaction diacetate is being produced from monoacetate. Both steps were regarded as reversible Michaelis-Menten reactions without inhibition. Apparent kinetic parameters for two consecutive reactions (V m constants for substrates and products and K m constants for forward and reverse reactions) were estimated for three examined acetyl acceptors and excellent fitting of experimental data (R 2 > 0.97) was achieved. Obtained results showed that derived kinetic model could be applicable for solvent-free esterifications of different flavonoid glycosides. As a result, it was valid for entire transesterification course (72 h of reaction) which, combined with complete conversions and green character of synthesis, represents firm basis for further process development.« less
Radiochromatographic assay of N-acyl-phosphatidylethanolamine-specific phospholipase D activity.

PubMed

Fezza, Filomena; Gasperi, Valeria; Mazzei, Cinzia; Maccarrone, Mauro

2005-04-01

A radiochromatographic method has been set up to assay the activity of N-acyl-phosphatidylethanolamine-specific phospholipase D (NAPE-PLD), based on reversed-phase high-performance liquid chromatography (HPLC) and online scintillation counting. The anandamide (N-arachidonoylethanolamine, AEA), product released by NAPE-PLD from the N-arachidonoyl-phosphatidylethanolamine (NArPE) substrate, was separated using a C18 column eluted with methanol-water-acetic acid and was quantified with an external standard method. Baseline separation of AEA and NArPE was completed in less than 15 min, with a detection limit of 0.5 fmol AEA at a signal-to-noise ratio of 4:1. The sensitivity and accuracy of the radiochromatographic procedure allowed detection and characterization of NAPE-PLD activity in very tiny tissue samples or in samples where the enzymatic activity is very low. With this method, we could determine the kinetic constants (i.e., apparent Michaelis-Menten constant (Km) of 40.0+/-5.6 microM and maximum velocity (Vmax) of 22.2+/-3.5 pmol/min per milligram protein toward NArPE) and the distribution of NAPE-PLD activity in brain areas and peripheral tissues of mouse. In addition, we could collect unprecedented evidence that compounds widely used in studies of the endocannabinoid system (e.g., AEA and congeners, receptor a(nta)gonists and inhibitors of AEA degradation) can also affect NAPE-PLD activity.
Effects of metal ions on the catalytic degradation of dicofol by cellulase.

PubMed

Zhai, Zihan; Yang, Ting; Zhang, Boya; Zhang, Jianbo

2015-07-01

A new technique whereby cellulase immobilized on aminated silica was applied to catalyze the degradation of dicofol, an organochlorine pesticide. In order to evaluate the performance of free and immobilized cellulase, experiments were carried out to measure the degradation efficiency. The Michaelis constant, Km, of the reaction catalyzed by immobilized cellulase was 9.16 mg/L, and the maximum reaction rate, Vmax, was 0.40 mg/L/min, while that of free cellulase was Km=8.18 mg/L, and Vmax=0.79 mg/L/min, respectively. The kinetic constants of catalytic degradation were calculated to estimate substrate affinity. Considering that metal ions may affect enzyme activity, the effects of different metal ions on the catalytic degradation efficiency were explored. The results showed that the substrate affinity decreased after immobilization. Monovalent metal ions had no effect on the reaction, while divalent metal ions had either positive or inhibitory effects, including activation by Mn2+, reversible competition with Cd2+, and irreversible inhibition by Pb2+. Ca2+ promoted the catalytic degradation of dicofol at low concentrations, but inhibited it at high concentrations. Compared with free cellulase, immobilized cellulase was affected less by metal ions. This work provided a basis for further studies on the co-occurrence of endocrine-disrupting chemicals and heavy metal ions in the environment. Copyright © 2015. Published by Elsevier B.V.
Oxidation of d-Amino Acids by a Particulate Enzyme from Pseudomonas aeruginosa

PubMed Central

Marshall, Vincent P.; Sokatch, John R.

1968-01-01

A particulate d-amino acid dehydrogenase has been partially purified from cell free extracts of Pseudomonas aeruginosa grown on dl-valine as the source of carbon and energy. A standard assay was developed which utilized 2,6-dichlorophenol-indophenol as the electron acceptor. The pH optimum for enzyme activity ranged from 6.0 to 8.0, depending on the amino acid assayed. The enzyme was most active with monoamino-monocarboxylic amino acids and histidine. The Michaelis constant for d-phenylalanine was found to be 1.3 × 10-3m d-phenylalanine. Constants could not be calculated for the other amino acids oxidized because anomalous plots of V as a function of V/S were obtained. Spectra of enzyme preparations reduced with d-valine or sodium hydrosulfite exhibited adsorption bands typical of the α, β, and γ bands of cytochromes as well as bleaching in the flavin region of the spectrum. When dl-valine was added to a medium with glycerol as the energy source, d-amino acid dehydrogenase was detected after the addition of valine and was produced at a rate directly proportional to the synthesis of total protein. The enzyme was formed when d-valine, l-valine, or dl-alanine was the source of carbon and energy, but not when glucose, glycerol, or succinate was the energy source. PMID:4384679
Fast evaluation of enantioselective drug metabolism by electrophoretically mediated microanalysis: application to fluoxetine metabolism by CYP2D6.

PubMed

Asensi-Bernardi, Lucía; Martín-Biosca, Yolanda; Escuder-Gilabert, Laura; Sagrado, Salvador; Medina-Hernández, María José

2013-12-01

In this work, a capillary electrophoretic methodology for the enantioselective in vitro evaluation of drugs metabolism is applied to the evaluation of fluoxetine (FLX) metabolism by cytochrome 2D6 (CYP2D6). This methodology comprises the in-capillary enzymatic reaction and the chiral separation of FLX and its major metabolite, norfluoxetine enantiomers employing highly sulfated β-CD and the partial filling technique. The methodology employed in this work is a fast way to obtain a first approach of the enantioselective in vitro metabolism of racemic drugs, with the additional advantage of an extremely low consumption of enzymes, CDs and all the reagents involved in the process. Michaelis-Menten kinetic parameters (Km and Vmax ) for the metabolism of FLX enantiomers by CYP2D6 have been estimated by nonlinear fitting of experimental data to the Michaelis-Menten equation. Km values have been found to be 30 ± 3 μM for S-FLX and 39 ± 5 μM for R-FLX. Vmax estimations were 28.6 ± 1.2 and 34 ± 2 pmol·min(-1) ·(pmol CYP)(-1) for S- and R-FLX, respectively. Similar results were obtained using a single enantiomer (R-FLX), indicating that the use of the racemate is a good option for obtaining enantioselective estimations. The results obtained show a slight enantioselectivity in favor of R-FLX. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Communication: Limitations of the stochastic quasi-steady-state approximation in open biochemical reaction networks

NASA Astrophysics Data System (ADS)

Thomas, Philipp; Straube, Arthur V.; Grima, Ramon

2011-11-01

It is commonly believed that, whenever timescale separation holds, the predictions of reduced chemical master equations obtained using the stochastic quasi-steady-state approximation are in very good agreement with the predictions of the full master equations. We use the linear noise approximation to obtain a simple formula for the relative error between the predictions of the two master equations for the Michaelis-Menten reaction with substrate input. The reduced approach is predicted to overestimate the variance of the substrate concentration fluctuations by as much as 30%. The theoretical results are validated by stochastic simulations using experimental parameter values for enzymes involved in proteolysis, gluconeogenesis, and fermentation.
Analysis of Mathematical Modelling on Potentiometric Biosensors

PubMed Central

Mehala, N.; Rajendran, L.

2014-01-01

A mathematical model of potentiometric enzyme electrodes for a nonsteady condition has been developed. The model is based on the system of two coupled nonlinear time-dependent reaction diffusion equations for Michaelis-Menten formalism that describes the concentrations of substrate and product within the enzymatic layer. Analytical expressions for the concentration of substrate and product and the corresponding flux response have been derived for all values of parameters using the new homotopy perturbation method. Furthermore, the complex inversion formula is employed in this work to solve the boundary value problem. The analytical solutions obtained allow a full description of the response curves for only two kinetic parameters (unsaturation/saturation parameter and reaction/diffusion parameter). Theoretical descriptions are given for the two limiting cases (zero and first order kinetics) and relatively simple approaches for general cases are presented. All the analytical results are compared with simulation results using Scilab/Matlab program. The numerical results agree with the appropriate theories. PMID:25969765
Analysis of mathematical modelling on potentiometric biosensors.

PubMed

Mehala, N; Rajendran, L

2014-01-01

A mathematical model of potentiometric enzyme electrodes for a nonsteady condition has been developed. The model is based on the system of two coupled nonlinear time-dependent reaction diffusion equations for Michaelis-Menten formalism that describes the concentrations of substrate and product within the enzymatic layer. Analytical expressions for the concentration of substrate and product and the corresponding flux response have been derived for all values of parameters using the new homotopy perturbation method. Furthermore, the complex inversion formula is employed in this work to solve the boundary value problem. The analytical solutions obtained allow a full description of the response curves for only two kinetic parameters (unsaturation/saturation parameter and reaction/diffusion parameter). Theoretical descriptions are given for the two limiting cases (zero and first order kinetics) and relatively simple approaches for general cases are presented. All the analytical results are compared with simulation results using Scilab/Matlab program. The numerical results agree with the appropriate theories.
Development of a novel non-radioactive cell-based method for the screening of SGLT1 and SGLT2 inhibitors using 1-NBDG.

PubMed

Chang, Hung-Chi; Yang, Su-Fu; Huang, Ching-Chun; Lin, Tzung-Sheng; Liang, Pi-Hui; Lin, Chun-Jung; Hsu, Lih-Ching

2013-08-01

Sodium-coupled glucose co-transporters SGLT1 and SGLT2 play important roles in intestinal absorption and renal reabsorption of glucose, respectively. Blocking SGLT2 is a novel mechanism for lowering the blood glucose level by inhibiting renal glucose reabsorption and selective SGLT2 inhibitors are under development for treatment of type 2 diabetes. Furthermore, it has been reported that perturbation of SGLT1 is associated with cardiomyopathy and cancer. Therefore, both SGLT1 and SGLT2 are potential therapeutic targets. Here we report the development of a non-radioactive cell-based method for the screening of SGLT inhibitors using COS-7 cells transiently expressing human SGLT1 (hSGLT1), CHO-K1 cells stably expressing human SGLT2 (hSGLT2), and a novel fluorescent d-glucose analogue 1-NBDG as a substrate. Our data indicate that 1-NBDG can be a good replacement for the currently used isotope-labeled SGLT substrate, (14)C-AMG. The Michaelis constant of 1-NBDG transport (0.55 mM) is similar to that of d-glucose (0.51 mM) and AMG (0.40 mM) transport through hSGLT1. The IC50 values of a SGLT inhibitor phlorizin for hSGLT1 obtained using 1-NBDG and (14)C-AMG were identical (0.11 μM) in our cell-based system. The IC50 values of dapagliflozin, a well-known selective SGLT2 inhibitor, for hSGLT2 and hSGLT1 determined using 1-NBDG were 1.86 nM and 880 nM, respectively, which are comparable to the published results obtained using (14)C-AMG. Compared to (14)C-AMG, the use of 1-NBDG is cost-effective, convenient and potentially more sensitive. Taken together, a non-radioactive system using 1-NBDG has been validated as a rapid and reliable method for the screening of SGLT1 and SGLT2 inhibitors.
Species difference in the mechanism of nonlinear pharmacokinetics of E2074, a novel sodium channel inhibitor, in rats, dogs, and monkeys.

PubMed

Nagaya, Yoko; Takenaka, Osamu; Kusano, Kazutomi; Yoshimura, Tsutomu

2013-05-01

New chemical entities often exhibit nonlinear pharmacokinetics (PK) profiles in experimental animals. However, the number of studies that have focused on species differences in nonlinear PK is very limited; thus, the aim of this study was to clarify the mechanism of the nonlinear PK of E2074 (2-[(2R)-2-fluoro-3-{(3r)-[(3-fluorobenzyl)oxy]-8-azabicyclo[3.2.1]oct-8-yl}propyl]-4,5-dimethyl-2,4-dihydro-3H-1,2,4-triazol-3-one), a novel sodium channel inhibitor, in rats, dogs, and monkeys. Nonlinear PK profiles with more than dose-proportional increases of Cmax and area under the plasma concentration curve were observed in all species after oral administration. The Michaelis-Menten constant (Km) values of hepatic microsomal metabolism were 7.23 and 0.41 μM in rats and dogs in vitro, respectively, which were lower than the unbound maximum plasma concentrations after oral administration in vivo, indicating that the nonlinear PK in rats and dogs was attributable to the saturation of hepatic metabolism. However, we do not believe that the saturation of hepatic metabolism was the mechanism of nonlinearity in monkeys because of the high Km value (42.44 μM) observed in liver microsomes. Intestinal metabolism was observed in monkey intestinal microsomes but not in rats and dogs, and the nonlinear PK in monkeys was diminished by inhibition of intestinal metabolism with a concomitant oral dose of ketoconazole. These results suggest that saturation of the intestinal metabolism is the potential mechanism of nonlinearity in monkeys. P-glycoprotein was not involved in the nonlinear PK profiles in any species. In conclusion, the mechanism of the nonlinear PK of E2074 is species dependent, with the saturation of hepatic metabolism in rats and dogs and that of intestinal metabolism in monkeys being the primary cause.
Receptor protein of Lysinibacillus sphaericus mosquito-larvicidal toxin displays amylomaltase activity.

PubMed

Sharma, Mahima; Gupta, Gagan D; Kumar, Vinay

2018-02-01

The activated binary toxin (BinAB) from Lysinibacillus sphaericus binds to surface receptor protein (Cqm1) on the midgut cell membrane and kills Culex quinquefasciatus larvae on internalization. Cqm1 is attached to cells via a glycosyl-phosphatidylinositol (GPI) anchor. It has been classified as a member of glycoside hydrolase family 13 of the CAZy database. Here, we report characterization of the ordered domain (residues 23-560) of Cqm1. Gene expressing Cqm1 of BinAB susceptible mosquito was chemically synthesized and the protein was purified using E. coli expression system. Values for the Michaelis-Menten kinetics parameters towards 4-nitrophenyl α-D-glucopyranoside (α-pNPG) substrate were estimated to be 0.44 mM (Km) and 1.9 s -1 (kcat). Thin layer chromatography experiments established Cqm1 as α-glucosidase competent to cleave α-1,4-glycosidic bonds of maltose and maltotriose with high glycosyltransferase activity to form glucose-oligomers. The observed hydrolysis and synthesis of glucose-oligomers is consistent with open and accessible active-site in the structural model. The protein also hydrolyses glycogen and sucrose. These activities suggest that Cqm1 may be involved in carbohydrate metabolism in mosquitoes. Further, toxic BinA component does not inhibit α-glucosidase activity of Cqm1, while BinB reduced the activity by nearly 50%. The surface plasmon resonance study reveals strong binding of BinB with Cqm1 (Kd, 9.8 nM). BinA interaction with Cqm1 however, is 1000-fold weaker. Notably the estimated Kd values match well with dissociation constants reported earlier with larvae brush border membrane fractions. The Cqm1 protein forms a stable dimer that is consistent with its apical localization in lipid rafts. Its melting temperature (T m ) as observed by thermofluor-shift assay is 51.5 °C and Ca 2+ provides structural stability to the protein. Copyright © 2017 Elsevier Ltd. All rights reserved.
Biosynthesis of agmatine in isolated mitochondria and perfused rat liver: studies with 15N-labelled arginine

PubMed Central

2005-01-01

An important but unresolved question is whether mammalian mitochondria metabolize arginine to agmatine by the ADC (arginine decarboxylase) reaction. 15N-labelled arginine was used as a precursor to address this question and to determine the flux through the ADC reaction in isolated mitochondria obtained from rat liver. In addition, liver perfusion system was used to examine a possible action of insulin, glucagon or cAMP on a flux through the ADC reaction. In mitochondria and liver perfusion, 15N-labelled agmatine was generated from external 15N-labelled arginine. The production of 15N-labelled agmatine was time- and dose-dependent. The time-course of [U-15N4]agmatine formation from 2 mM [U-15N4]arginine was best fitted to a one-phase exponential curve with a production rate of approx. 29 pmol·min−1·(mg of protein)−1. Experiments with an increasing concentration (0– 40 mM) of [guanidino-15N2]arginine showed a Michaelis constant Km for arginine of 46 mM and a Vmax of 3.7 nmol·min−1·(mg of protein)−1 for flux through the ADC reaction. Experiments with broken mitochondria showed little changes in Vmax or Km values, suggesting that mitochondrial arginine uptake had little effect on the observed Vmax or Km values. Experiments with liver perfusion demonstrated that over 95% of the effluent agmatine was derived from perfusate [guanidino-15N2]arginine regardless of the experimental condition. However, the output of 15N-labelled agmatine (nmol·min−1·g−1) increased by approx. 2-fold (P<0.05) in perfusions with cAMP. The findings of the present study provide compelling evidence that mitochondrial ADC is present in the rat liver, and suggest that cAMP may stimulate flux through this pathway. PMID:15656789
Phosphoryl transfer reaction snapshots in crystals: Insights into the mechanism of protein kinase a catalytic subunit

DOE PAGES

Das, Amit; Gerlits, Oksana O.; Heller, William T.; ...

2015-06-19

To study the catalytic mechanism of phosphorylation catalyzed by cAMP-dependent protein kinase (PKA) a structure of the enzyme-substrate complex representing the Michaelis complex is of specific interest as it can shed light on the structure of the transition state. However, all previous crystal structures of the Michaelis complex mimics of the PKA catalytic subunit (PKAc) were obtained with either peptide inhibitors or ATP analogs. Here we utilized Ca 2+ ions and sulfur in place of the nucleophilic oxygen in a 20-residue pseudo-substrate peptide (CP20) and ATP to produce a close mimic of the Michaelis complex. In the ternary reactant complex,more » the thiol group of Cys-21 of the peptide is facing Asp-166 and the sulfur atom is positioned for an in-line phosphoryl transfer. Replacement of Ca 2+ cations with Mg 2+ ions resulted in a complex with trapped products of ATP hydrolysis: phosphate ion and ADP. As a result, the present structural results in combination with the previously reported structures of the transition state mimic and phosphorylated product complexes complete the snapshots of the phosphoryl transfer reaction by PKAc, providing us with the most thorough picture of the catalytic mechanism to date.« less

Cycling of Dissolved Organic Phosphorus and Alkaline Phosphatase Activity in Euphotic Zone of the Western North Pacific

NASA Astrophysics Data System (ADS)

Suzumura, M.

2010-12-01

Phosphorus is an essential nutrient for marine organisms. In oligotrophic environments, concentrations of dissolved inorganic phosphate (SRP), the most bioavailable form of phosphorus, are low and have been hypothesized to constrain the primary productivity. Evidence has been found that dissolved organic phosphorus (DOP) supports a significant fraction of primary production through hydrolytic remineralization of DOP to SRP by alkaline phosphatase (APA). In this study, DOP biogeochemistry was investigated at three locations of the open-ocean environment in the Kuroshio region and at a semi-eutrophic coastal site of the western North Pacific. Concentrations of SRP, DOP and hydrolyzable ester-P were measured in the euphotic zone. Kinetic parameters of APA were determined using a fluorogenic substrate, including potential maximum velocity (Vmax), apparent Michaelis-Menten half-saturation constant (Km), and turnover time (TA) of APA hydrolyzable DOP. SRP concentrations were quite low (≤ 10 nM) in the surface seawater and rapidly increased below the chlorophyll a maximum layer (CML). DOP concentration ranged from 29 to 223 nM. Above the CML, DOP composed a major fraction accounting for 60-100% of dissolved total P. A significant linear relationship was found between the concentrations of SRP and hydrolyzable ester-P (R2 = 0.83, P < 0.01). This suggests active utilization of ester-P under phosphate-depleted conditions. In the Kuroshio region, Vmax of APA exhibited the highest value at the surface water (0 m) and decreased rapidly with depth, while at the coastal site the peak value was found at CML. TA of hydrolyzable DOP was quite variable among the locations and increased with depth especially below CML. The estimated values of in situ hydrolysis rate were much lower (2-34%) than the potential Vmax which was determined with the addition of an excess amount of the substrate. The results suggest that marine microbes can efficiently and rapidly utilize hydrolyzable DOP under phosphate-depleted conditions and that there is still room in the in situ APA activity. Utilization of DOP, however, is likely regulated by the ambient concentrations of hydrolyzable ester-P lower than the apparent Km.
Evaluation of thrombin inhibitory activity of catechins by online capillary electrophoresis-based immobilized enzyme microreactor and molecular docking.

PubMed

Li, Qiao-Qiao; Yang, Feng-Qing; Wang, Yin-Zhen; Wu, Zhao-Yu; Xia, Zhi-Ning; Chen, Hua

2018-08-01

An online capillary electrophoresis (CE)-based thrombin (THR) immobilized enzyme microreactor (IMER) method was established to screen THR inhibitors in this study. S-2366 was used as chromogenic substrate for determination of THR activity and other kinetic constants. After continuously run for 50 times, the prepared IMER could still remain 89% of the initial immobilized enzyme activity. The Michaelis-Menten constant (K m ) of immobilized THR was measured as 0.514 mmol/L and the half-maximal inhibitory concentration (IC 50 ) and inhibition constant (K i ) of argatroban on THR were determined as 78.07 and 26.53 nmol/L, respectively, which indicated that CE-based THR IMER was successfully established and could be applied to screen THR inhibitors. Then the prepared IMER was used to investigate the inhibitory potency on THR of four main catechins in green tea including epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG), and epigallocatechin gallate (EGCG). The results showed that ECG and EGCG had good THR inhibition activity and their inhibition rates at concentration of 200 μmol/L were 53.2 ± 3.8% and 55.8 ± 2.6%, respectively, which was in consistent with the results of microplate reader assay. Additionally, molecular docking results showed that the benzopyran groups of ECG and EGCG were inserted into the THR active pocket and interacted with residues LYS60F, TRP60D, TRY60A, IEU99, GLY216, HIS57 and SER195, but EC and EGC did not. Therefore, the developed CE-based THR IMER is reliable method for measuring THR inhibitory activity of natural inhibitors. Copyright © 2018 Elsevier B.V. All rights reserved.
The reductive half-reaction of xanthine dehydrogenase from Rhodobacter capsulatus: the role of Glu232 in catalysis.

PubMed

Hall, James; Reschke, Stefan; Cao, Hongnan; Leimkühler, Silke; Hille, Russ

2014-11-14

The kinetic properties of an E232Q variant of the xanthine dehydrogenase from Rhodobacter capsulatus have been examined to ascertain whether Glu(232) in wild-type enzyme is protonated or unprotonated in the course of catalysis at neutral pH. We find that kred, the limiting rate constant for reduction at high [xanthine], is significantly compromised in the variant, a result that is inconsistent with Glu(232) being neutral in the active site of the wild-type enzyme. A comparison of the pH dependence of both kred and kred/Kd from reductive half-reaction experiments between wild-type and enzyme and the E232Q variant suggests that the ionized Glu(232) of wild-type enzyme plays an important role in catalysis by discriminating against the monoanionic form of substrate, effectively increasing the pKa of substrate by two pH units and ensuring that at physiological pH the neutral form of substrate predominates in the Michaelis complex. A kinetic isotope study of the wild-type R. capsulatus enzyme indicates that, as previously determined for the bovine and chicken enzymes, product release is principally rate-limiting in catalysis. The disparity in rate constants for the chemical step of the reaction and product release, however, is not as great in the bacterial enzyme as compared with the vertebrate forms. The results indicate that the bacterial and bovine enzymes catalyze the chemical step of the reaction to the same degree and that the faster turnover observed with the bacterial enzyme is due to a faster rate constant for product release than is seen with the vertebrate enzyme. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
Diffusion-controlled interface kinetics-inclusive system-theoretic propagation models for molecular communication systems

NASA Astrophysics Data System (ADS)

Chude-Okonkwo, Uche A. K.; Malekian, Reza; Maharaj, B. T.

2015-12-01

Inspired by biological systems, molecular communication has been proposed as a new communication paradigm that uses biochemical signals to transfer information from one nano device to another over a short distance. The biochemical nature of the information transfer process implies that for molecular communication purposes, the development of molecular channel models should take into consideration diffusion phenomenon as well as the physical/biochemical kinetic possibilities of the process. The physical and biochemical kinetics arise at the interfaces between the diffusion channel and the transmitter/receiver units. These interfaces are herein termed molecular antennas. In this paper, we present the deterministic propagation model of the molecular communication between an immobilized nanotransmitter and nanoreceiver, where the emission and reception kinetics are taken into consideration. Specifically, we derived closed-form system-theoretic models and expressions for configurations that represent different communication systems based on the type of molecular antennas used. The antennas considered are the nanopores at the transmitter and the surface receptor proteins/enzymes at the receiver. The developed models are simulated to show the influence of parameters such as the receiver radius, surface receptor protein/enzyme concentration, and various reaction rate constants. Results show that the effective receiver surface area and the rate constants are important to the system's output performance. Assuming high rate of catalysis, the analysis of the frequency behavior of the developed propagation channels in the form of transfer functions shows significant difference introduce by the inclusion of the molecular antennas into the diffusion-only model. It is also shown that for t > > 0 and with the information molecules' concentration greater than the Michaelis-Menten kinetic constant of the systems, the inclusion of surface receptors proteins and enzymes in the models makes the system act like a band-stop filter over an infinite frequency range.
CODATA recommended values of the fundamental constants

NASA Astrophysics Data System (ADS)

Mohr, Peter J.; Taylor, Barry N.

2000-11-01

A review is given of the latest Committee on Data for Science and Technology (CODATA) adjustment of the values of the fundamental constants. The new set of constants, referred to as the 1998 values, replaces the values recommended for international use by CODATA in 1986. The values of the constants, and particularly the Rydberg constant, are of relevance to the calculation of precise atomic spectra. The standard uncertainty (estimated standard deviation) of the new recommended value of the Rydberg constant, which is based on precision frequency metrology and a detailed analysis of the theory, is approximately 1/160 times the uncertainty of the 1986 value. The new set of recommended values as well as a searchable bibliographic database that gives citations to the relevant literature is available on the World Wide Web at physics.nist.gov/constants and physics.nist.gov/constantsbib, respectively. .
Comparative kinetics and reciprocal inhibition of nitrate and nitrite uptake in roots of uninduced and induced barley (Hordeum vulgare L.) seedlings

NASA Technical Reports Server (NTRS)

Aslam, M.; Travis, R. L.; Huffaker, R. C.

1992-01-01

Nitrate and NO2- transport by roots of 8-day-old uninduced and induced intact barley (Hordeum vulgare L. var CM 72) seedlings were compared to kinetic patterns, reciprocal inhibition of the transport systems, and the effect of the inhibitor, p-hydroxymercuribenzoate. Net uptake of NO3- and NO2- was measured by following the depletion of the ions from the uptake solutions. The roots of uninduced seedlings possessed a low concentration, saturable, low Km, possibly a constitutive uptake system, and a linear system for both NO3- and NO2-. The low Km system followed Michaelis-Menten kinetics and approached saturation between 40 and 100 micromolar, whereas the linear system was detected between 100 and 500 micromolar. In roots of induced seedlings, rates for both NO3- and NO2- uptake followed Michaelis-Menten kinetics and approached saturation at about 200 micromolar. In induced roots, two kinetically identifiable transport systems were resolved for each anion. At the lower substrate concentrations, less than 10 micromolar, the apparent low Kms of NO3- and NO2- uptake were 7 and 9 micromolar, respectively, and were similar to those of the low Km system in uninduced roots. At substrate concentrations between 10 and 200 micromolar, the apparent high Km values of NO3- uptake ranged from 34 to 36 micromolar and of NO2- uptake ranged from 41 to 49 micromolar. A linear system was also found in induced seedlings at concentrations above 500 micromolar. Double reciprocal plots indicated that NO3- and NO2- inhibited the uptake of each other competitively in both uninduced and induced seedlings; however, Ki values showed that NO3- was a more effective inhibitor than NO2-. Nitrate and NO2- transport by both the low and high Km systems were greatly inhibited by p-hydroxymercuribenzoate, whereas the linear system was only slightly inhibited.
Stimulatory effect of calcium on metabolism and its sensitivity to pH in kidney mitochondria.

PubMed

Drewnowska, K; Schoolwerth, A C

1994-07-01

The relationship between mitochondrial matrix free Ca2+ concentration ([Ca2+]m) and pH was evaluated by incubating isolated rat kidney mitochondria with different extramitochondrial Ca2+ concentrations ([Ca2+]e) at medium pH (pHe) 7.0 and 7.4. [Ca2+]m was monitored using the fluorescent signal from mitochondria loaded with the Ca2+ indicator fura 2. The changes in [Ca2+]m were compared with alpha-ketoglutarate dehydrogenase (alpha-KGDH) flux, measured as O2 consumption (nmol.min-1.mg protein-1) from 185 microM alpha-ketoglutarate (alpha-KG). The apparent dissociation constant of the matrix fluorescent probe for Ca2+ was determined in each experiment and was 323 +/- 45 nM (n = 14). When mitochondria were exposed to [Ca2+]e below 160 nM, [Ca2+]m was greater at pHe 7.0 than at pHe 7.4. However, above 160 nM [Ca2+]e, [Ca2+]m plateaued at pHe 7.0 but rose progressively at pHe 7.4. Increasing [Ca2+]m by consecutive additions of Ca2+ to the medium had a significantly more pronounced acceleratory effect on alpha-KG oxidation at pHe 7.0 than at pHe 7.4. Kinetic analysis of alpha-KGDH revealed a 45% decrease in the Michaelis constant (Km) for alpha-KG at pHe 7.0, but the Km was unchanged at pHe 7.4 with elevation of [Ca2+]m from 32 to 751 nM. Maximal velocity (Vmax) increased significantly at both pHe values. Half-maximal alpha-KG oxidation occurred at [Ca2+]m of 76 +/- 11 nM and 105 +/- 31 nM at pHe 7.0 and 7.4, respectively. These studies demonstrate a direct, pH-sensitive correlation between [Ca2+]e and [Ca2+]m; [Ca2+]m changed over a range that may regulate alpha-KGDH flux in intact kidney mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)
Influence of substrate structure on turnover of the organic cation/H+ exchanger of the renal luminal membrane.

PubMed

Wright, S H; Wunz, T M

1998-08-01

We examined the influence of organic cation (OC) structure on the rate of turnover of the OC/H+ exchanger in rabbit renal brush-border membrane vesicles (BBMV). The rate of efflux of [14C]tetraethylammonium ([14C]TEA) from BBMV, measured in the presence of an inwardly directed chemical gradient for test agent, provided an indirect measure of activity of the OC/H+(OC) exchanger. The trans-stimulation of [14C]TEA efflux from BBMV was a saturable function of increasing extravesicular concentration of both unlabeled TEA and tetramethylammonium (TMA), with an apparent Michaelis constant (Kt) for the interaction of these compounds with the OC/H+(OC) exchanger of 25 microM and 1 mM, respectively. The effect on [14C]TEA efflux of saturating extravesicular concentrations of a series of n-tetraalkylammonium compounds was examined. Whereas the short-chain compounds TMA and TEA markedly stimulated [14C]TEA efflux (by 830% and 690%, respectively), the long-chain compounds tetrapropylammonium and tetrabutylammonium were less effective, increasing efflux by only 40% and 120%, respectively. When the exchanger was saturated with tetrapentylammonium, mediated efflux of [14C]TEA was reduced. Increasing alkyl chain length was also correlated with an increase in the inhibitory effect (as measured by the apparent inhibition constant, Ki, or the IC50 value) that these compounds had against transport of [14C]TEA by the OC/H+(OC) exchanger; i.e., there was a correlation between decreasing IC50 and decreasing turnover of the OC/H+(OC) exchanger. This same correlation was observed for a broader set of test agents of diverse molecular structure, including a series of n-tetraalkylammonium and -phosphonium compounds and the OCs, choline, N1-methyl nicotinamide, 1-methyl-4-phenylpyridinium, and amiloride. Because high affinity of substrates for the OC/H+(OC) exchanger is correlated with increasing substrate hydrophobicity, we conclude that the interaction of hydrophobic OCs with the renal OC/H+(OC) exchanger results in the formation of a substrate-exchanger complex that has a comparatively low rate of turnover.
A century of enzyme kinetic analysis, 1913 to 2013.

PubMed

Johnson, Kenneth A

2013-09-02

This review traces the history and logical progression of methods for quantitative analysis of enzyme kinetics from the 1913 Michaelis and Menten paper to the application of modern computational methods today. Following a brief review of methods for fitting steady state kinetic data, modern methods are highlighted for fitting full progress curve kinetics based upon numerical integration of rate equations, including a re-analysis of the original Michaelis-Menten full time course kinetic data. Finally, several illustrations of modern transient state kinetic methods of analysis are shown which enable the elucidation of reactions occurring at the active sites of enzymes in order to relate structure and function. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Enzymatic production and characterization of manno-oligosaccharides from Gleditsia sinensis galactomannan gum.

PubMed

Jian, Hong-Lei; Zhu, Li-Wei; Zhang, Wei-Ming; Sun, Da-Feng; Jiang, Jian-Xin

2013-04-01

Enzymatic hydrolysis of Gleditsia sinensis gum was performed to produce manno-oligosaccharides having functional applications as dietary fiber and prebiotics. The optimum hydrolysis conditions, including enzyme loading, temperature and time, from response surface methodology were 8.1 U/g, 57.4 °C and 34.1 h, respectively. The yield of DP 1-5 oligosaccharides was 75.9% (29.1 g/L). The Michaelis-Menten kinetics and molecular weight distribution were determined. The obtained oligosaccharides were further separated by HPLC and SEC, and the galactose distribution of G. sinensis gum was elucidated. Results indicated that G. sinensis gum has potential to produce value-added oligosaccharides in food industries. Copyright © 2013 Elsevier B.V. All rights reserved.
Cyanide removal by Chinese vegetation--quantification of the Michaelis-Menten kinetics.

PubMed

Yu, Xiaozhang; Zhou, Puhua; Zhou, Xishi; Liu, Yunda

2005-07-01

Little is known about metabolism rates of environmental chemicals by vegetation. A good model compound to study the variation of rates among plant species is cyanide. Vascular plants possess an enzyme system that detoxifies cyanide by converting it to the amino acid asparagine. Knowledge of the kinetic parameters, the half-saturation constant (Km) and the maximum metabolic capacity (vmax), is very useful for enzyme characterization and biochemical purposes. The goal of this study is to find the enzyme kinetics (K(M) and vmax) during cyanide metabolism in the presence of Chinese vegetation, to provide quantitative data for engineered phytoremediation, and to investigate the variation of metabolic rates of plants. Detached leaves (1.0 g fresh weight) from 12 species out of 9 families were kept in glass vessels with 100 mL of aqueous solution spiked with potassium cyanide at 23 degrees C for 28 h. Four different treatment concentrations of cyanide were used, ranging from 0.44 to 7.69 mg CN/L. The disappearance of cyanide from the aqueous solution was analyzed spectrophotometrically. Realistic values of the half-saturation constant (KM) and the maximum metabolic capacity (vmax) were estimated by a computer program using non-linear regression treatments. As a comparison, Lineweaver-Burk plots were also used to estimate the kinetic parameters. The values obtained for K(M) and vmax varied with plant species. Using non-linear regression treatments, values of vmax and K(M) were found in a range between 6.68 and 21.91 mg CN/kg/h and 0.90 to 3.15 mg CN/L, respectively. The highest vmax was by Chinese elder (Sambucus chinensis), followed by upright hedge-parsley (Torilis japonica). The lowest Vmax was demonstrated by the hybrid willow (Salix matssudana x alba). However, the highest K(M) was found in the water lily (Nymphea teragona), followed by the poplar (Populus deltoides Marsh). The lowest K(M) was demonstrated by corn (Zea mays L.). The values of vmax were normally distributed with a mean of 13 mg CN/kg/h. Significant removal of cyanide from aqueous solution was observed in the presence of plant materials without phytotoxicity, even at high doses of cyanide. This gives rise to the conclusion that the Chinese plant species used in this study are all able to efficiently metabolize cyanide, although with different maximum metabolic capacities. A second conclusion is that the variation of metabolism rates between species is small. All these plants had a similar K(M), indicating the same enzyme is active in all plants. Detoxification of cyanide with trees seems to be a feasible option for cleaning soils and water contaminated with cyanide. For phytoremediation projects, screening appropriate plant species adapted to local conditions should be seriously considered. More chemicals should be investigated to find common principles of the metabolism of environmental chemicals by plants.
Part-2: Analytical Expressions of Concentrations of Glucose, Oxygen, and Gluconic Acid in a Composite Membrane for Closed-Loop Insulin Delivery for the Non-steady State Conditions.

PubMed

Mehala, N; Rajendran, L; Meena, V

2017-02-01

A mathematical model developed by Abdekhodaie and Wu (J Membr Sci 335:21-31, 2009), which describes a dynamic process involving an enzymatic reaction and diffusion of reactants and product inside glucose-sensitive composite membrane has been discussed. This theoretical model depicts a system of non-linear non-steady state reaction diffusion equations. These equations have been solved using new approach of homotopy perturbation method and analytical solutions pertaining to the concentrations of glucose, oxygen, and gluconic acid are derived. These analytical results are compared with the numerical results, and limiting case results for steady state conditions and a good agreement is observed. The influence of various kinetic parameters involved in the model has been presented graphically. Theoretical evaluation of the kinetic parameters like the maximal reaction velocity (V max ) and Michaelis-Menten constants for glucose and oxygen (K g and K ox ) is also reported. This predicted model is very much useful for designing the glucose-responsive composite membranes for closed-loop insulin delivery.
DOE Office of Scientific and Technical Information (OSTI.GOV)

Tam, Tsz Kin; Chen, Baowei; Lei, Chenghong

NAD/NADH is a coenzyme found in all living cells, carrying electrons from one reaction to another. We report on characterizations of in situ regeneration of NADH via lipoamide dehydrogenase (LD)-catalyzed electron transfer reaction to regenerate NADH using UV-vis spectroelectrochemistry. The Michaelis-Menten constant (Km) and maximum velocity (Vmax) of NADH regeneration were measured as 0.80 {+-} 0.15 mM and 1.91 {+-} 0.09 {micro}M s-1 in a 1-mm thin-layer spectroelectrochemical cell using gold gauze as the working electrode at the applied potential -0.75 V (vs. Ag/AgCl). The electrocatalytic reduction of the NAD system was further coupled with the enzymatic conversion of pyruvatemore » to lactate by lactate dehydrogenase to examine the coenzymatic activity of the regenerated NADH. Although the reproducible electrocatalytic reduction of NAD into NADH is known to be difficult compared to the electrocatalytic oxidation of NADH, our spectroelectrochemical results indicate that the in situ regeneration of NADH via LD-catalyzed electron transfer reaction is fast and sustainable and can be potentially applied to many NAD/NADH-dependent enzyme systems.« less
Efficacy of wood charcoal and its modified form as packing media for biofiltration of isoprene.

PubMed

Srivastva, Navnita; Singh, Ram S; Dubey, Suresh K

2017-07-01

The efficacy of wood charcoal (WC) and nutrient-enriched wood charcoal (NWC) as biofilter packing media were assessed for isoprene biodegradation in a bioreactor comprising bioscrubber and a biofilter connected in series and inoculated with Pseudomonas sp. The bioreactors using WC and NWC exhibited >90% removal efficiency and around 369 g m -3 h -1 elimination capacity at around 404 g m -3 h -1 inlet loading rate. In both the bioreactors, the biofilter component showed better degradation capacity compared to the bioscrubber unit. The kinetic parameters, maximum elimination capacity, EC max ; substrate constant, K s and EC max /K s for Michaelis-Menten model were evaluated. The lower K s for the WC packed bioreactor indicated that EC max achieved, was faster compared to others, while higher EC max and EC max /K s for the NWC packed bioreactor suggests its superiority in isoprene abatement in the continuous mode. A comparison of the available published information on biofiltration of isoprene reflected polyurethane foam as the superior packing media. Copyright © 2017 Elsevier Ltd. All rights reserved.
Direct electrochemistry of nitrate reductase from the fungus Neurospora crassa.

PubMed

Kalimuthu, Palraj; Ringel, Phillip; Kruse, Tobias; Bernhardt, Paul V

2016-09-01

We report the first direct (unmediated) catalytic electrochemistry of a eukaryotic nitrate reductase (NR). NR from the filamentous fungus Neurospora crassa, is a member of the mononuclear molybdenum enzyme family and contains a Mo, heme and FAD cofactor which are involved in electron transfer from NAD(P)H to the (Mo) active site where reduction of nitrate to nitrite takes place. NR was adsorbed on an edge plane pyrolytic graphite (EPG) working electrode. Non-turnover redox responses were observed in the absence of nitrate from holo NR and three variants lacking the FAD, heme or Mo cofactor. The FAD response is due to dissociated cofactor in all cases. In the presence of nitrate, NR shows a pronounced cathodic catalytic wave with an apparent Michaelis constant (KM) of 39μM (pH7). The catalytic cathodic current increases with temperature from 5 to 35°C and an activation enthalpy of 26kJmol(-1) was determined. In spite of dissociation of the FAD cofactor, catalytically activity is maintained. Copyright © 2016. Published by Elsevier B.V.
MOFzyme: Intrinsic protease-like activity of Cu-MOF.

PubMed

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

2014-10-24

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

NASA Astrophysics Data System (ADS)

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

2014-10-01

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

PubMed Central

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

2014-01-01

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

PubMed Central

Ou, Yangguang; Wu, Juanfang; Sandberg, Mats

2014-01-01

This review covers recent advances in sampling fluid from the extracellular space of brain tissue by electroosmosis (EO). Two techniques, EO sampling with a single fused-silica capillary and EO push–pull perfusion, have been developed. These tools were used to investigate the function of membrane-bound enzymes with outward-facing active sites, or ectoenzymes, in modulating the activity of the neuropeptides leu-enkephalin and galanin in organotypic-hippocampal-slice cultures (OHSCs). In addition, the approach was used to determine the endogenous concentration of a thiol, cysteamine, in OHSCs. We have also investigated the degradation of coenzyme A in the extracellular space. The approach provides information on ectoenzyme activity, including Michaelis constants, in tissue, which, as far as we are aware, has not been done before. On the basis of computational evidence, EO push–pull perfusion can distinguish ectoenzyme activity with a ~100 µm spatial resolution, which is important for studies of enzyme kinetics in adjacent regions of the rat hippocampus. PMID:25168111
Morphine-induced kinetic alterations of choline acetyltransferase of the rat caudate nucleus

PubMed Central

Datta, K.; Wajda, I. J.

1972-01-01

1. In order to explain the decrease of choline acetyltransferase (2.3.1.6.) activity observed in the caudate nucleus of morphine-treated rats, partially purified preparations of the enzyme were used in kinetic studies, with choline as substrate. 2. The apparent Michaelis constant for the enzyme obtained from normal rats was found to be 0·9 mM choline; this value doubled when the animals were killed one hour after a single injection of morphine (30 mg/kg). When the rats were injected daily for 4 or 15 days, and killed one hour after the last injection, the apparent Km value was 2·1 mM in each case. Prolonged daily treatment with morphine, followed by 48 h withdrawal, or by administration of 4 mg/kg of naloxone (given half an hour after the last injection of morphine) resulted in apparent Km values of 1·3-1·5 mM of choline, suggesting a gradual return to the lower, normal substrate requirement. Vmax changes were insignificant. 3. The effect of morphine added in vitro to different enzyme preparations was also studied. The Km values of 0·9 mM, in the enzyme isolated from normal rats, increased to 2·0 after incubation in vitro with 12·5 mM morphine. Similar increases were found in enzymes obtained from rats 48 h after the withdrawal of morphine or from rats injected with naloxone after prolonged morphine treatment. The high apparent Km values, found in enzyme obtained from animals killed one hour after the last dose of morphine, did not change upon incubation with 12·5 mM morphine. A similar pattern of Km changes was noticed after incubation with 25 mM acetylcholine. 4. An increase of 32% in acetylcholine (ACh) level was found in the caudate nucleus one hour after subcutaneous injection of 30 mg/kg of morphine. Return to normal values was observed when morphine was administered daily. After two to three weeks of daily treatment and subsequent withdrawal from morphine for 48 h, the levels of ACh were normal. If the daily treated rats were given naloxone within half an hour of the last injection of morphine, and killed 30 min later, the levels of ACh remained normal. 5. Fifty per cent inhibition of enzyme activity was observed upon in vitro incubation with 75 mM acetylcholine, or with 25 mM morphine. The same degree of inhibition was noticed when the enzyme was obtained from normal or from morphine-treated rats. PMID:5041452

Large variation in the Rubisco kinetics of diatoms reveals diversity among their carbon-concentrating mechanisms.

PubMed

Young, Jodi N; Heureux, Ana M C; Sharwood, Robert E; Rickaby, Rosalind E M; Morel, François M M; Whitney, Spencer M

2016-05-01

While marine phytoplankton rival plants in their contribution to global primary productivity, our understanding of their photosynthesis remains rudimentary. In particular, the kinetic diversity of the CO2-fixing enzyme, Rubisco, in phytoplankton remains unknown. Here we quantify the maximum rates of carboxylation (k cat (c)), oxygenation (k cat (o)), Michaelis constants (K m) for CO2 (K C) and O2 (K O), and specificity for CO2 over O2 (SC/O) for Form I Rubisco from 11 diatom species. Diatom Rubisco shows greater variation in K C (23-68 µM), SC/O (57-116mol mol(-1)), and K O (413-2032 µM) relative to plant and algal Rubisco. The broad range of K C values mostly exceed those of C4 plant Rubisco, suggesting that the strength of the carbon-concentrating mechanism (CCM) in diatoms is more diverse, and more effective than previously predicted. The measured k cat (c) for each diatom Rubisco showed less variation (2.1-3.7s(-1)), thus averting the canonical trade-off typically observed between K C and k cat (c) for plant Form I Rubisco. Uniquely, a negative relationship between K C and cellular Rubisco content was found, suggesting variation among diatom species in how they allocate their limited cellular resources between Rubisco synthesis and their CCM. The activation status of Rubisco in each diatom was low, indicating a requirement for Rubisco activase. This work highlights the need to better understand the correlative natural diversity between the Rubisco kinetics and CCM of diatoms and the underpinning mechanistic differences in catalytic chemistry among the Form I Rubisco superfamily. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.
Large variation in the Rubisco kinetics of diatoms reveals diversity among their carbon-concentrating mechanisms

PubMed Central

Young, Jodi N.; Heureux, Ana M.C.; Sharwood, Robert E.; Rickaby, Rosalind E.M.; Morel, François M.M.; Whitney, Spencer M.

2016-01-01

While marine phytoplankton rival plants in their contribution to global primary productivity, our understanding of their photosynthesis remains rudimentary. In particular, the kinetic diversity of the CO2-fixing enzyme, Rubisco, in phytoplankton remains unknown. Here we quantify the maximum rates of carboxylation (k cat c), oxygenation (k cat o), Michaelis constants (K m) for CO2 (K C) and O2 (K O), and specificity for CO2 over O2 (SC/O) for Form I Rubisco from 11 diatom species. Diatom Rubisco shows greater variation in K C (23–68 µM), SC/O (57–116mol mol−1), and K O (413–2032 µM) relative to plant and algal Rubisco. The broad range of K C values mostly exceed those of C4 plant Rubisco, suggesting that the strength of the carbon-concentrating mechanism (CCM) in diatoms is more diverse, and more effective than previously predicted. The measured k cat c for each diatom Rubisco showed less variation (2.1–3.7s−1), thus averting the canonical trade-off typically observed between K C and k cat c for plant Form I Rubisco. Uniquely, a negative relationship between K C and cellular Rubisco content was found, suggesting variation among diatom species in how they allocate their limited cellular resources between Rubisco synthesis and their CCM. The activation status of Rubisco in each diatom was low, indicating a requirement for Rubisco activase. This work highlights the need to better understand the correlative natural diversity between the Rubisco kinetics and CCM of diatoms and the underpinning mechanistic differences in catalytic chemistry among the Form I Rubisco superfamily. PMID:27129950
pH regulation in barnacle muscle fibers: dependence on extracellular sodium and bicarbonate.

PubMed

Boron, W F; McCormick, W C; Roos, A

1981-01-01

Intracellular pH (pHi) regulation was studied in barnacle muscle fibers with pH-sensitive microelectrodes. The cells were acid loaded, and the subsequent recovery of pHi was monitored. The rate of recovery was reduced by one-third when external Na+ ([Na+]o) was replaced by Li+, but recovery was completely abolished when Na+ was replaced by choline or N-methyl-D-glucamine. In other experiments, varying amounts of Na+ were replaced by choline, and the acid extrusion rate, derived from the recovery rate of pHi, was calculated at a single value of pHi, 6.80. The dependence of the acid extrusion rate on [Na+]o could be described by Michaelis-Menten kinetics; at pHo (extracellular) = 8.0 and [HCO3-]o (extracellular) = 10 mM, the apparent Km and Vmax were 59 mM and 1.3 mmol x l(-1) x min-1. When [HCO3-]o was reduced to 2.5 mM at the same pHo, Km did not change significantly, but Vmax was substantially reduced. On the other hand, when pHo was reduced to 7.4 at constant [HCO3-]o, Vmax changed only slightly, but Km increased substantially. In similar experiments, we examined the dependence of the acid extrusion rate on [HCO3-]o. At pHo = 8.0 and [Na+]o = 440 mM, the apparent Km and Vmax were 4.1 mM and 2.1 mmol x 1-1 x min-1. When pHo was reduced to 7.4, Vmax was not altered, but Km substantially increased. The kinetic data are discussed in terms of the role of pHo, [Na+]o, and [HCO3-]o in the pHi-regulating system.
Heat-resistant cytosolic malate dehydrogenases (cMDHs) of thermophilic intertidal snails (genus Echinolittorina): protein underpinnings of tolerance to body temperatures reaching 55°C.

PubMed

Liao, Ming-Ling; Zhang, Shu; Zhang, Guang-Ya; Chu, Yun-Meng; Somero, George N; Dong, Yun-Wei

2017-06-01

Snails of the genus Echinolittorina are among the most heat-tolerant animals; they experience average body temperatures near 41-44°C in summer and withstand temperatures up to at least 55°C. Here, we demonstrate that heat stability of function (indexed by the Michaelis-Menten constant of the cofactor NADH, K M NADH ) and structure (indexed by rate of denaturation) of cytosolic malate dehydrogenases (cMDHs) of two congeners ( E. malaccana and E. radiata ) exceeds values previously found for orthologs of this protein from less thermophilic species. The ortholog of E. malaccana is more heat stable than that of E. radiata , in keeping with the congeners' thermal environments. Only two inter-congener differences in amino acid sequence in these 332 residue proteins were identified. In both cases (positions 48 and 114), a glycine in the E. malaccana ortholog is replaced by a serine in the E. radiata protein. To explore the relationship between structure and function and to characterize how amino acid substitutions alter stability of different regions of the enzyme, we used molecular dynamics simulation methods. These computational methods allow determination of thermal effects on fine-scale movements of protein components, for example, by estimating the root mean square deviation in atom position over time and the root mean square fluctuation for individual residues. The minor changes in amino acid sequence favor temperature-adaptive change in flexibility of regions in and around the active sites. Interspecific differences in effects of temperature on fine-scale protein movements are consistent with the differences in thermal effects on binding and rates of heat denaturation. © 2017. Published by The Company of Biologists Ltd.
Catalytic properties of the isolated diaphorase fragment of the NAD-reducing [NiFe]-hydrogenase from Ralstonia eutropha.

PubMed

Lauterbach, Lars; Idris, Zulkifli; Vincent, Kylie A; Lenz, Oliver

2011-01-01

The NAD+-reducing soluble hydrogenase (SH) from Ralstonia eutropha H16 catalyzes the H₂-driven reduction of NAD+, as well as reverse electron transfer from NADH to H+, in the presence of O₂. It comprises six subunits, HoxHYFUI₂, and incorporates a [NiFe] H+/H₂ cycling catalytic centre, two non-covalently bound flavin mononucleotide (FMN) groups and an iron-sulfur cluster relay for electron transfer. This study provides the first characterization of the diaphorase sub-complex made up of HoxF and HoxU. Sequence comparisons with the closely related peripheral subunits of Complex I in combination with UV/Vis spectroscopy and the quantification of the metal and FMN content revealed that HoxFU accommodates a [2Fe2S] cluster, FMN and a series of [4Fe4S] clusters. Protein film electrochemistry (PFE) experiments show clear electrocatalytic activity for both NAD+ reduction and NADH oxidation with minimal overpotential relative to the potential of the NAD+/NADH couple. Michaelis-Menten constants of 56 µM and 197 µM were determined for NADH and NAD+, respectively. Catalysis in both directions is product inhibited with K(I) values of around 0.2 mM. In PFE experiments, the electrocatalytic current was unaffected by O₂, however in aerobic solution assays, a moderate superoxide production rate of 54 nmol per mg of protein was observed, meaning that the formation of reactive oxygen species (ROS) observed for the native SH can be attributed mainly to HoxFU. The results are discussed in terms of their implications for aerobic functioning of the SH and possible control mechanism for the direction of catalysis.
Identification of essential active-site residues in the cyanogenic beta-glucosidase (linamarase) from cassava (Manihot esculenta Crantz) by site-directed mutagenesis.

PubMed Central

Keresztessy, Z; Brown, K; Dunn, M A; Hughes, M A

2001-01-01

The coding sequence of the mature cyanogenic beta-glucosidase (beta-glucoside glucohydrolase, EC 3.2.1.21; linamarase) was cloned into the vector pYX243 modified to contain the SUC2 yeast secretion signal sequence and expressed in Saccharomyces cerevisiae. The recombinant enzyme is active, glycosylated and showed similar stability to the plant protein. Michaelis constants for hydrolysis of the natural substrate, linamarin (K(m)=1.06 mM) and the synthetic p-nitrophenyl beta-D-glucopyranoside (PNP-Glc; K(m)=0.36 mM), as well as apparent pK(a) values of the free enzyme and the enzyme-substrate complexes (pK(E)(1)=4.4-4.8, pK(E)(2)=6.7-7.2, pK(ES)(1)=3.9-4.4, pK(ES)(2)=8.3) were very similar to those of the plant enzyme. Site-directed mutagenesis was carried out to study the function of active-site residues based on a homology model generated for the enzyme using the MODELLER program. Changing Glu-413 to Gly destroyed enzyme activity, consistent with it being the catalytic nucleophile. The Gln-339Glu mutation also abolished activity, confirming a function in positioning the catalytic diad. The Ala-201Val mutation shifted the pK(a) of the acid/base catalyst Glu-198 from 7.22 to 7.44, reflecting a change in its hydrophobic environment. A Phe-269Asn change increased K(m) for linamarin hydrolysis 16-fold (16.1 mM) and that for PNP-Glc only 2.5-fold (0.84 mM), demonstrating that Phe-269 contributes to the cyanogenic specificity of the cassava beta-glucosidase. PMID:11139381
Competitive inhibition of carcinogen-activating CYP1A1 and CYP1B1 enzymes by a standardized complex mixture of PAH extracted from coal tar

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

Mahadevan, B.; Marston, C.P.; Luch, A.

2007-03-15

A complex mixture of polycyclic aromatic hydrocarbons (PAH) extracted from coal tar, the Standard Reference Material (SRM) 1597, was recently shown to decrease the levels of DNA binding of the 2 strong carcinogens benzo(a)pyrene (BP) and dibenzo(a,l)pyrene (DBP) in the human mammary carcinoma-derived cell line MCF-7. The present study was designed to further elucidate the biochemical mechanisms involved in this inhibition process. We examined the effects of SRM 1597 on the metabolic activation of BP and DBP toward DNA-binding derivatives in Chinese hamster cells expressing either human cytochrome P450 (CYP) 1A1 or CYP1B1. The data obtained from biochemical experiments revealedmore » that SRM 1597 competitively inhibited the activity of both human enzymes as analyzed by 7-ethoxyresorufin O-deethylation assays. While the Michaelis-Menten constant (K-M) was {lt} 0.4 {mu}M in the absence of SRM 1597, this value increased up to 1.12 (CYP1A1) or 4.45 {mu}M (CYP1B1) in the presence of 0.1 {mu} g/ml SRM 1597. Hence the inhibitory effects of the complex mixture on human CYP1B1 were much stronger when compared to human CYP1A1 Taken together, the decreases in PAH-DNA adduct formation on co-treatment with SRM 1597 revealed inhibitory effects on the CYP enzymes that convert carcinogenic PAH into DNA-binding metabolites. The implications for the tumorigenicity of complex environmental PAR mixtures are discussed.« less
Rapid transcapillary exchange and unidirectional neuronal uptake of noradrenaline in the perfused rabbit heart.

PubMed Central

Mann, G E; Yudilevich, D L

1984-01-01

Capillary permeability and cellular uptake of noradrenaline by the isolated artificially perfused rabbit heart was measured using rapid (less than 30 s) single-circulation tracer-dilution techniques. In a single coronary circulation capillary extractions of L-[14C]noradrenaline and D-[3H]mannitol (extracellular reference) relative to an intravascular marker, 125I-labelled albumin, were similar and above 60%. The 'apparent' volume of distribution for tracer noradrenaline was 2.5-fold larger than that measured for D-mannitol (0.32 ml g-1) suggesting cellular uptake of the amine. Unidirectional noradrenaline uptake was estimated by directly comparing coronary sinus dilution profiles of L-[3H]noradrenaline and D-[14C]mannitol. Michaelis-Menten saturation kinetics based on a single-entry system were determined (Km = 2.8 +/- 1.5 microM, Vmax = 2.1 +/- 0.5 nmol min-1 g-1, n = 4) by perfusing hearts with varying concentrations of L-noradrenaline (1-10 microM). Various known inhibitors of noradrenaline uptake were investigated to determine whether uptake was mediated by neuronal (uptake1) and/or extraneuronal (uptake2) mechanisms. Desipramine (5 microM), imipramine (5 microM) and metaraminol (2 microM) resulted in a 66-94% inhibition of noradrenaline influx. In comparison, the steroids, 17 beta-oestradiol (1 microM) and corticosterone (10 microM), and the noradrenaline metabolite normetanephrine (5 microM) caused virtually no inhibitory effects. The beta-adrenergic antagonist propranolol (5 microM) was also relatively ineffective. These results together with the kinetic constants estimated suggest that the rapid noradrenaline uptake reflects transport into adrenergic neurones lying in the coronary interstitium. The high resolution of this paired-tracer dilution technique has permitted a 'non-invasive' study of neuronal uptake mechanisms and its application may be of clinical value. PMID:6425496
Oxidation of monohydric phenol substrates by tyrosinase. An oximetric study.

PubMed

Naish-Byfield, S; Riley, P A

1992-11-15

The purity of commercially available mushroom tyrosinase was investigated by non-denaturing PAGE. Most of the protein in the preparation migrated as a single band under these conditions. This band contained both tyrosinase and dopa oxidase activity. No other activity of either classification was found in the preparation. Oxygen consumption by tyrosinase during oxidation of the monohydric phenol substrates tyrosine and 4-hydroxyanisole (4HA) was monitored by oximetry in order to determine the stoichiometry of the reactions. For complete oxidation, the molar ratio of oxygen: 4HA was 1:1. Under identical conditions, oxidation of tyrosine required 1.5 mol of oxygen/mol of tyrosine. The additional oxygen uptake during tyrosine oxidation is due to the internal cyclization of dopaquinone to form cyclodopa, which undergoes a redox reaction with dopaquinone to form dopachrome and dopa, which is then oxidized by the enzyme, leading to an additional 0.5 mol of oxygen/mol of original substrate. Oxygen consumption for complete oxidation of 200 nmol of 4HA was constant over a range of concentrations of tyrosinase of 33-330 units/ml of substrate. The maximum rate of reaction was directly proportional to the concentration of tyrosinase, whereas the length of the lag phase decreased non-linearly with increasing tyrosinase concentration. Activation of the enzyme by exposure to citrate was not seen, nor was the lag phase abolished by exposure of the enzyme to low pH. Michaelis-Menten analysis of tyrosinase in which the lag phase is abolished by pre-exposure of the enzyme to a low concentration of dithiothreitol gave Km values for tyrosine and 4HA of 153 and 20 microM respectively.
Administration of progesterone after trauma and hemorrhagic shock prevents hepatocellular injury.

PubMed

Kuebler, Joachim F; Yokoyama, Yukihiro; Jarrar, Doraid; Toth, Balazs; Rue, Loring W; Bland, Kirby I; Wang, Ping; Chaudry, Irshad H

2003-07-01

Administration of a single dose of progesterone following trauma and hemorrhage in progesterone-deficient rats would ameliorate the inflammatory response and hepatocellular damage. A university laboratory. Ovariectomized female Sprague-Dawley rats (250-350 g; Charles River Laboratories, Wilmington, Mass) underwent a 5-cm midline laparotomy (ie, induction of soft tissue trauma), were bled to a mean arterial blood pressure of 35 mm Hg for about 90 minutes, and then were resuscitated using Ringer lactate solution. Progesterone (25 mg/kg of body weight) or vehicle was administered subcutaneously at the end of resuscitation. In additional animals, Kupffer cells were isolated following trauma, hemorrhage, and resuscitation and treated in vitro with progesterone, lipopolysaccharide, or both. Six hours following resuscitation, plasma tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) levels and liver myeloperoxidase activity were determined. Hepatocellular function (maximum velocity of indocyanine green clearance [Vmax] and the efficiency of the active transport or Michaelis-Menten constant [Km]) and plasma levels of transaminases were measured 20 hours after resuscitation. Kupffer cell IL-6 and TNF-alpha production were assessed. Plasma levels of TNF-alpha, IL-6, aspartate aminotransferase, and alanine aminotransferase, as well as hepatic myeloperoxidase activity were increased, whereas indocyanine green clearance was depressed in vehicle-treated rats following trauma-hemorrhage. Animals treated with progesterone showed significantly reduced levels of the TNF-alpha, IL-6, and transaminases as well as reduced myeloperoxidase activity in the liver. Progesterone-treated animals showed increased Vmax and Kmax values for indocyanine green. In vitro treatment of Kupffer cells with progesterone decreased TNF-alpha production but did not affect the production of IL-6. Progesterone administration following trauma-hemorrhage ameliorates the proinflammatory response and, subsequently, the hepatocellular injury via direct action on immunocompetent cells.
Removal of cyanide by woody plants.

PubMed

Larsen, Morten; Trapp, Stefan; Pirandello, Alessandro

2004-01-01

Hydrogen cyanide is a high volume production chemical that causes severe environmental problems. The toxicity of potassium cyanide (KCN) to basket willow trees (Salix viminalis) was tested. In aqueous solution, 2 mg CN l(-1) as KCN depressed the transpiration after 72 h about 50%. Trees exposed to 0.4 mg CN l(-1) in aqueous solution showed initially a depression of transpiration, but recovered. Doses of 8 and 20 mg CN l(-1) in aqueous solution were quickly mortal to the trees. At the end of the test, almost all cyanide had disappeared from the solutions. Levels of cyanide in plants were related to the toxicity, with no elevated levels of cyanide in plants exposed to 0.4 mg CN l(-1). Willows grown in sand survived 423.5 h irrigation with 20 mg CN l(-1). Willows grown in sand irrigated with 50 mg CN l(-1) died within a few days. The roots of the surviving willows were able to consume about 10 mg CN kg fresh weight(-1)h(-1). Vascular plants possess the enzymes beta-cyanoalanine synthase and beta-cyanoalanine hydrolase, which convert free cyanide to the amino acid asparagine. The in vivo capacity of woody plants (willow, poplar, elder, rose, birch) to remove cyanide was evaluated. Tests were performed with detached leaves and roots in KCN solutions of different concentrations. The highest removal capacity was obtained for basket willow hybrids (Salix viminalis x schwerinii). The Michaelis-Menten kinetics was determined. Realistic values of the half-saturation constant, K(M), were between 0.6 and 1.7 mg CN l(-1); the maximum metabolic capacity, v(max), was around 9.3 mg CN kg fresh weight(-1)h(-1). The removal of cyanide by plants might be useful in phytoremediation and treatment of wastewater from gold mining.
Erythrocyte glucose-6-phosphate dehydrogenase from Brazilian opossum Didelphis marsupialis.

PubMed

Barretto, O C de O; Oshiro, M; Oliveira, R A G; Fedullo, J D L; Nonoyama, K

2006-05-01

In a comparative study of erythrocyte metabolism of vertebrates, the specific activity of glucose-6-phosphate dehydrogenase (G6PD) of the Brazilian opossum Didelphis marsupialis in a hemolysate was shown to be high, 207 +/- 38 IU g-1 Hb-1 min-1 at 37 degrees C, compared to the human erythrocyte activity of 12 +/- 2 IU g-1 Hb-1 min-1 at 37 degrees C. The apparent high specific activity of the mixture led us to investigate the physicochemical properties of the opossum enzyme. We report that reduced glutathione (GSH) in the erythrocytes was only 50% higher than in human erythrocytes, a value lower than expected from the high G6PD activity since GSH is maintained in a reduced state by G6PD activity. The molecular mass, determined by G-200 Sephadex column chromatography at pH 8.0, was 265 kDa, which is essentially the same as that of human G6PD (260 kDa). The Michaelis-Menten constants (Km: 55 microM) for glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate (Km: 3.3 microM) were similar to those of the human enzyme (Km: 50-70 and Km: 2.9-4.4, respectively). A 450-fold purification of the opossum enzyme was achieved and the specific activity of the purified enzyme, 90 IU/mg protein, was actually lower than the 150 IU/mg protein observed for human G6PD. We conclude that G6PD after purification from the hemolysate of D. marsupialis does not have a high specific activity. Thus, it is quite probable that the red cell hyperactivity reported may be explained by increased synthesis of G6PD molecules per unit of hemoglobin or to reduced inactivation in the RBC hemolysate.
Chitosan-immobilized pectinolytics with novel catalytic features and fruit juice clarification potentialities.

PubMed

Irshad, Muhammad; Murtza, Aimen; Zafar, Muddassar; Bhatti, Khizar Hayat; Rehman, Abdul; Anwar, Zahid

2017-11-01

Biological macromolecules are primarily composed of complex polysaccharides that strengthen microbial growth for the production of industrially relevant enzymes. The presence of polysaccharides in the form of the disrupted cell wall and cell materials are among major challenges in the fruit juice industry. The breakdown of such biological macromolecules including cellulose and pectin is vital for the juices processing. In this background, pectinolytic enzymes including polygalacturonase (PG), pectin lyase (PL), and pectin methylesterase (PME) were isolated from Aspergillus ornatus, statistically optimized and purified via ammonium sulfate fractionation (ASF), dialysis, and Sephadex G-100 gel permeation chromatography. After passing through Sephadex G-100 column, PG, PL, and PME were 2.60-fold, 3.30-fold, and 4.52-fold purified with specific activities of 475.2U/mg, 557.1U/mg, and 205.7U/mg. The active PG, PL, and PME, each separately, were surface immobilized using various concentrations of chitosan and dextran polyaldehyde as a macromolecular crosslinking agent. Prior to exploit for juice clarification purposes, various parameters including pH, thermal and Michaelis-Menten kinetic constants of purified and chitosan-immobilized fractions were investigated. A considerable improvement in the pH and thermal profiles was recorded after immobilization. However, the negligible difference between the K m and V max values of purified free and chitosan-immobilized fractions revealed that the conformational flexibility of pectinolytics was retained as such. A significant color and turbidity reductions were recorded after 60min treatment with CTS-PG, followed by CTS-PME, and CTS-PL. It can be concluded that the clarification of apples, mango, peach, and apricot juices was greatly affected by CTS-PG, CTS-PME, and CTS-PL treatments rendering them as potential candidatures for food industry applications. Copyright © 2017 Elsevier B.V. All rights reserved.
ACTION OF A COMPLEX RADIATION FLUX ON ERYTHROCYTE PHOSPHOMONOESTERASE (in Rumanian)

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

Buruiana, L.M.; Hadarag, El.; Dema, A.

To study the effect of radiation on the enzyme, erythrocytes were irradiated in the reactor of the Institute of Atomic Physics of the Romanian Academy of Sciences, Bucharest, in which the intensity of the various radiation components is: thermal neutrons 2.3 x 10/sup 7//cm/sup 2//sec, epithermal neutrons 7.1 x 10/sup 5//cm/sup 2//sec, fast neutrons 4.0 x 10/sup 7//cm/sup 2// sec, and gamma radiation 0.06 r/sec. In general, irradiation lowered the enzyme activity of solutions of the enzyme from horse erythrocytes, this reduction depending on the duration of irradiation and the initial enzyme activity. Kinetics of the nonirradiated and irradiated enzymemore » with respect to its substrate, alpha -glycerophosphate, were studied at various temperatures and substrate concentrations, according to the formulations of Lineweaver and Burk and the Michaelis constant (K/sub m/) was determined. The value of K/sub m/ was 0.0294 and 0.10 mole/l after 30 and 60 min irradiation, respectively, in contrast to 0.04 mole/l for the native enzyme. The corresponding hydrolysis rates at a substrate concentration of 0.50 g/100 ml were 0.036, 0.025, and 0.045, as g P per 100 ml erythrocytes at 37 deg C. Impairment of quality of the enzyme during irradiation was shown by the progressive increase in activation energy, which rose from 8955 cal/mole in native enzyme to 11500 and 11666 cal/mole in solutions of enzyme irradiated for 15 and 30 min, respectively. Although the above data apply to the equine enzyme only, similar changes in kinetics were observed following irradiation of the enzyme in bovine erythrocytes. (BBB)« less
Recommended Values of the Fundamental Physical Constants: A Status Report

PubMed Central

Taylor, Barry N.; Cohen, E. Richard

1990-01-01

We summarize the principal advances made in the fundamental physical constants field since the completion of the 1986 CODATA least-squares adjustment of the constants and discuss their implications for both the 1986 set of recommended values and the next least-squares adjustment. In general, the new results lead to values of the constants with uncertainties 5 to 7 times smaller than the uncertainties assigned the 1986 values. However, the changes in the values themselves are less than twice the 1986 assigned one-standard-deviation uncertainties and thus are not highly significant. Although much new data has become available since 1986, three new results dominate the analysis: a value of the Planck constant obtained from a realization of the watt; a value of the fine-structure constant obtained from the magnetic moment anomaly of the electron; and a value of the molar gas constant obtained from the speed of sound in argon. Because of their dominant role in determining the values and uncertainties of many of the constants, it is highly desirable that additional results of comparable uncertainty that corroborate these three data items be obtained before the next adjustment is carried out. Until then, the 1986 CODATA set of recommended values will remain the set of choice. PMID:28179787
Estimating reaction rate coefficients within a travel-time modeling framework.

PubMed

Gong, R; Lu, C; Wu, W-M; Cheng, H; Gu, B; Watson, D; Jardine, P M; Brooks, S C; Criddle, C S; Kitanidis, P K; Luo, J

2011-01-01

A generalized, efficient, and practical approach based on the travel-time modeling framework is developed to estimate in situ reaction rate coefficients for groundwater remediation in heterogeneous aquifers. The required information for this approach can be obtained by conducting tracer tests with injection of a mixture of conservative and reactive tracers and measurements of both breakthrough curves (BTCs). The conservative BTC is used to infer the travel-time distribution from the injection point to the observation point. For advection-dominant reactive transport with well-mixed reactive species and a constant travel-time distribution, the reactive BTC is obtained by integrating the solutions to advective-reactive transport over the entire travel-time distribution, and then is used in optimization to determine the in situ reaction rate coefficients. By directly working on the conservative and reactive BTCs, this approach avoids costly aquifer characterization and improves the estimation for transport in heterogeneous aquifers which may not be sufficiently described by traditional mechanistic transport models with constant transport parameters. Simplified schemes are proposed for reactive transport with zero-, first-, nth-order, and Michaelis-Menten reactions. The proposed approach is validated by a reactive transport case in a two-dimensional synthetic heterogeneous aquifer and a field-scale bioremediation experiment conducted at Oak Ridge, Tennessee. The field application indicates that ethanol degradation for U(VI)-bioremediation is better approximated by zero-order reaction kinetics than first-order reaction kinetics. Copyright © 2010 The Author(s). Journal compilation © 2010 National Ground Water Association.
Numerical modeling of coupled nitrification-denitrification in sediment perfusion cores from the hyporheic zone of the Shingobee River, MN

USGS Publications Warehouse

Sheibley, R.W.; Jackman, A.P.; Duff, J.H.; Triska, F.J.

2003-01-01

Nitrification and denitrification kinetics in sediment perfusion cores were numerically modeled and compared to experiments on cores from the Shingobee River MN, USA. The experimental design incorporated mixing groundwater discharge with stream water penetration into the cores, which provided a well-defined, one-dimensional simulation of in situ hydrologic conditions. Ammonium (NH+4) and nitrate (NO-3) concentration gradients suggested the upper region of the cores supported coupled nitrification-denitrification, where groundwater-derived NH+4 was first oxidized to NO-3 then subsequently reduced via denitrification to N2. Nitrification and denitrification were modeled using a Crank-Nicolson finite difference approximation to a one-dimensional advection-dispersion equation. Both processes were modeled using first-order reaction kinetics because substrate concentrations (NH+4 and NO-3) were much smaller than published Michaelis constants. Rate coefficients for nitrification and denitrification ranged from 0.2 to 15.8 h-1 and 0.02 to 8.0 h-1, respectively. The rate constants followed an Arrhenius relationship between 7.5 and 22 ??C. Activation energies for nitrification and denitrification were 162 and 97.3 kJ/mol, respectively. Seasonal NH+4 concentration patterns in the Shingobee River were accurately simulated from the relationship between perfusion core temperature and NH+4 flux to the overlying water. The simulations suggest that NH+4 in groundwater discharge is controlled by sediment nitrification that, consistent with its activation energy, is strongly temperature dependent. ?? 2003 Elsevier Ltd. All rights reserved.
Pharmacokinetics of aniracetam and its metabolites in rats.

PubMed

Ogiso, T; Iwaki, M; Tanino, T; Ikeda, K; Paku, T; Horibe, Y; Suzuki, H

1998-05-01

The pharmacokinetics of aniracetam (AP), a new cognitive performance enhancer, and its main metabolites was investigated after intravenous (iv) and oral administrations to rat. The plasma levels of AP, 4-p-anisamidobutyric acid (ABA), and p-anisic acid (AA) were determined simultaneously by the HPLC method. The plasma concentrations of the parent drug and ABA quickly declined in a biexponential manner, with rapid terminal decay and a small mean residence time. However, AA yielded nonlinearly high levels at the initial times and the plasma concentrations of 2-pyrrolidinone (PD) were sustained over a relatively long time. When AA was administered intravenously, nonlinearity of the plasma concentrations was also found at higher doses. To describe the time course of the plasma levels of AP and its metabolites after iv administration, a pharmacokinetic model with seven compartments was applied, which included 10 first-order rate constants and one Michaelis-Menten constant. An approximate fit was obtained between the observed and calculated curves based on the model, except for the plasma concentrations of ABA. The plasma concentration-time profiles of AP and its metabolites following oral administration of AP (50 and 100 mg/kg) were similar to those after iv dosing, with the exception of PD, which showed much lower plasma levels than those after iv administration. Elimination of AP and ABA was rapid after oral dosing, and the bioavailability of AP was extremely small (11.4 and 8.6%). As a result, AP was largely metabolized to ABA, AA, and PD in rat.
Amyloglucosidase enzymatic reactivity inside lipid vesicles

PubMed Central

Li, Mian; Hanford, Michael J; Kim, Jin-Woo; Peeples, Tonya L

2007-01-01

Efficient functioning of enzymes inside liposomes would open new avenues for applications in biocatalysis and bioanalytical tools. In this study, the entrapment of amyloglucosidase (AMG) (EC 3.2.1.3) from Aspergillus niger into dipalmitoylphosphatidylcholine (DPPC) multilamellar vesicles (MLVs) and large unilamellar vesicles (LUVs) was investigated. Negative-stain, freeze-fracture, and cryo-transmission electron microscopy images verified vesicle formation in the presence of AMG. Vesicles with entrapped AMG were isolated from the solution by centrifugation, and vesicle lamellarity was identified using fluorescence laser confocal microscopy. The kinetics of starch hydrolysis by AMG was modeled for two different systems, free enzyme in aqueous solution and entrapped enzyme within vesicles in aqueous suspension. For the free enzyme system, intrinsic kinetics were described by a Michaelis-Menten kinetic model with product inhibition. The kinetic constants, Vmax and Km, were determined by initial velocity measurements, and Ki was obtained by fitting the model to experimental data of glucose concentration-time curves. Predicted concentration-time curves using these kinetic constants were in good agreement with experimental measurements. In the case of the vesicles, the time-dependence of product (glucose) formation was experimentally determined and simulated by considering the kinetic behavior of the enzyme and the permeation of substrate into the vesicle. Experimental results demonstrated that entrapped enzymes were much more stable than free enyzme. The entrapped enzyme could be recycled with retention of 60% activity after 3 cycles. These methodologies can be useful in evaluating other liposomal catalysis operations. PMID:18271982
Kinetics of DCE and VC mineralization under methanogenic and Fe(III)- reducing conditions

USGS Publications Warehouse

Bradley, P.M.; Chapelle, F.H.

1997-01-01

The kinetics of anaerobic mineralization of DCE and VC under mathanogenic and Fe(III)-reducing conditions as a function of dissolved contaminant concentration were evaluated. Microorganisms indigenous to creek bed sediments, where groundwater contaminated with chlorinated ethenes continuously discharges, demonstrated significant mineralization of DCE and VC under methanogenic and Fe(III)- reducing conditions. Over 37 days, the recovery of [1,214C]VC radioactivity as 14CO2 ranged from 5% to 44% and from 8% to 100% under methanogenic and Fe(III)-reducing conditions, respectively. The recovery of [1,2-14C]DCE radioactivity as 14CO2 ranged from 4% to 14% and did not vary significantly between methanogenic and Fe(III)reducing conditions. VC mineralization was described by Michaelis- Menten kinetics. Under methanogenic conditions, V(max) was 0.19 ?? 0.01 ??mol L-1 d-1 and the half-saturation constant, k(m), was 7.6 ?? 1.7 ??M. Under Fe(III)-reducing conditions, V(max) was 0.76 ?? 0.07 ??mol L-1 d-1 and k(m) was 1.3 ?? 0.5 ??M. In contrast, DCE mineralization could be described by first-order kinetics. The first-order degradation rate constant for DCE mineralization was 0.6 ?? 0.2% d-1 under methanogenic and Fe(III)-reducing conditions. The results indicate that the kinetics of chlorinated ethane mineralization can vary significantly with the specific contaminant and the predominant redox conditions under which mineralization occurs.

A novel nitromethane biosensor based on biocompatible conductive redox graphene-chitosan/hemoglobin/graphene/room temperature ionic liquid matrix.

PubMed

Wang, Lu; Zhang, Xiuhua; Xiong, Huayu; Wang, Shengfu

2010-11-15

A novel amperometric biosensor for nitromethane (CH(3)NO(2)) based on immobilization of graphene (GR), chitosan (CS), hemoglobin (Hb) and room temperature ionic liquid (IL) on a glassy carbon electrode (GCE) was developed for the first time. The surface morphologies of a set of representative membranes were characterized by means of scanning electron microscopy (SEM). The electrochemical performance of the biosensor was evaluated by cyclic voltammetry (CV) and chronoamperometry. A pair of stable and well-defined redox peaks of Hb with a formal potential of -0.240 V was observed at the GR-CS/Hb/GR/IL/GCE. The effects of phosphate buffer pH, scan rate, and temperature on the biosensor were investigated to provide optimum analytical performance. Moreover, several electrochemical parameters, e.g., the heterogeneous electron transfer rate constant (k(s)), were calculated in detail. The presence of both GR and IL not only dramatically facilitated the electron transfer of Hb, but also greatly enhanced electrocatalytic activity towards CH(3)NO(2). The apparent Michaelis-Menten constant was down to 0.16 μM, indicating that the biosensor possessed high affinity to CH(3)NO(2). Besides this, the proposed biosensor exhibited fast amperometric response (<5s), low detection limit (6.0 × 10(-10)M), and excellent long-time storage stability for the determination of CH(3)NO(2). Copyright © 2010 Elsevier B.V. All rights reserved.
Function of muscle-type lactate dehydrogenase and citrate synthase of the Galápagos marine iguana, Amblyrhynchus cristatus, in relation to temperature.

PubMed

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

2008-05-01

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

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

Gong, R; Lu, C; Luo, Jian

A generalized, efficient, and practical approach based on the travel-time modeling framework is developed to estimate in situ reaction rate coefficients for groundwater remediation in heterogeneous aquifers. The required information for this approach can be obtained by conducting tracer tests with injection of a mixture of conservative and reactive tracers and measurements of both breakthrough curves (BTCs). The conservative BTC is used to infer the travel-time distribution from the injection point to the observation point. For advection-dominant reactive transport with well-mixed reactive species and a constant travel-time distribution, the reactive BTC is obtained by integrating the solutions to advective-reactive transportmore » over the entire travel-time distribution, and then is used in optimization to determine the in situ reaction rate coefficients. By directly working on the conservative and reactive BTCs, this approach avoids costly aquifer characterization and improves the estimation for transport in heterogeneous aquifers which may not be sufficiently described by traditional mechanistic transport models with constant transport parameters. Simplified schemes are proposed for reactive transport with zero-, first-, nth-order, and Michaelis-Menten reactions. The proposed approach is validated by a reactive transport case in a two-dimensional synthetic heterogeneous aquifer and a field-scale bioremediation experiment conducted at Oak Ridge, Tennessee. The field application indicates that ethanol degradation for U(VI)-bioremediation is better approximated by zero-order reaction kinetics than first-order reaction kinetics.« less
Quantitative analysis of immobilized penicillinase using enzyme-modified AlGaN/GaN field-effect transistors.

PubMed

Müntze, Gesche Mareike; Baur, Barbara; Schäfer, Wladimir; Sasse, Alexander; Howgate, John; Röth, Kai; Eickhoff, Martin

2015-02-15

Penicillinase-modified AlGaN/GaN field-effect transistors (PenFETs) are utilized to systematically investigate the covalently immobilized enzyme penicillinase under different experimental conditions. We demonstrate quantitative evaluation of covalently immobilized penicillinase layers on pH-sensitive field-effect transistors (FETs) using an analytical kinetic PenFET model. This kinetic model is explicitly suited for devices with thin enzyme layers that are not diffusion-limited, as it is the case for the PenFETs discussed here. By means of the kinetic model it was possible to extract the Michaelis constant of covalently immobilized penicillinase as well as relative transport coefficients of the different species associated with the enzymatic reaction which, exempli gratia, give information about the permeability of the enzymatic layer. Based on this analysis we quantify the reproducibility and the stability of the analyzed PenFETs over the course of 33 days as well as the influence of pH and buffer concentration on the properties of the enzymatic layer. Thereby the stability measurements reveal a Michalis constant KM of (67 ± 13)μM while the chronological development of the relative transport coefficients suggests a detachment of physisorbed penicillinase during the first two weeks since production. Our results show that AlGaN/GaN PenFETs prepared by covalent immobilization of a penicillinase enzyme layer present a powerful tool for quantitative analysis of enzyme functionality. Copyright © 2014 Elsevier B.V. All rights reserved.
Immobilization of myoglobin on Au nanoparticle-decorated carbon nanotube/polytyramine composite as a mediator-free H2O2 and nitrite biosensor

PubMed Central

Vilian, A. T. Ezhil; Veeramani, Vediyappan; Chen, Shen-Ming; Madhu, Rajesh; Kwak, Cheol Hwan; Huh, Yun Suk; Han, Young-Kyu

2015-01-01

A novel composite film was designed for use as a highly selective mediator-free amperometric biosensor, and a method was created for accomplishing direct electrochemistry of myoglobin on a multi-walled carbon nanotube and tyramine-modified composite decorated with Au nanoparticles on a glassy carbon electrode. The ultraviolet-visible and electrochemical impedance spectroscopy results showed that myoglobin retained its native conformation in the interaction with Au-PTy-f-MWCNT. The surface coverage of Mb-heme-Fe(II)/(III) immobilized on Au-PTy-f-MWCNT and the heterogeneous electron-transfer rate constant were 2.12 × 10−9 mol cm−2 and 4.86 s−1, respectively, indicating a higher loading capacity of the nanocomposite for direct electron transfer of Mb onto the electrode surface. The proposed Mb/Au-PTy-f-MWCNT biofilm exhibited excellent electrocatalytic behavior toward the reduction of H2O2 and the oxidation of nitrite with linear ranges of 2 to 5000 μM and 1 to 8000 μM and lower detection limits of 0.01 μM and 0.002 μM, respectively. An apparent Michaelis-Menten constant of 0.12 mM indicated that the Mb immobilized on the Au-PTy-f-MWCNT film retained its native activity. This biosensor can be successfully applied to detect H2O2 and nitrite in disinfectant cream, eye drops, pickle juice, and milk samples. PMID:26672985
Transport mechanism for lovastatin acid in bovine kidney NBL-1 cells: kinetic evidences imply involvement of monocarboxylate transporter 4.

PubMed

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

2003-08-27

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

PubMed

Chen, Wei-Yu; Liao, Chung-Min

2012-11-01

The purpose of this study was to link toxicokinetics/toxicodynamics (TK/TD) and bioavailability-based metal uptake kinetics to assess arsenic (As) uptake and bioaccumulation in three common farmed species of tilapia (Oreochromis mossambicus), milkfish (Chanos chanos), and freshwater clam (Corbicula fluminea). We developed a mechanistic framework by linking damage assessment model (DAM) and bioavailability-based Michaelis-Menten model for describing TK/TD and As uptake mechanisms. The proposed model was verified with published acute toxicity data. The estimated TK/TD parameters were used to simulate the relationship between bioavailable As uptake and susceptibility probability. The As toxicity was also evaluated based on a constructed elimination-recovery scheme. Absorption rate constants were estimated to be 0.025, 0.016, and 0.175 mL g(-1) h(-1) and As uptake rate constant estimates were 22.875, 63.125, and 788.318 ng g(-1) h(-1) for tilapia, milkfish, and freshwater clam, respectively. Here we showed that a potential trade-off between capacities of As elimination and damage recovery was found among three farmed species. Moreover, the susceptibility probability can also be estimated by the elimination-recovery relations. This study suggested that bioavailability-based uptake kinetics and TK/TD-based DAM could be integrated for assessing metal uptake and toxicity in aquatic organisms. This study is useful to quantitatively assess the complex environmental behavior of metal uptake and implicate to risk assessment of metals in aquaculture systems.
Stochastic theory of large-scale enzyme-reaction networks: Finite copy number corrections to rate equation models

NASA Astrophysics Data System (ADS)

Thomas, Philipp; Straube, Arthur V.; Grima, Ramon

2010-11-01

Chemical reactions inside cells occur in compartment volumes in the range of atto- to femtoliters. Physiological concentrations realized in such small volumes imply low copy numbers of interacting molecules with the consequence of considerable fluctuations in the concentrations. In contrast, rate equation models are based on the implicit assumption of infinitely large numbers of interacting molecules, or equivalently, that reactions occur in infinite volumes at constant macroscopic concentrations. In this article we compute the finite-volume corrections (or equivalently the finite copy number corrections) to the solutions of the rate equations for chemical reaction networks composed of arbitrarily large numbers of enzyme-catalyzed reactions which are confined inside a small subcellular compartment. This is achieved by applying a mesoscopic version of the quasisteady-state assumption to the exact Fokker-Planck equation associated with the Poisson representation of the chemical master equation. The procedure yields impressively simple and compact expressions for the finite-volume corrections. We prove that the predictions of the rate equations will always underestimate the actual steady-state substrate concentrations for an enzyme-reaction network confined in a small volume. In particular we show that the finite-volume corrections increase with decreasing subcellular volume, decreasing Michaelis-Menten constants, and increasing enzyme saturation. The magnitude of the corrections depends sensitively on the topology of the network. The predictions of the theory are shown to be in excellent agreement with stochastic simulations for two types of networks typically associated with protein methylation and metabolism.
The CODATA 2017 values of h, e, k, and N A for the revision of the SI

NASA Astrophysics Data System (ADS)

Newell, D. B.; Cabiati, F.; Fischer, J.; Fujii, K.; Karshenboim, S. G.; Margolis, H. S.; de Mirandés, E.; Mohr, P. J.; Nez, F.; Pachucki, K.; Quinn, T. J.; Taylor, B. N.; Wang, M.; Wood, B. M.; Zhang, Z.

2018-04-01

Sufficient progress towards redefining the International System of Units (SI) in terms of exact values of fundamental constants has been achieved. Exact values of the Planck constant h, elementary charge e, Boltzmann constant k, and Avogadro constant N A from the CODATA 2017 Special Adjustment of the Fundamental Constants are presented here. These values are recommended to the 26th General Conference on Weights and Measures to form the foundation of the revised SI.
Kinetics of the hydrolysis of N-benzoyl-l-serine methyl ester catalysed by bromelain and by papain. Analysis of modifier mechanisms by lattice nomography, computational methods of parameter evaluation for substrate-activated catalyses and consequences of postulated non-productive binding in bromelain- and papain-catalysed hydrolyses

PubMed Central

Wharton, Christopher W.; Cornish-Bowden, Athel; Brocklehurst, Keith; Crook, Eric M.

1974-01-01

1. N-Benzoyl-l-serine methyl ester was synthesized and evaluated as a substrate for bromelain (EC 3.4.22.4) and for papain (EC 3.4.22.2). 2. For the bromelain-catalysed hydrolysis at pH7.0, plots of [S0]/vi (initial substrate concn./initial velocity) versus [S0] are markedly curved, concave downwards. 3. Analysis by lattice nomography of a modifier kinetic mechanism in which the modifier is substrate reveals that concave-down [S0]/vi versus [S0] plots can arise when the ratio of the rate constants that characterize the breakdown of the binary (ES) and ternary (SES) complexes is either less than or greater than 1. In the latter case, there are severe restrictions on the values that may be taken by the ratio of the dissociation constants of the productive and non-productive binary complexes. 4. Concave-down [S0]/vi versus [S0] plots cannot arise from compulsory substrate activation. 5. Computational methods, based on function minimization, for determination of the apparent parameters that characterize a non-compulsory substrate-activated catalysis are described. 6. In an attempt to interpret the catalysis by bromelain of the hydrolysis of N-benzoyl-l-serine methyl ester in terms of substrate activation, the general substrate-activation model was simplified to one in which only one binary ES complex (that which gives rise directly to products) can form. 7. In terms of this model, the bromelain-catalysed hydrolysis of N-benzoyl-l-serine methyl ester at pH7.0, I=0.1 and 25°C is characterized by Km1 (the dissociation constant of ES)=1.22±0.73mm, k (the rate constant for the breakdown of ES to E+products, P)=1.57×10−2±0.32×10−2s−1, Ka2 (the dissociation constant that characterizes the breakdown of SES to ES and S)=0.38±0.06m, and k′ (the rate constant for the breakdown of SES to E+P+S)=0.45±0.04s−1. 8. These parameters are compared with those in the literature that characterize the bromelain-catalysed hydrolysis of α-N-benzoyl-l-arginine ethyl ester and of α-N-benzoyl-l-arginine amide; Km1 and k for the serine ester hydrolysis are somewhat similar to Km and kcat. for the arginine amide hydrolysis and Kas and k′ for the serine ester hydrolysis are somewhat similar to Km and kcat. for the arginine ester hydrolysis. 9. A previous interpretation of the inter-relationships of the values of kcat. and Km for the bromelain-catalysed hydrolysis of the arginine ester and amide substrates is discussed critically and an alternative interpretation involving substantial non-productive binding of the arginine amide substrate to bromelain is suggested. 10. The parameters for the bromelain-catalysed hydrolysis of the serine ester substrate are tentatively interpreted in terms of non-productive binding in the binary complex and a decrease of this type of binding by ternary complex-formation. 11. The Michaelis parameters for the papain-catalysed hydrolysis of the serine ester substrate (Km=52±4mm, kcat.=2.80±0.1s−1 at pH7.0, I=0.1, 25.0°C) are similar to those for the papain-catalysed hydrolysis of methyl hippurate. 12. Urea and guanidine hydrochloride at concentrations of 1m have only small effects on the kinetic parameters for the hydrolysis of the serine ester substrate catalysed by bromelain and by papain. PMID:4455211
Calcium homeostasis in the outer segments of retinal rods from the tiger salamander.

PubMed Central

Lagnado, L; Cervetto, L; McNaughton, P A

1992-01-01

1. The processes regulating intracellular calcium in the outer segments of salamander rods have been investigated. The main preparation used was the isolated rod loaded with the Ca(2+)-sensitive photoprotein aequorin, from which outer segment membrane current and free [Ca2+]i could be recorded simultaneously. Two other preparations were also used: outer segment membrane current was recorded from intact, isolated rods using a suction pipette, and from detached outer segments using a whole-cell pipette. 2. Measurements of free intracellular [Ca2+] in Ringer solution were obtained from two aequorin-loaded rods. Mean [Ca2+]i in darkness was 0.41 microM, and after a bright flash [Ca2+]i fell to below detectable levels ( < 0.3 microM). No release of intracellular Ca2+ by a bright flash of light could be detected ( < 0.2 microM). 3. Application of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) caused an increase in the size of the light-sensitive current and a rise in [Ca2+]i, but application of IBMX either when the light-sensitive channels had been closed by a bright light or in the absence of external Ca2+ caused no detectable rise in [Ca2+]i. It is concluded that IBMX increases [Ca2+]i by opening light-sensitive channels, and does not release Ca2+ from stores within the outer segment. 4. Removal of external Na+ caused a rise in [Ca2+]i to around 2 microM and completely suppressed the light-sensitive current. 5. The Na(+)-Ca2+, K+ exchange current in aequorin-loaded rods was activated in first-order manner by internal free calcium, with a mean Michaelis constant, KCa, of 1.6 microM. 6. The KCa of the Na(+)-Ca2+, K+ exchange was increased by elevating internal [Na+]. 7. The Michaelis relation between [Ca2+]i and the activity of the Na(+)-Ca2+, K+ exchange was used to calculate the change in [Ca2+]i occurring during the response to a bright light. In aequorin-loaded rods in Ringer solution the mean change in free [Ca2+]i after a bright flash was 0.34 microM. In these rods 10% of the dark current was carried by Ca2+. 8. Most of the calcium entering the outer segment was taken up rapidly and reversibly by buffer systems. The time constant of equilibration between free and rapidly bound Ca2+ was less than 20 ms. No slow component of calcium uptake was detected. 9. Two components of calcium buffering could be distinguished in the outer segments of aequorin-loaded rods.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:1282928
Nonlinear least-squares data fitting in Excel spreadsheets.

PubMed

Kemmer, Gerdi; Keller, Sandro

2010-02-01

We describe an intuitive and rapid procedure for analyzing experimental data by nonlinear least-squares fitting (NLSF) in the most widely used spreadsheet program. Experimental data in x/y form and data calculated from a regression equation are inputted and plotted in a Microsoft Excel worksheet, and the sum of squared residuals is computed and minimized using the Solver add-in to obtain the set of parameter values that best describes the experimental data. The confidence of best-fit values is then visualized and assessed in a generally applicable and easily comprehensible way. Every user familiar with the most basic functions of Excel will be able to implement this protocol, without previous experience in data fitting or programming and without additional costs for specialist software. The application of this tool is exemplified using the well-known Michaelis-Menten equation characterizing simple enzyme kinetics. Only slight modifications are required to adapt the protocol to virtually any other kind of dataset or regression equation. The entire protocol takes approximately 1 h.
Nitrate decreases xanthine oxidoreductase-mediated nitrite reductase activity and attenuates vascular and blood pressure responses to nitrite.

PubMed

Damacena-Angelis, Célio; Oliveira-Paula, Gustavo H; Pinheiro, Lucas C; Crevelin, Eduardo J; Portella, Rafael L; Moraes, Luiz Alberto B; Tanus-Santos, Jose E

2017-08-01

Nitrite and nitrate restore deficient endogenous nitric oxide (NO) production as they are converted back to NO, and therefore complement the classic enzymatic NO synthesis. Circulating nitrate and nitrite must cross membrane barriers to produce their effects and increased nitrate concentrations may attenuate the nitrite influx into cells, decreasing NO generation from nitrite. Moreover, xanthine oxidoreductase (XOR) mediates NO formation from nitrite and nitrate. However, no study has examined whether nitrate attenuates XOR-mediated NO generation from nitrite. We hypothesized that nitrate attenuates the vascular and blood pressure responses to nitrite either by interfering with nitrite influx into vascular tissue, or by competing with nitrite for XOR, thus inhibiting XOR-mediated NO generation. We used two independent vascular function assays in rats (aortic ring preparations and isolated mesenteric arterial bed perfusion) to examine the effects of sodium nitrate on the concentration-dependent responses to sodium nitrite. Both assays showed that nitrate attenuated the vascular responses to nitrite. Conversely, the aortic responses to the NO donor DETANONOate were not affected by sodium nitrate. Further confirming these results, we found that nitrate attenuated the acute blood pressure lowering effects of increasing doses of nitrite infused intravenously in freely moving rats. The possibility that nitrate could compete with nitrite and decrease nitrite influx into cells was tested by measuring the accumulation of nitrogen-15-labeled nitrite ( 15 N-nitrite) by aortic rings using ultra-performance liquid chromatography tandem mass-spectrometry (UPLC-MS/MS). Nitrate exerted no effect on aortic accumulation of 15 N-nitrite. Next, we used chemiluminescence-based NO detection to examine whether nitrate attenuates XOR-mediated nitrite reductase activity. Nitrate significantly shifted the Michaelis Menten saturation curve to the right, with a 3-fold increase in the Michaelis constant. Together, our results show that nitrate inhibits XOR-mediated NO production from nitrite, and this mechanism may explain how nitrate attenuates the vascular and blood pressure responses to nitrite. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.
Diverse Data Sets Can Yield Reliable Information through Mechanistic Modeling: Salicylic Acid Clearance.

PubMed

Raymond, G M; Bassingthwaighte, J B

This is a practical example of a powerful research strategy: putting together data from studies covering a diversity of conditions can yield a scientifically sound grasp of the phenomenon when the individual observations failed to provide definitive understanding. The rationale is that defining a realistic, quantitative, explanatory hypothesis for the whole set of studies, brings about a "consilience" of the often competing hypotheses considered for individual data sets. An internally consistent conjecture linking multiple data sets simultaneously provides stronger evidence on the characteristics of a system than does analysis of individual data sets limited to narrow ranges of conditions. Our example examines three very different data sets on the clearance of salicylic acid from humans: a high concentration set from aspirin overdoses; a set with medium concentrations from a research study on the influences of the route of administration and of sex on the clearance kinetics, and a set on low dose aspirin for cardiovascular health. Three models were tested: (1) a first order reaction, (2) a Michaelis-Menten (M-M) approach, and (3) an enzyme kinetic model with forward and backward reactions. The reaction rates found from model 1 were distinctly different for the three data sets, having no commonality. The M-M model 2 fitted each of the three data sets but gave a reliable estimates of the Michaelis constant only for the medium level data (K m = 24±5.4 mg/L); analyzing the three data sets together with model 2 gave K m = 18±2.6 mg/L. (Estimating parameters using larger numbers of data points in an optimization increases the degrees of freedom, constraining the range of the estimates). Using the enzyme kinetic model (3) increased the number of free parameters but nevertheless improved the goodness of fit to the combined data sets, giving tighter constraints, and a lower estimated K m = 14.6±2.9 mg/L, demonstrating that fitting diverse data sets with a single model improves confidence in the results. This modeling effort is also an example of reproducible science available at html://www.physiome.org/jsim/models/webmodel/NSR/SalicylicAcidClearance.
Organic anion transporter 1 (OAT1/SLC22A6) enhances bioluminescence based on d-luciferin-luciferase reaction in living cells by facilitating the intracellular accumulation of d-luciferin.

PubMed

Furuya, Takahito; Takehara, Issey; Shimura, Asuka; Kishimoto, Hisanao; Yasujima, Tomoya; Ohta, Kinya; Shirasaka, Yoshiyuki; Yuasa, Hiroaki; Inoue, Katsuhisa

2018-01-15

Bioluminescence (BL) imaging based on d-luciferin (d-luc)-luciferase reaction allows noninvasive and real-time monitoring of luciferase-expressing cells. Because BL intensity depends on photons generated through the d-luc-luciferase reaction, an approach to increase intracellular levels of d-luc could improve the detection sensitivity. In the present study, we showed that organic anion transporter 1 (OAT1) is useful, as a d-luc transporter, in boosting the BL intensity in luciferase-expressing cells. Functional screening of several transporters showed that the expression of OAT1 in HEK293 cells stably expressing Pyrearinus termitilluminans luciferase (HEK293/eLuc) markedly enhanced BL intensity in the presence of d-luc. When OAT1 was transiently expressed in HEK293 cells, intracellular accumulation of d-luc was higher than that in control cells, and the specific d-luc uptake mediated by OAT1 was saturable with a Michaelis constant (K m ) of 0.23 μM. The interaction between OAT1 and d-luc was verified using 6-carboxyfluorescein, a typical substrate of OAT1, which showed that d-luc inhibited the uptake of 6-carboxyfluorescein mediated by OAT1. BL intensity was concentration-dependent at steady states in HEK293/eLuc cells stably expressing OAT1, and followed Michaelis-Menten kinetics with an apparent K m of 0.36 μM. In addition, the enhanced BL was significantly inhibited by OAT1-specific inhibitors. Thus, OAT1-mediated transport of d-luc could be a rate-limiting step in the d-luc-luciferase reaction. Furthermore, we found that expressing OAT1 in HEK293/eLuc cells implanted subcutaneously in mice also significantly increased the BL after intraperitoneal injection of d-luc. Our findings suggest that because OAT1 is capable of transporting d-luc, it can also be used to improve visualization and monitoring of luciferase-expressing cells. Copyright © 2017 Elsevier Inc. All rights reserved.
Purification and properties of aryl acylamidase from Pseudomonas fluorescens ATCC 39004.

PubMed

Hammond, P M; Price, C P; Scawen, M D

1983-05-16

Aryl acylamidase has been purified from a strain of Pseudomonas fluorescens ATCC 39004, selected from soil on the basis of its ability to utilise acylanilide compounds as a sole source of carbon. The enzyme was purified to homogeneity by a combination of ion-exchange, hydrophobic and gel-permeation chromatography. A relative molecular mass of about 52 500 was estimated by gel filtration. The native enzyme was shown to be a monomeric protein by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The enzyme was maximally active at a pH of 8.6 and at a temperature of 45 degrees C. The enzyme shows Michaelis-Menten kinetics; Km values for nitroacetanilide (69 microM) and hydroxyacetanilide (6.1 microM) were low, indicating that the enzyme has a very high affinity for both substrates.
Evaluation of the effectiveness factor along immobilized enzyme fixed-bed reactors: design of a reactor with naringinase covalently immobilized into glycophase-coated porous glass

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

Manjon, A.; Iborra, J.L.; Gomez, J.L.

A design equation is presented for packed-bed reactors containing immobilized enzymes in spherical porous particles with internal diffusion effects and obeying reversible one-intermediate Michaelis-Menten kinetics. The equation is also able to explain irreversible and competitive product inhibition kinetics. It allows the axial substrate profiles to be calculated and the dependence of the effectiveness factor along the reactor length to be continuously evaluated. The design equation was applied to explain the behavior of naringinase immobilized in Glycophase-coated porous glass operating in a packed-bed reactor and hydrolyzing both p-nitrophenyl-alpha-L-rhamnoside and naringin. The theoretically predicted results were found to fit well with experimentallymore » measured values. (Refs. 28).« less
More Nuts and Bolts of Michaelis-Menten Enzyme Kinetics

ERIC Educational Resources Information Center

Lechner, Joseph H.

2011-01-01

Several additions to a classroom activity are proposed in which an "enzyme" (the student) converts "substrates" (nut-bolt assemblies) into "products" (separated nuts and bolts) by unscrewing them. (Contains 1 table.)
The origins of enzyme kinetics.

PubMed

Cornish-Bowden, Athel

2013-09-02

The equation commonly called the Michaelis-Menten equation is sometimes attributed to other authors. However, although Victor Henri had derived the equation from the correct mechanism, and Adrian Brown before him had proposed the idea of enzyme saturation, it was Leonor Michaelis and Maud Menten who showed that this mechanism could also be deduced on the basis of an experimental approach that paid proper attention to pH and spontaneous changes in the product after formation in the enzyme-catalysed reaction. By using initial rates of reaction they avoided the complications due to substrate depletion, product accumulation and progressive inactivation of the enzyme that had made attempts to analyse complete time courses very difficult. Their methodology has remained the standard approach to steady-state enzyme kinetics ever since. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Structures of bacterial polynucleotide kinase in a Michaelis complex with GTP•Mg2+ and 5'-OH oligonucleotide and a product complex with GDP•Mg2+ and 5'-PO4 oligonucleotide reveal a mechanism of general acid-base catalysis and the determinants of phosphoacceptor recognition.

PubMed

Das, Ushati; Wang, Li Kai; Smith, Paul; Jacewicz, Agata; Shuman, Stewart

2014-01-01

Clostridium thermocellum polynucleotide kinase (CthPnk), the 5' end-healing module of a bacterial RNA repair system, catalyzes reversible phosphoryl transfer from an NTP donor to a 5'-OH polynucleotide acceptor. Here we report the crystal structures of CthPnk-D38N in a Michaelis complex with GTP•Mg(2+) and a 5'-OH oligonucleotide and a product complex with GDP•Mg(2+) and a 5'-PO4 oligonucleotide. The O5' nucleophile is situated 3.0 Å from the GTP γ phosphorus in the Michaelis complex, where it is coordinated by Asn38 and is apical to the bridging β phosphate oxygen of the GDP leaving group. In the product complex, the transferred phosphate has undergone stereochemical inversion and Asn38 coordinates the 5'-bridging phosphate oxygen of the oligonucleotide. The D38N enzyme is poised for catalysis, but cannot execute because it lacks Asp38-hereby implicated as the essential general base catalyst that abstracts a proton from the 5'-OH during the kinase reaction. Asp38 serves as a general acid catalyst during the 'reverse kinase' reaction by donating a proton to the O5' leaving group of the 5'-PO4 strand. The acceptor strand binding mode of CthPnk is distinct from that of bacteriophage T4 Pnk.

Direct measurement of catalase activity in living cells and tissue biopsies

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

Scaglione, Christine N.; Xu, Qijin; Ramanujan, V. Krishnan, E-mail: Ramanujanv@csmc.edu

Spatiotemporal regulation of enzyme-substrate interactions governs the decision-making steps in biological systems. Enzymes, being functional units of every living cell, contribute to the macromolecular stability of cell survival, proliferation and hence are vital windows to unraveling the biological complexity. Experimental measurements capturing this dynamics of enzyme-substrate interactions in real time add value to this understanding. Furthermore these measurements, upon validation in realistic biological specimens such as clinical biopsies – can further improve our capability in disease diagnostics and treatment monitoring. Towards this direction, we describe here a novel, high-sensitive measurement system for measuring diffusion-limited enzyme-substrate kinetics in real time. Usingmore » catalase (enzyme) and hydrogen peroxide (substrate) as the example pair, we demonstrate that this system is capable of direct measurement of catalase activity in vitro and the measured kinetics follows the classical Michaelis-Menten reaction kinetics. We further demonstrate the system performance by measuring catalase activity in living cells and in very small amounts of liver biopsies (down to 1 μg total protein). Catalase-specific enzyme activity is demonstrated by genetic and pharmacological tools. Finally we show the clinically-relevant diagnostic capability of our system by comparing the catalase activities in liver biopsies from young and old mouse (liver and serum) samples. We discuss the potential applicability of this system in clinical diagnostics as well as in intraoperative surgical settings. - Highlights: • A novel, direct measurement of Catalase enzyme activity via, oxygen sensing method. • Steady-stateprofiles of Catalase activity follow the Michaelis-Menten Kinetics. • Catalase-specific activity demonstrated using genetic and pharmacological tools. • Overcomes limitations of spectroscopic methods and indirect calorimetric approaches. • Clear demonstration of the applicability in cancer cells and aging animal tissues.« less
Effect of fuel concentration on cargo transport by a team of Kinesin motors

NASA Astrophysics Data System (ADS)

Takshak, Anjneya; Mishra, Nirvantosh; Kulkarni, Aditi; Kunwar, Ambarish

2017-02-01

Eukaryotic cells employ specialized proteins called molecular motors for transporting organelles and vesicles from one location to another in a regulated and directed manner. These molecular motors often work collectively in a team while transporting cargos. Molecular motors use cytoplasmic ATP as fuel, which is hydrolyzed to generate mechanical force. While the effect of ATP concentration on cargo transport by single Kinesin motor function is well understood, it is still unexplored, both theoretically and experimentally, how ATP concentration would affect cargo transport by a team of Kinesin motors. For instance, how does fuel concentration affect the travel distances and travel velocities of cargo? How cooperativity of Kinesin motors engaged on a cargo is affected by ATP concentration? To answer these questions, here we develop mechano-chemical models of cargo transport by a team of Kinesin motors. To develop these models we use experimentally-constrained mechano-chemical model of a single Kinesin motor as well as earlier developed mean-field and stochastic models of load sharing for cargo transport. Thus, our new models for cargo transport by a team of Kinesin motors include fuel concentration explicitly, which was not considered in earlier models. We make several interesting predictions which can be tested experimentally. For instance, the travel distances of cargos are very large at limited ATP concentrations in spite of very small travel velocity. Velocities of cargos driven by multiple Kinesin have a Michaelis-Menten dependence on ATP concentration. Similarly, cooperativity among the engaged Kinesin motors on the cargo shows a Michaelis-Menten type dependence, which attains a maximum value near physiological ATP concentrations. Our new results can be potentially useful in controlling artificial nano-molecular shuttles precisely for targeted delivery in various nano-technological applications.
Projection rule for complex-valued associative memory with large constant terms

NASA Astrophysics Data System (ADS)

Kitahara, Michimasa; Kobayashi, Masaki

Complex-valued Associative Memory (CAM) has an inherent property of rotation invariance. Rotation invariance produces many undesirable stable states and reduces the noise robustness of CAM. Constant terms may remove rotation invariance, but if the constant terms are too small, rotation invariance does not vanish. In this paper, we eliminate rotation invariance by introducing large constant terms to complex-valued neurons. We have to make constant terms sufficiently large to improve the noise robustness. We introduce a parameter to control the amplitudes of constant terms into projection rule. The large constant terms are proved to be effective by our computer simulations.
Catalytic efficiency is a better predictor of arsenic toxicity to soil alkaline phosphatase.

PubMed

Wang, Ziquan; Tian, Haixia; Lu, Guannan; Zhao, Yiming; Yang, Rui; Megharaj, Mallavarapu; He, Wenxiang

2018-02-01

Arsenic (As) is an inhibitor of phosphatase, however, in the complex soil system, the substrate concentration effect and the mechanism of As inhibition of soil alkaline phosphatase (ALP) and its kinetics has not been adequately studied. In this work, we investigated soil ALP activity in response to As pollution at different substrate concentrations in various types of soils and explored the inhibition mechanism using the enzyme kinetics. The results showed that As inhibition of soil ALP activity was substrate concentration-dependent. Increasing substrate concentration decreased inhibition rate, suggesting reduced toxicity. This dependency was due to the competitive inhibition mechanism of As to soil ALP. The kinetic parameters, maximum reaction velocity (V max ) and Michaelis constant (K m ) in unpolluted soils were 0.012-0.267mMh -1 and 1.34-3.79mM respectively. The competitive inhibition constant (K ic ) was 0.17-0.70mM, which was lower than K m , suggesting higher enzyme affinity for As than for substrate. The ecological doses, ED 10 and ED 50 (concentration of As that results in 10% and 50% inhibition on enzyme parameter) for inhibition of catalytic efficiency (V max /K m ) were lower than those for inhibition of enzyme activity at different substrate concentrations. This suggests that the integrated kinetic parameter, catalytic efficiency is substrate concentration independent and more sensitive to As than ALP activity. Thus, catalytic efficiency was proposed as a more reliable indicator than ALP activity for risk assessment of As pollution. Copyright © 2017 Elsevier Inc. All rights reserved.
Purification and characterization of creatine kinase isozymes from the nurse shark Ginglymostoma cirratum.

PubMed

Gray, K A; Grossman, S H; Summers, D D

1986-01-01

Creatine kinase from nurse shark brain and muscle has been purified to apparent homogeneity. In contrast to creatine kinases from most other vertebrate species, the muscle isozyme and the brain isozyme from nurse shark migrate closely in electrophoresis and, unusually, the muscle isozyme is anodal to the brain isozyme. The isoelectric points are 5.3 and 6.2 for the muscle and brain isozymes, respectively. The purified brain preparation also contains a second active protein with pI 6.0. The amino acid content of the muscle isozyme is compared with other isozymes of creatine kinase using the Metzger Difference Index as an estimation of compositional relatedness. All comparisons show a high degree of compositional similarity including arginine kinase from lobster muscle. The muscle isozyme is marginally more resistant to temperature inactivation than the brain isozyme; the muscle protein does not exhibit unusual stability towards high concentrations of urea. Kinetic analysis of the muscle isozyme reveals Michaelis constants of 1.6 mM MgATP, 12 mM creatine, 1.2 mM MgADP and 50 mM creatine phosphate. Dissociation constants for the same substrate from the binary and ternary enzyme-substrate complex do not differ significantly, indicating limited cooperatively in substrate binding. Enzyme activity is inhibited by small planar anions, most severely by nitrate. Shark muscle creatine kinase hybridizes in vitro with rabbit muscle or monkey brain creatine kinase; shark brain isozyme hybridizes with monkey brain or rabbit brain creatine kinase. Shark muscle and shark brain isozymes, under a wide range of conditions, failed to produce a detectable hybrid.
Carbon nanotube mat as mediator-less glucose sensor electrode.

PubMed

Ryu, Jongeun; Kim, Hansang; Lee, Sangeui; Hahn, H Thomas; Lashmore, David

2010-02-01

In this paper, the direct electron transfer of glucose oxidase (GOx) on carbon nanotube (CNT) mat electrode is demonstrated. Because of the electrical conductivity and mechanical strength of CNT mat, it can be used as an electrode as well as a catalyst support. Therefore, the preparation process for the CNT mat based sensor electrode is simpler than that of the conventional CNT dispersed sensor electrodes. GOx was covalently immobilized on the oxidized CNT mat, which is connected to a wire by using silver paste and epoxy glue. Attenuated Total Reflectance Fourier Transform-Infrared (ATR-FTIR) result shows transmittance peaks at 1637 cm(-1) and 1525 cm(-1) which are corresponding to the band I and II of amide. Cyclic voltammetric shows a pair of well-defined redox peaks with the average formal potential of -0.425 V (vs. Ag/AgCl reference electrode) in the phosphate buffered saline solution (1 x PBS, pH 7.4). Calculated electron transfer rate constant and the surface density of GOx were 1.71 s(-1) and (3.27 +/- 0.20) x 10(-13) mol/cm2, respectively. Cyclic voltammograms of GOx-CNT mat in glucose solution show that the immobilized GOx retains its catalytic activity to glucose. The amperometric sensor response showed a linear dependence on the glucose concentration in the range of 0.2 mM to 2.18 mM with a detection sensitivity of 4.05 microA mM(-1) cm(-2). The Michaelis-Menten constant of the immobilized GOx was calculated to be 2.18 mM.
Rational design of reversible inhibitors for trehalose 6-phosphate phosphatases.

PubMed

Liu, Chunliang; Dunaway-Mariano, Debra; Mariano, Patrick S

2017-03-10

In some organisms, environmental stress triggers trehalose biosynthesis that is catalyzed collectively by trehalose 6-phosphate synthase, and trehalose 6-phosphate phosphatase (T6PP). T6PP catalyzes the hydrolysis of trehalose 6-phosphate (T6P) to trehalose and inorganic phosphate and is a promising target for the development of antibacterial, antifungal and antihelminthic therapeutics. Herein, we report the design, synthesis and evaluation of a library of aryl d-glucopyranoside 6-sulfates to serve as prototypes for small molecule T6PP inhibitors. Steady-state kinetic techniques were used to measure inhibition constants (K i ) of a panel of structurally diverse T6PP orthologs derived from the pathogens Brugia malayi, Ascaris suum, Mycobacterium tuberculosis, Shigella boydii and Salmonella typhimurium. The binding affinities of the most active inhibitor of these T6PP orthologs, 4-n-octylphenyl α-d-glucopyranoside 6-sulfate (9a), were found to be in the low micromolar range. The K i of 9a with the B. malayi T6PP ortholog is 5.3 ± 0.6 μM, 70-fold smaller than the substrate Michaelis constant. The binding specificity of 9a was demonstrated using several representative sugar phosphate phosphatases from the HAD enzyme superfamily, the T6PP protein fold family of origin. Lastly, correlations drawn between T6PP active site structure, inhibitor structure and inhibitor binding affinity suggest that the aryl d-glucopyranoside 6-sulfate prototypes will find future applications as a platform for development of tailored second-generation T6PP inhibitors. Copyright © 2017 Elsevier Masson SAS. All rights reserved.
Improved ethanol production by engineered Saccharomyces cerevisiae expressing a mutated cellobiose transporter during simultaneous saccharification and fermentation.

PubMed

Lee, Won-Heong; Jin, Yong-Su

2017-03-10

Although simultaneous saccharification and fermentation (SSF) of cellulosic biomass can offer efficient hydrolysis of cellulose through alleviating feed-back inhibition of cellulases by glucose, supplementation of β-glucosidase is necessary because most fermenting microorganisms cannot utilize cellobiose. Previously, we observed that SSF of cellulose by an engineered Saccharomyces cerevisiae expressing a cellobiose transporter (CDT-1) and an intracellular β-glucosidase (GH1-1) without β-glucosidase could not be performed as efficiently as the traditional SSF with extracellular β-glucosidase. However, we improved the ethanol production from SSF of cellulose by employing a further engineered S. cerevisiae expressing a mutant cellobiose transporter [CDT-1 (F213L) exhibiting higher V MAX than CDT-1] and GH1-1 in this study. Furthermore, limitation of cellobiose formation by reducing the amounts of cellulases mixture in SSF could lead the further engineered strain to produce ethanol considerably better than the parental strain with β-glucosidase. Probably, better production of ethanol by the further engineered strain seemed to be due to a higher affinity to cellobiose, which might be attributed to not only 2-times lower Monod constant (K S ) for cellobiose than K S of the parental strain for glucose but also 5-times lower K S than Michaelis-Menten constant (K M ) of the extracellular β-glucosidase for glucose. Our results suggest that modification of the cellobiose transporter in the engineered yeast to transport lower level of cellobiose enables a more efficient SSF for producing ethanol from cellulose. Copyright © 2017 Elsevier B.V. All rights reserved.
Buprofezin Is Metabolized by CYP353D1v2, a Cytochrome P450 Associated with Imidacloprid Resistance in Laodelphax striatellus.

PubMed

Elzaki, Mohammed Esmail Abdalla; Miah, Mohammad Asaduzzaman; Han, Zhaojun

2017-11-29

CYP353D1v2 is a cytochrome P450 related to imidacloprid resistance in Laodelphax striatellus . This work was conducted to examine the ability of CYP353D1v2 to metabolize other insecticides. Carbon monoxide difference spectra analysis indicates that CYP353D1v2 was successfully expressed in insect cell Sf9. The catalytic activity of CYP353D1v2 relating to degrading buprofezin, chlorpyrifos, and deltamethrin was tested by measuring substrate depletion and analyzing the formation of metabolites. The results showed the nicotinamide-adenine dinucleotide phosphate (NADPH)-dependent depletion of buprofezin (eluting at 8.7 min) and parallel formation of an unknown metabolite (eluting 9.5 min). However, CYP353D1v2 is unable to metabolize deltamethrin and chlorpyrifos. The recombinant CYP353D1v2 protein efficiently catalyzed the model substrate p -nitroanisole with a maximum velocity of 9.24 nmol/min/mg of protein and a Michaelis constant of Km = 6.21 µM. In addition, imidacloprid was metabolized in vitro by the recombinant CYP353D1v2 microsomes (catalytic constant Kcat) 0.064 pmol/min/pmol P450, Km = 6.41 µM. The mass spectrum of UPLC-MS analysis shows that the metabolite was a product of buprofezin, which was buprofezin sulfone. This result provided direct evidence that L. striatellus cytochrome P450 CYP353D1v2 is capable of metabolizing imidacloprid and buprofezin.
Buprofezin Is Metabolized by CYP353D1v2, a Cytochrome P450 Associated with Imidacloprid Resistance in Laodelphax striatellus

PubMed Central

Elzaki, Mohammed Esmail Abdalla; Miah, Mohammad Asaduzzaman; Han, Zhaojun

2017-01-01

CYP353D1v2 is a cytochrome P450 related to imidacloprid resistance in Laodelphax striatellus. This work was conducted to examine the ability of CYP353D1v2 to metabolize other insecticides. Carbon monoxide difference spectra analysis indicates that CYP353D1v2 was successfully expressed in insect cell Sf9. The catalytic activity of CYP353D1v2 relating to degrading buprofezin, chlorpyrifos, and deltamethrin was tested by measuring substrate depletion and analyzing the formation of metabolites. The results showed the nicotinamide–adenine dinucleotide phosphate (NADPH)-dependent depletion of buprofezin (eluting at 8.7 min) and parallel formation of an unknown metabolite (eluting 9.5 min). However, CYP353D1v2 is unable to metabolize deltamethrin and chlorpyrifos. The recombinant CYP353D1v2 protein efficiently catalyzed the model substrate p-nitroanisole with a maximum velocity of 9.24 nmol/min/mg of protein and a Michaelis constant of Km = 6.21 µM. In addition, imidacloprid was metabolized in vitro by the recombinant CYP353D1v2 microsomes (catalytic constant Kcat) 0.064 pmol/min/pmol P450, Km = 6.41 µM. The mass spectrum of UPLC-MS analysis shows that the metabolite was a product of buprofezin, which was buprofezin sulfone. This result provided direct evidence that L. striatellus cytochrome P450 CYP353D1v2 is capable of metabolizing imidacloprid and buprofezin. PMID:29186030
A hybrid biocatalyst consisting of silver nanoparticle and naphthalenethiol self-assembled monolayer prepared for anchoring glucose oxidase and its use for an enzymatic biofuel cell

NASA Astrophysics Data System (ADS)

Christwardana, Marcelinus; Kim, Do-Heyoung; Chung, Yongjin; Kwon, Yongchai

2018-01-01

A novel hybrid biocatalyst is synthesized by the enzyme composite consisting of silver nanoparticle (AgNP), naphthalene-thiol based couplers (Naph-SH) and glucose oxidase (GOx), which is then bonded with the supporter consisting of polyethyleneimine (PEI) and carbon nanotube (CNT) (CNT/PEI/AgNPs/Naph-SH/GOx) to facilitate glucose oxidation reaction (GOR). Here, the AgNPs play a role in obstructing denaturation of the GOx molecules from the supporter because of Ag-thiol bond, while the PEIs have the AgNPs keep their states without getting ionized by hydrogen peroxide produced during anodic reaction. The Naph-SHs also prevent ionization of the AgNP by forming self-assembled monolayer on their surface. Such roles of each component enable the catalyst to form (i) hydrophobic interaction between the GOx molecules and supporter and (ii) π-conjugated electron pathway between the GOx molecules and AgNP, promoting electron transfer. Catalytic nature of the catalyst is characterized by measuring catalytic activity and performance of enzymatic biofuel cell (EBC) using the catalyst. Regarding the catalytic activity, the catalyst leads to high electron transfer rate constant (9.6 ± 0.4 s-1), low Michaelis-Menten constant (0.51 ± 0.04 mM), and low charge transfer resistance (7.3 Ω cm2) and high amount of immobilized GOx (54.6%), while regarding the EBC performance, high maximum power density (1.46 ± 0.07 mW cm-2) with superior long-term stability result are observed.
Effects of tetraethylammonium on potassium currents in a molluscan neurons

PubMed Central

1981-01-01

The effects of tetraethylammonium (TEA) on the delayed K+ current and on the Ca2+-activated K+ current of the Aplysia pacemaker neurons R-15 and L-6 were studied. The delayed outward K+ current was measured in Ca2+-free ASW containing tetrodotoxin (TTX), using brief depolarizing clamp pulses. External TEA blocks the delayed K+ current reversibly in a dose-dependent manner. The experimental results are well fitted with a Michaelis-Menten expression, assuming a one-to-one reaction between TEA and a receptor site, with an apparent dissociation constant of 6.0 mM. The block depends on membrane voltage and is reduced at positive membrane potentials. The Ca2+-activated K+ current was measured in Ca2+- free artificial seawater (ASW) containing TTX, using internal Ca2+ ion injection to directly activate the K+ conductance. External TEA and a number of other quaternary ammonium ions block the Ca2+-activated K+ current reversibly in a dose-dependent manner. TEA is the most effective blocker, with an apparent dissociation constant, for a one-to- one reaction with a receptor site, of 0.4 mM. The block decreases with depolarization. The Ca2+-activated K+ current was also measured after intracellular iontophoretic TEA injection. Internal TEA blocks the Ca2+- activated K+ current (but the block is only apparent at positive membrane potentials), is increased by depolarization, and is irreversible. The effects of external and internal TEA can be seen in measurements of the total outward K+ current at different membrane potentials in normal ASW. PMID:6265594
Imidazoline derivative templated synthesis of broccoli-like Bi2S3 and its electrocatalysis towards the direct electrochemistry of hemoglobin.

PubMed

Chen, Xiaoqian; Wang, Qingxiang; Wang, Liheng; Gao, Feng; Wang, Wei; Hu, Zhengshui

2015-04-15

A broccoli-like bismuth sulfide (bBi2S3) was synthesized via a solvothermal method using a self-made imidazoline derivative of 2-undecyl-1-dithioureido-ethyl-imidazoline as the soft template. The morphology and chemical constitution of the product were characterized by scanning electron microscope (SEM), transmission electron microscope (TEM) and X-ray diffraction (XRD). Electrochemical characterization experiments show that the bBi2S3 has the higher specific surface area and standard heterogeneous electron transfer rate constant than the rod-like Bi2S3 (rBi2S3). Hemoglobin (Hb) was then chosen as a protein model to investigate the electrocatalytic property of the synthesized bBi2S3. The results show that Hb entrapped in the composite film of chitosan and bBi2S3 displays an excellent direct electrochemistry, and retains its biocatalytic activity toward the electro-reduction of hydrogen peroxide. The current response in the amperometry shows a linear response to H2O2 concentrations in the range from 0.4 to 4.8µM with high sensitivity (444µAmM(-1)) and low detection limit (0.096µM). The Michaelis-Menten constant (KM(app)) of the fabricated bioelectrode for H2O2 was determined as low as 1µM. These results demonstrate that the synthesized bBi2S3 offers a new path for the immobilization of redox-active protein and the construction of the third-generation biosensors. Copyright © 2014 Elsevier B.V. All rights reserved.
4-n-butylresorcinol, a depigmenting agent used in cosmetics, reacts with tyrosinase.

PubMed

Garcia-Jimenez, Antonio; Teruel-Puche, Jose Antonio; Ortiz-Ruiz, Carmen Vanessa; Berna, Jose; Tudela, Jose; Garcia-Canovas, Francisco

2016-08-01

4-n-Butylresorcinol (BR) is considered the most potent inhibitor of tyrosinase, which is why it is used in cosmetics as a depigmenting agent. However, this work demonstrates that BR is a substrate of this enzyme. The Em (met-tyrosinase) form is not active on BR, but Eox (oxy-tyrosinase) can act on this molecule, hydroxylating it to o-diphenol. In turn, this is oxidized to an o-quinone, which isomerizes to a red p-quinone. Thus, for tyrosinase to act on this compound, a mechanism to generate Eox in the medium is required, which can be achieved by means of hydrogen peroxide or ascorbic acid. A kinetic analysis of the proposed mechanism allows its kinetic characterization: catalytic constant kcatBR (8.49 ± 0.20 s(-1) ) and Michaelis-constant KMBR (60.26 ± 8.76 μM). These findings are compared with those for other monophenolic substrates of tyrosinase. Studies of BR docking to the Em form of the enzyme show that the hydroxyl group in C-1 position is oriented toward the copper atom A (CuA), as in it is L-tyrosine. As regards Eox , BR is oriented with the carbon in C-6 position ready to be hydroxylated. The reaction of BR originates o-quinones, which isomerize to p-quinones, which in turn, could react with thiol compounds, a finding that could have important implications for pharmacology and the cosmetic industry. © 2016 IUBMB Life, 68(8):663-672, 2016. © 2016 International Union of Biochemistry and Molecular Biology.
p-Aminohippurate transport in airways: competitive inhibition.

PubMed

Cloutier, M M; Guernsey, L

1992-05-01

p-Aminohippurate (PAH) transport in canine tracheal epithelium occurs by a HCO3- -PAH exchange process that is located on the luminal membrane and is inhibited by stilbene derivatives. The effects of increasing concentrations of other organic anions, including probenecid (10-250 microM), dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP; 10-1,000 microM), phenol red (10-250 microM), and urate (25-500 microM), and the organic cation tetraethylammonium bromide (TEA; 250 microM) on PAH transport were examined in canine tracheal epithelium mounted in Ussing chambers. Neither phenol red, urate, nor TEA had any effect on electrophysiological properties or unidirectional or net PAH fluxes. In contrast, beginning at 10 microM, both probenecid and cAMP produced significant decreases in unidirectional and net PAH absorption without change in unidirectional PAH secretion. The initial change in net PAH absorption occurred in the absence of any change in electrophysiological properties. Higher concentrations of both probenecid and cAMP produced further decreases in net PAH absorption and significant changes in electrophysiological properties. Probenecid and cAMP increased the apparent Michaelis constant for PAH absorption without affecting maximum transport rate. The inhibitory constant for probenecid was 1.01 +/- 0.06 x 10(-4) M (mean +/- SE) and for cAMP was 5.18 +/- 0.20 x 10(-4) M. We conclude that PAH transport in canine tracheal epithelium demonstrates competitive inhibition by other organic anions and substrate specificity. We also conclude that the affinity of the exchange transport system is higher for probenecid than for PAH and cAMP.
Characterization of a uronate dehydrogenase from Thermobispora bispora for production of glucaric acid from hemicellulose substrate.

PubMed

Li, Yaxian; Xue, Yemin; Cao, Zhigang; Zhou, Tao; Alnadari, Fawze

2018-06-23

A thermostable uronate dehydrogenase Tb-UDH from Thermobispora bispora was over-expressed in Escherichia coli using the T7 polymerase expression system. The Tb-UDH was purified by metal affinity chromatography, and gave a single band on SDS-PAGE. The maximum activity on glucuronic acid was found at 60 °C and pH 7.0. The purified enzyme retained over 58% of its activity after holding a pH ranging from 7.0 to 7.5 for 1 h at 60 °C. The K m and V max values of the purified Tb-UDH for Glucuronic acid (GluUA) were 0.165 mM and 117.7 U mg -1 , respectively, those for galacturonic acid (GalUA) were 0.115 mM and 104.2 U mg -1 , respectively, and those for NAD + were 0.120 mM and 133.3 U mg -1 , respectively; the turnover number (k cat ) with GluUA as a substrate was higher than that with GalUA; however, the Michaelis constant (K m ) for GalUA was lower than that for GluUA. After 60 min of incubation at 50 °C, Tb-UDH exhibited a conversion ratio for glucuronic acid to the glucaric acid of 84% on chemical reagent and 81.3% on hydrolysates from breech xylans formed by xylanase and α-glucuronidase. This work shows that biocatalytic routes have great potential for the conversion of hemicellulose substrate into value-added products derived from renewable biomass. TOC GRAPHIC: (A) The structure of the xylan is described and the site of action of the xylan degrading enzyme is indicated. (B) The effect of substrate concentration on recombinant Tb-UDH activity when galacturonic acid was used as substrate. (C) SDS-PAGE analysis of E. coli BL21 (DE3) harboring pET-20b(+) and pET-20b-Tb-UDH. (D) Oxidative conversion of glucuronic acid from a beechwood xylan to glucaric acid.
Use of mushroom tyrosinase to introduce michaelis-menten enzyme kinetics to biochemistry students.

PubMed

Flurkey, William H; Inlow, Jennifer K

2017-05-01

An inexpensive enzyme kinetics laboratory exercise for undergraduate biochemistry students is described utilizing tyrosinase from white button mushrooms. The exercise can be completed in one or two three-hour lab sessions. The optimal amounts of enzyme, substrate (catechol), and inhibitor (kojic acid) are first determined, and then kinetic data is collected in the absence and presence of the inhibitor. A Microsoft Excel template is used to plot the data and to fit the Michaelis-Menten equation to the data to determine the kinetic parameters V max and K m . The exercise is designed to clarify and reinforce concepts covered in an accompanying biochemistry lecture course. It has been used with positive results in an upper-level biochemistry laboratory course for junior/senior students majoring in chemistry or biology. © 2016 by The International Union of Biochemistry and Molecular Biology, 45(3):270-276, 2017. © 2016 The International Union of Biochemistry and Molecular Biology.
Pop-it beads to introduce catalysis of reaction rate and substrate depletion effects.

PubMed

Gehret, Austin U

2017-03-04

A kinesthetic classroom activity was designed to help students understand enzyme activity and catalysis of reaction rate. Students served the role of enzymes by manipulating Pop-It Beads as the catalytic event. This activity illuminates the relationship between reaction rate and reaction progress by allowing students to experience first-hand the effect of substrate depletion on catalyzed reaction rate. Preliminary findings based on survey results and exam performance suggest the activity could prove beneficial to students in the targeted learning outcomes. Unique to previous kinesthetic approaches that model Michaelis-Menten kinetics, this activity models the effects of substrate depletion on catalyzed reaction rate. Therefore, it could prove beneficial for conveying the reasoning behind the initial rate simplification used in Michaelis-Menten kinetics. © 2016 by The International Union of Biochemistry and Molecular Biology, 45(2):179-183, 2017. © 2016 The International Union of Biochemistry and Molecular Biology.
Patterns induced by super cross-diffusion in a predator-prey system with Michaelis-Menten type harvesting.

PubMed

Liu, Biao; Wu, Ranchao; Chen, Liping

2018-04-01

Turing instability and pattern formation in a super cross-diffusion predator-prey system with Michaelis-Menten type predator harvesting are investigated. Stability of equilibrium points is first explored with or without super cross-diffusion. It is found that cross-diffusion could induce instability of equilibria. To further derive the conditions of Turing instability, the linear stability analysis is carried out. From theoretical analysis, note that cross-diffusion is the key mechanism for the formation of spatial patterns. By taking cross-diffusion rate as bifurcation parameter, we derive amplitude equations near the Turing bifurcation point for the excited modes by means of weakly nonlinear theory. Dynamical analysis of the amplitude equations interprets the structural transitions and stability of various forms of Turing patterns. Furthermore, the theoretical results are illustrated via numerical simulations. Copyright © 2018. Published by Elsevier Inc.
Plant Photosynthesis-Irradiance Curve Responses to Pollution Show Non-Competitive Inhibited Michaelis Kinetics

PubMed Central

Lin, Maozi; Wang, Zhiwei; He, Lingchao; Xu, Kang; Cheng, Dongliang; Wang, Genxuan

2015-01-01

Photosynthesis-irradiance (PI) curves are extensively used in field and laboratory research to evaluate the photon-use efficiency of plants. However, most existing models for PI curves focus on the relationship between the photosynthetic rate (Pn) and photosynthetically active radiation (PAR), and do not take account of the influence of environmental factors on the curve. In the present study, we used a new non-competitive inhibited Michaelis-Menten model (NIMM) to predict the co-variation of Pn, PAR, and the relative pollution index (I). We then evaluated the model with published data and our own experimental data. The results indicate that the Pn of plants decreased with increasing I in the environment and, as predicted, were all fitted well by the NIMM model. Therefore, our model provides a robust basis to evaluate and understand the influence of environmental pollution on plant photosynthesis. PMID:26561863

Insulin stimulation of glucose transport in isolated rat adipocytes. Functional evidence for insulin activation of intrinsic transporter activity within the plasma membrane.

PubMed Central

Hyslop, P A; Kuhn, C E; Sauerheber, R D

1985-01-01

We examined the effects of the membrane-impermeant amino-group-modifying agent fluorescein isothiocyanate (FITC) on the basal and insulin-stimulated hexose-transport activity of isolated rat adipocytes. Pre-treatment of cells with FITC causes irreversible inhibition of transport measured in subsequently washed cells. Transport activity was inhibited by approx. 50% with 2 mM-FITC in 8 min. The cells respond to insulin, after FITC treatment and removal, and the fold increase in transport above the basal value caused by maximal concentrations of insulin was independent of the concentration of FITC used for pre-treatment over the range 0-2 mM, where basal activity was progressively inhibited. The ability of FITC to modify selectively hexose transporters accessible only to the external milieu was evaluated by two methods. (1) Free intracellular FITC, and the distribution of FITC bound to cellular components, were assessed after dialysis of the homogenate and subcellular fractionation on sucrose gradients by direct spectroscopic measurement of fluorescein. Most (98%) of the FITC was associated with the non-diffusible fractions. Equilibrium sucrose-density-gradient centrifugation of the homogenate demonstrated that the subcellular distribution of the bound FITC correlated with the density distribution of a plasma-membrane marker, but not markers for Golgi, endoplasmic reticulum, mitochondria or protein. Exposing the cellular homogenate, rather than the intact cell preparation, to 2 mM-FITC resulted in a 4-5-fold increase in total bound FITC, and the density-distribution profile more closely resembled the distribution of total protein. (2) Incubation of hexokinase preparations with FITC rapidly and irreversibly inactivates this protein. However, both intracellular hexokinase total activity and its apparent Michaelis constant for glucose were unaffected in FITC-treated intact cells. Further control experiments demonstrated that FITC pre-treatment of cells had no effect on the intracellular ATP concentration or the dose-response curve of insulin stimulation of hexose transport. Since the fold increase of hexose transport induced by insulin is constant over the range of inhibition of surface-labelled hexose transporters, we suggest that insulin-induced insertion of additional transporters into the plasma membrane may not be the major locus of acceleration of hexose transport by the hormone. PMID:3910027
Galaxy Formation Efficiency and the Multiverse Explanation of the Cosmological Constant with EAGLE Simulations

NASA Astrophysics Data System (ADS)

Barnes, Luke A.; Elahi, Pascal J.; Salcido, Jaime; Bower, Richard G.; Lewis, Geraint F.; Theuns, Tom; Schaller, Matthieu; Crain, Robert A.; Schaye, Joop

2018-04-01

Models of the very early universe, including inflationary models, are argued to produce varying universe domains with different values of fundamental constants and cosmic parameters. Using the cosmological hydrodynamical simulation code from the EAGLE collaboration, we investigate the effect of the cosmological constant on the formation of galaxies and stars. We simulate universes with values of the cosmological constant ranging from Λ = 0 to Λ0 × 300, where Λ0 is the value of the cosmological constant in our Universe. Because the global star formation rate in our Universe peaks at t = 3.5 Gyr, before the onset of accelerating expansion, increases in Λ of even an order of magnitude have only a small effect on the star formation history and efficiency of the universe. We use our simulations to predict the observed value of the cosmological constant, given a measure of the multiverse. Whether the cosmological constant is successfully predicted depends crucially on the measure. The impact of the cosmological constant on the formation of structure in the universe does not seem to be a sharp enough function of Λ to explain its observed value alone.
Galaxy formation efficiency and the multiverse explanation of the cosmological constant with EAGLE simulations

NASA Astrophysics Data System (ADS)

Barnes, Luke A.; Elahi, Pascal J.; Salcido, Jaime; Bower, Richard G.; Lewis, Geraint F.; Theuns, Tom; Schaller, Matthieu; Crain, Robert A.; Schaye, Joop

2018-07-01

Models of the very early Universe, including inflationary models, are argued to produce varying universe domains with different values of fundamental constants and cosmic parameters. Using the cosmological hydrodynamical simulation code from the EAGLE collaboration, we investigate the effect of the cosmological constant on the formation of galaxies and stars. We simulate universes with values of the cosmological constant ranging from Λ = 0 to Λ0 × 300, where Λ0 is the value of the cosmological constant in our Universe. Because the global star formation rate in our Universe peaks at t = 3.5 Gyr, before the onset of accelerating expansion, increases in Λ of even an order of magnitude have only a small effect on the star formation history and efficiency of the universe. We use our simulations to predict the observed value of the cosmological constant, given a measure of the multiverse. Whether the cosmological constant is successfully predicted depends crucially on the measure. The impact of the cosmological constant on the formation of structure in the universe does not seem to be a sharp enough function of Λ to explain its observed value alone.
Lipase-catalyzed synthesis of palmitanilide: Kinetic model and antimicrobial activity study.

PubMed

Liu, Kuan-Miao; Liu, Kuan-Ju

2016-01-01

Enzymatic syntheses of fatty acid anilides are important owing to their wide range of industrial applications in detergents, shampoo, cosmetics, and surfactant formulations. The amidation reaction of Mucor miehei lipase Lipozyme IM20 was investigated for direct amidation of triacylglycerol in organic solvents. The process parameters (reaction temperature, substrate molar ratio, enzyme amount) were optimized to achieve the highest yield of anilide. The maximum yield of palmitanilide (88.9%) was achieved after 24 h of reaction at 40 °C at an enzyme concentration of 1.4% (70 mg). Kinetics of lipase-catalyzed amidation of aniline with tripalmitin has been investigated. The reaction rate could be described in terms of the Michaelis-Menten equation with a Ping-Pong Bi-Bi mechanism and competitive inhibition by both the substrates. The kinetic constants were estimated by using non-linear regression method using enzyme kinetic modules. The enzyme operational stability study showed that Lipozyme IM20 retained 38.1% of the initial activity for the synthesis of palmitanilide (even after repeated use for 48 h). Palmitanilide, a fatty acid amide, exhibited potent antimicrobial activity toward Bacillus cereus. Copyright © 2015 Elsevier Inc. All rights reserved.
Effect of tin oxide nanoparticle binding on the structure and activity of α-amylase from Bacillus amyloliquefaciens

NASA Astrophysics Data System (ADS)

Jahir Khan, Mohammad; Qayyum, Shariq; Alam, Fahad; Husain, Qayyum

2011-11-01

Proteins adsorbed on nanoparticles (NPs) are being used in biotechnology, biosensors and drug delivery. However, understanding the effect of NPs on the structure of proteins is still in a nascent state. In the present paper tin oxide (SnO2) NPs were synthesized by the reaction of SnCl4·5H2O in methanol via the sol-gel method and characterized by x-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR) and transmission electron microscopy (TEM). The binding of these SnO2-NPs with α-amylase was investigated by using UV-vis, fluorescence and circular dichroism (CD) spectroscopic techniques. A strong quenching of tryptophan fluorescence intensity in α-amylase was observed due to formation of a ground state complex with SnO2-NPs. Far-UV CD spectra showed that the secondary structure of α-amylase was changed in the presence of NPs. The Michaelis-Menten constant (Km), was found to be 26.96 and 28.45 mg ml - 1, while Vmax was 4.173 and 3.116 mg ml - 1 min - 1 for free and NP-bound enzyme, respectively.
The P-type ATPase CtpG preferentially transports Cd2+ across the Mycobacterium tuberculosis plasma membrane.

PubMed

López, Marcela; Quitian, Laudy-Viviana; Calderón, Martha-Nancy; Soto, Carlos-Y

2018-04-01

P 1B -type ATPases are involved in heavy metal transport across the plasma membrane. Some Mycobacterium tuberculosis P-type ATPases are induced during infection, suggesting that this type of transporter could play a critical role in mycobacterial survival. To date, the ion specificity of M. tuberculosis heavy metal-transporting P 1B -ATPases is not well understood. In this work, we observed that, although divalent heavy metal cations such as Cu 2+ , Co 2+ , Ni 2+ , Zn 2+ Cd 2+ and Pb 2+ stimulate the ATPase activity of the putative P 1B -type ATPase CtpG in the plasma membrane, whole cells of M. smegmatis expressing CtpG only tolerate high levels of Cd 2+ and Cu 2+ . As indicator of the catalytic constant, Michaelis-Menten kinetics showed that CtpG embedded in the mycobacterial cell membrane has a V max /K m ratio 7.4-fold higher for Cd 2+ than for Cu 2+ ions. Thus, although CtpG can accept different substrates in vitro, this P-type ATPase transports Cd 2+ more efficiently than other heavy metal cations across the mycobacterial plasma membrane.
Glutathione peroxidase: fact and fiction.

PubMed

Flohé, L

The present knowledge of glutathione (GSH) peroxidase is briefly reviewed: GSH peroxidase has a molecular weight of about 85,000, consists of four apparently-identical subunits and contains four g atom of selenium/mol. The enzyme-bound selenium can undergo a substrate-induced redox change and is obviously essential for activity. In accordance with the assumption that a selenol group is reversibly oxidized during catalysis, ping-pong kinetics are observed. Limiting maximum velocities and Michaelis constants, indicating the formation of an enzyme-substrate complex, are not detectable. The enzyme is highly specific for GSH but reacts with many hydroperoxides. It can be deduced from the kinetic analysis of GSH peroxidase that in physiological conditions removal of hydroperoxide is largely independent of fluctuations in the cellular concentration of GSH. However, the system will abruptly collapse if the rate of hydroperoxide formation exceeds that of regeneration of GSH. By these considerations, the pathophysiological manifestation of disorders in GSH metabolism and pentose-phosphate shunt may be explained. With regard to its low specificity for hydroperoxides, GSH peroxidase could be involved in various metabolic events such as H2O2 removal in compartments low in catalase, hydroperoxide-mediated mutagenesis, protection of unsaturated lipids in biomembranes, prostaglandin biosynthesis, and regulation of prostacyclin formation.
The mechanism of zinc uptake by cultured rat liver cells.

PubMed Central

Taylor, J A; Simons, T J

1994-01-01

1. The initial rate of 65Zn uptake into cultured rat hepatocytes has been measured over a range of Zn2+ concentrations from 3 x 10(-10) M to 5 x 10(-6) M. Histidine and albumin were used to buffer Zn2+ ions at concentrations below 1 x 10(-6) M. 2. The results suggest there are two mechanisms for Zn2+ uptake; a high-affinity, saturable pathway, with a maximum velocity (Vmax) of 20-30 pmol (mg protein)-1 min-1 and a Michaelis-Menten constant (Km) of about 2 x 10(-9) M Zn2+ (with histidine), and a low-affinity, linear pathway, that only makes a significant contribution to Zn2+ uptake at Zn2+ concentrations above 1 x 10(-6) M. 3. Transport via the high-affinity pathway is dependent on the concentration of Zn2+ ions and not on the concentrations of Zn(2+)-ligand complexes, suggesting that Zn2+ is the transported species. 4. The affinity of the saturable pathway for Zn2+ is slightly lower in the presence of albumin, with a Km of about 1.3 x 10(-8) M. The reason for this is uncertain. PMID:8014898
Trypsin inhibitor screening in traditional Chinese medicine by using an immobilized enzyme microreactor in capillary and molecular docking study.

PubMed

Cheng, Mengxia; Chen, Zilin

2017-08-01

A trypsin immobilized enzyme microreactor was successfully prepared in capillary for studying enzyme kinetics of trypsin and online screening of trypsin inhibitors from traditional Chinese medicine through capillary electrophoresis. Trypsin was immobilized on the inner wall at the inlet of the capillary treated with polydopamine. The rest of the capillary was used as a separation channel. The parameters including the separation efficiency and the activity of immobilized trypsin were comprehensively evaluated. Under the optimal conditions, online screening of trypsin inhibitors each time can be carried out within 6 min. The Michaelis-Menten constant of immobilized trypsin was calculated to be 0.50 mM, which indicated high affinity of the immobilized trypsin for the substrate. The half-maximal inhibitory concentration of known inhibitor of benzamidine hydrochloride hydrate as a model inhibitor was 13.32 mM. The proposed method was successfully applied to screen trypsin inhibitors from 15 compounds of traditional Chinese medicine. It has been found that baicalin showed inhibitory potency. Molecular docking study well supported the experimental result by exhibiting molecular interaction between enzyme and inhibitors. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Highly-sensitive cholesterol biosensor based on platinum-gold hybrid functionalized ZnO nanorods.

PubMed

Wang, Chengyan; Tan, Xingrong; Chen, Shihong; Yuan, Ruo; Hu, Fangxin; Yuan, Dehua; Xiang, Yun

2012-05-30

A novel scheme for the fabrication of gold/platinum hybrid functionalized ZnO nanorods (Pt-Au@ZnONRs) and multiwalled carbon nanotubes (MWCNTs) modified electrode is presented and its application for cholesterol biosensor is investigated. Firstly, Pt-Au@ZnONRs was prepared by the method of chemical synthesis. Then, the Pt-Au@ZnONRs suspension was dropped on the MWCNTs modified glass carbon electrode, and followed with cholesterol oxidase (ChOx) immobilization by the adsorbing interaction between the nano-material and ChOx as well as the electrostatic interaction between ZnONRs and ChOx molecules. The combination of MWCNTs and Pt-Au@ZnONRs provided a favorable environment for ChOx and resulted in the enhanced analytical response of the biosensor. The resulted biosensor exhibited a linear response to cholesterol in the wide range of 0.1-759.3 μM with a low detection limit of 0.03 μM and a high sensitivity of 26.8 μA mM(-1). The calculated apparent Michaelis constant K(M)(app) was 1.84 mM, indicating a high affinity between ChOx and cholesterol. Copyright © 2012 Elsevier B.V. All rights reserved.
Purification, immobilization and characterization of tannase from Penicillium variable.

PubMed

Sharma, Shashi; Agarwal, Lata; Saxena, Rajendra Kumar

2008-05-01

Tannase from Penicillium variable IARI 2031 was purified by a two-step purification strategy comprising of ultra-filtration using 100 kDa molecular weight cutoff and gel-filtration using Sephadex G-200. A purification fold of 135 with 91% yield of tannase was obtained. The enzyme has temperature and pH optima of 50 degrees C and 5 degrees C, respectively. However, the functional temperature range is from 25 to 80 degrees C and functional pH range is from 3.0 to 8.0. This tannase could successfully be immobilized on Amberlite IR where it retains about 85% of the initial catalytic activity even after ninth cycle of its use. Based on the Michaelis-Menten constant (Km) of tannase, tannic acid is the best substrate with Km of 32 mM and Vmax of 1.11 micromol ml(-1)min(-1). Tannase is inhibited by phenyl methyl sulphonyl fluoride (PMSF) and N-ethylmaleimide retaining only 28.1% and 19% residual activity indicating that this enzyme belongs to the class of serine hydrolases. Tannase in both crude and crude lyophilized forms is stable for one year retaining more than 60% residual activity.
Identification and cloning of a gene encoding tannase (tannin acylhydrolase) from Lactobacillus plantarum ATCC 14917(T).

PubMed

Iwamoto, Kazuaki; Tsuruta, Hiroki; Nishitaini, Yosuke; Osawa, Ro

2008-09-01

The gene tanLpl, encoding a novel tannase enzyme (TanLpl), has been cloned from Lactobacillus plantarum ATCC 14917(T). This is the first report of a tannase gene cloned from a bacterial source other than from Staphylococcus lugdunensis, which has been reported elsewhere. The open reading frame of tanLpl, spanning 1410 bp, encoded a 469-amino-acid protein that showed 28.8% identity to the tannase of S. lugdunensis with several commonly conserved sequences. These sequences could not be found in putative tannases reported for other bacteria and fungi. TanLpl was expressed in Escherichia coli DH5alpha from a pGEM-T expression system and purified. SDS-PAGE analysis indicated that purified TanLpl was a monomer polypeptide of approximately 50 kDa in size. Subsequent enzymatic characterization revealed that TanLpl was most active in an alkaline pH range at 40 degrees C, which was quite different from that observed for a fungal tannase of Aspergillus oryzae. In addition, the Michaelis-Menten constant of TanLpl was markedly lower than that of A. oryzae tannase. The evidence suggests that TanLpl should be classified into a novel family of tannases.
In vitro characterization of the NAD+ synthetase NadE1 from Herbaspirillum seropedicae.

PubMed

Laskoski, Kerly; Santos, Adrian R S; Bonatto, Ana C; Pedrosa, Fábio O; Souza, Emanuel M; Huergo, Luciano F

2016-05-01

Nicotinamide adenine dinucleotide synthetase enzyme (NadE) catalyzes the amination of nicotinic acid adenine dinucleotide (NaAD) to form NAD(+). This reaction represents the last step in the majority of the NAD(+) biosynthetic routes described to date. NadE enzymes typically use either glutamine or ammonium as amine nitrogen donor, and the reaction is energetically driven by ATP hydrolysis. Given the key role of NAD(+) in bacterial metabolism, NadE has attracted considerable interest as a potential target for the development of novel antibiotics. The plant-associative nitrogen-fixing bacteria Herbaspirillum seropedicae encodes two putative NadE, namely nadE1 and nadE2. The nadE1 gene is linked to glnB encoding the signal transduction protein GlnB. Here we report the purification and in vitro characterization of H. seropedicae NadE1. Gel filtration chromatography analysis suggests that NadE1 is an octamer. The NadE1 activity was assayed in vitro, and the Michaelis-Menten constants for substrates NaAD, ATP, glutamine and ammonium were determined. Enzyme kinetic and in vitro substrate competition assays indicate that H. seropedicae NadE1 uses glutamine as a preferential nitrogen donor.
Modelling the removal of p-TSA (para-toluenesulfonamide) during rapid sand filtration used for drinking water treatment.

PubMed

Meffe, Raffaella; Kohfahl, Claus; Holzbecher, Ekkehard; Massmann, Gudrun; Richter, Doreen; Dünnbier, Uwe; Pekdeger, Asaf

2010-01-01

A finite element model was set-up to determine degradation rate constants for p-TSA during rapid sand filtration (RSF). Data used for the model originated from a column experiment carried out in the filter hall of a drinking water treatment plant in Berlin (Germany). Aerated abstracted groundwater was passed through a 1.6m long column-shaped experimental sand filter applying infiltration rates from 2 to 6mh(-1). Model results were fitted to measured profiles and breakthrough curves of p-TSA for different infiltration rates using both first-order reaction kinetics and Michaelis-Menten kinetics. Both approaches showed that degradation rates varied both in space and time. Higher degradation rates were observed in the upper part of the column, probably related to higher microbial activity in this zone. Measured and simulated breakthrough curves revealed an adaption phase with lower degradation rates after infiltration rates were changed, followed by an adapted phase with more elevated degradation rates. Irrespective of the mathematical approach and the infiltration rate, degradation rates were very high, probably owing to the fact that filter sands have been in operation for decades, receiving high p-TSA concentrations with the raw water.
A Conductometric Indium Oxide Semiconducting Nanoparticle Enzymatic Biosensor Array

PubMed Central

Lee, Dongjin; Ondrake, Janet; Cui, Tianhong

2011-01-01

We report a conductometric nanoparticle biosensor array to address the significant variation of electrical property in nanomaterial biosensors due to the random network nature of nanoparticle thin-film. Indium oxide and silica nanoparticles (SNP) are assembled selectively on the multi-site channel area of the resistors using layer-by-layer self-assembly. To demonstrate enzymatic biosensing capability, glucose oxidase is immobilized on the SNP layer for glucose detection. The packaged sensor chip onto a ceramic pin grid array is tested using syringe pump driven feed and multi-channel I–V measurement system. It is successfully demonstrated that glucose is detected in many different sensing sites within a chip, leading to concentration dependent currents. The sensitivity has been found to be dependent on the channel length of the resistor, 4–12 nA/mM for channel lengths of 5–20 μm, while the apparent Michaelis-Menten constant is 20 mM. By using sensor array, analytical data could be obtained with a single step of sample solution feeding. This work sheds light on the applicability of the developed nanoparticle microsensor array to multi-analyte sensors, novel bioassay platforms, and sensing components in a lab-on-a-chip. PMID:22163696
Purification and Characterization of an Aminopeptidase from Lactococcus lactis subsp. cremoris AM2.

PubMed

Neviani, E; Boquien, C Y; Monnet, V; Thanh, L P; Gripon, J C

1989-09-01

An aminopeptidase was purified from cell extracts of Lactococcus lactis subsp. cremoris AM2 by ion-exchange chromatography. After electrophoresis of the purified enzyme in the presence or absence of sodium dodecyl sulfate, one protein band was detected. The enzyme was a 300-kilodalton hexamer composed of identical subunits not linked by disulfide bridges. Activity was optimal at 40 degrees C and pH 7 and was inhibited by classical thiol group inhibitors. The aminopeptidase hydrolyzed naphthylamide-substituted amino acids, as well as dipeptides and tripeptides. Longer protein chains such as the B chain of insulin were hydrolyzed, but at a much slower rate. The Michaelis constant (K(m)) and the maximal rate of hydrolysis (V(max)) were, respectively, 4.5 mM and 3,600 pkat/mg for the substrate l-histidyl-beta-naphthylamide. Amino acid analysis showed that the enzyme contained low levels of hydrophobic residues. The partial N-terminal sequence of the first 19 residues of the mature enzyme was determined. Polyclonal antibodies were obtained from the purified enzyme, and after immunoblotting, there was no cross-reaction between these antibodies and other proteins in the crude extract.
Modeling cereal starch hydrolysis during simultaneous saccharification and lactic acid fermentation; case of a sorghum-based fermented beverage, gowé.

PubMed

Mestres, Christian; Bettencourt, Munanga de J C; Loiseau, Gérard; Matignon, Brigitte; Grabulos, Joël; Achir, Nawel

2017-10-01

Gowé is an acidic beverage obtained after simultaneous saccharification and fermentation (SSF) of sorghum. A previous paper focused on modeling the growth of lactic acid bacteria during gowé processing. This paper focuses on modeling starch amylolysis to build an aggregated SSF model. The activity of α-amylase was modeled as a function of temperature and pH, and the hydrolysis rates of both native and soluble starch were modeled via a Michaelis-Menten equation taking into account the maltose and glucose inhibition constants. The robustness of the parameter estimators was ensured by step by step identification in sets of experiments conducted with different proportions of native and gelatinized starch by modifying the pre-cooking temperature. The aggregated model was validated on experimental data and showed that both the pre-cooking and fermentation parameters, particularly temperature, are significant levers for controlling not only acid and sugar contents but also the expected viscosity of the final product. This generic approach could be used as a tool to optimize the sanitary and sensory quality of fermentation of other starchy products. Copyright © 2017 Elsevier Ltd. All rights reserved.
[Adaptive specific features of energy metabolism in fish ontogenesis].

PubMed

Ozerniuk, N D

2011-01-01

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

Barik, Md. Abdul, E-mail: abdulnpl@gmail.com; Dutta, Jiten Ch.

We have reported fabrication and characterization of polyaniline (PANI)/zinc oxide (ZnO) membrane-based junctionless carbon nanotube field effect transistor deposited on indium tin oxide glass plate for the detection of cholesterol (0.5–22.2 mM). Cholesterol oxidase (ChOx) has been immobilized on the PANI/ZnO membrane by physical adsorption technique. Electrical response has been recorded using digital multimeter (Agilent 3458A) in the presence of phosphate buffer saline of 50 mM, pH 7.0, and 0.9% NaCl contained in a glass pot. The results of response studies for cholesterol reveal linearity as 0.5–16.6 mM and improved sensitivity of 60 mV/decade in good agreement with Nernstian limit ∼59.2 mV/decade. The life timemore » of this sensor has been found up to 5 months and response time of 1 s. The limit of detection with regression coefficient (r) ∼ 0.998 and Michaelis-Menten constant (K{sub m}) were found to be ∼0.25 and 1.4 mM, respectively, indicating high affinity of ChOx to cholesterol. The results obtained in this work show negligible interference with glucose and urea.« less
An Effect of Dexamethasone on Adenosine 3′,5′ -Monophosphate Content and Adenosine 3′,5′ -Monophosphate Phosphodiesterase Activity of Cultured Hepatoma Cells

PubMed Central

Manganiello, Vincent; Vaughan, Martha

1972-01-01

The effect of dexamethasone on adenosine 3′,5′-monophosphate (cAMP) phosphodiesterase activity in cultured HTC hepatoma cells was investigated. Homogenates of these cells contain phosphodiesterase activity with two apparent Michaelis constants for cAMP (2-5 μm and 50 μm). At all substrate concentrations tested, phosphodiesterase activity was decreased 25-40% in cells incubated for 36 hr or more with 1 μm dexamethasone. Acid phosphatase activity in the same cells was not decreased. α-Methyl testosterone, 1 μm, was without effect on phosphodiesterase activity. Incubation for 10 min with epinephrine plus theophylline increased the cAMP content of the HTC cells 3- to 6-fold. In cells incubated for 72 hr with dexamethasone, the basal concentration of cAMP was slightly increased and the increment produced by epinephrine plus theophylline was markedly increased. We believe that in many cells the so-called permissive effects of steroid hormones on cAMP mediated processes may be due to an effect of these hormones on cAMP phosphodiesterase activity similar to that observed in HTC cells incubated with dexamethasone. PMID:4341439

Highly sensitive and stable electrochemical sulfite biosensor incorporating a bacterial sulfite dehydrogenase.

PubMed

Kalimuthu, Palraj; Tkac, Jan; Kappler, Ulrike; Davis, Jason J; Bernhardt, Paul V

2010-09-01

This paper describes a highly sensitive electrochemical (voltammetric) determination of sulfite using a combination of Starkeya novella sulfite dehydrogenase (SDH), horse heart cytochrome c (cyt c), and a self-assembled monolayer of 11-mercaptoundecanol (MU) cast on a gold electrode. The biosensor was optimized in terms of pH and the ratio of cyt c/SDH. The electrocatalytic oxidation current of sulfite increased linearly from 1 to 6 microM at the enzyme-modified electrode with a correlation coefficient of 0.9995 and an apparent Michaelis constant (K(M,app)) of 43 microM. Using an amperometric method, the low detection limit for sulfite at the enzyme-modified electrode was 44 pM (signal-to-noise ratio = 3). The modified electrode retained a stable response for 3 days while losing only ca. 4% of its initial sensitivity during a 2 week storage period in 50 mM Tris buffer solution at 4 degrees C. The enzyme electrode was successfully used for the determination of sulfite in beer and white wine samples. The results of these electrochemical analyses agreed well with an independent spectrophotometric method using Ellman's reagent, but the detection limit was far superior using the electrochemical method.
Temperature measurement in a gas turbine engine combustor

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

DeSilva, Upul

A method and system for determining a temperature of a working gas passing through a passage to a turbine section of a gas turbine engine. The method includes identifying an acoustic frequency at a first location in the engine upstream from the turbine section, and using the acoustic frequency for determining a first temperature value at the first location that is directly proportional to the acoustic frequency and a calculated constant value. A second temperature of the working gas is determined at a second location in the engine and, using the second temperature, a back calculation is performed to determinemore » a temperature value for the working gas at the first location. The first temperature value is compared to the back calculated temperature value to change the calculated constant value to a recalculated constant value. Subsequent first temperature values at the first location may be determined based on the recalculated constant value.« less
Inhibition of Human Cytochrome P450 2c8-catalyzed Amodiaquine N-desethylation: Effect of Five Traditionally and Commonly Used Herbs

PubMed Central

Muthiah, Yasotha Devi; Ong, Chin Eng; Sulaiman, Siti Amrah; Ismail, Rusli

2016-01-01

Background: In Southeast Asia and many parts of the world, herbal products are increasingly used in parallel with modern medicine. Objective: This study aimed to investigate the effects of herbs commonly used in Southeast Asia on activity of cytochrome P450 2C8 (CYP2C8), an important human hepatic enzyme in drug metabolism. Materials and Methods: The selected herbs, such as Eurycoma longifolia Jack (ELJ), Labisia pumila (LP), Echinacea purpurea (EP), Andrographis paniculata (AP), and Ginkgo biloba (GB), were subjected to inhibition studies using an in vitro CYP2C8 activity marker, amodiaquine N-desethylase assay. Inhibition parameters, inhibitory concentration 50% (IC50), and Ki values were determined to study the potency and mode of inhibition. Results: All herbs inhibited CYP2C8 with the following order of potency: LP > ELJ > GB > AP > EP. LP and ELJ inhibited potently at Ki's of 2 and 4 times the Ki of quercetin, the positive control. The inhibition by LP was uncompetitive in nature as compared to competitive or mixed type inhibition observed with other herbs. GB exhibited moderate inhibitory effect at a Ki6 times larger than quercetin Ki. AP and EP, on the other hand, showed only weak inhibition. Conclusion: The herbs we chose represented the more commonly used herbs in Southeast Asia where collision of tradition and modernization in healthcare, if not properly managed, may lead to therapeutic misadventures. We conclude that concurrent consumption of some herbs, in particular, LP and ELJ, may have relevance in drug-herb interactions via CYP2C8 inhibition in vivo. SUMMARY Herbs are increasingly used in parallel with modern medicines nowadays. In this study five commonly used herbs in Southeast Asia region, ELJ, LP, EP, AP and GB, were investigated for their in vitro inhibitory potency on CYP2C8, an important drug-metaboliz-ing human hepatic enzyme. All herbs inhibited CYP2C8 activity marker, amodiaquine N-desethylation, with potency order of LP > ELJ > GB >AP > EP. LP, ELJ and GB exhibited Ki values of 2, 4 and 6 times the Ki of quercetin, the positive control, indicating potent to moderate degree of enzyme inhibition. AP and EP, on the other hand, showed only weak inhibition. In summary, concurrent consumption of some herbs especially LP and ELJ may have relevance in drug-herb interactions via CYP2C8 inhibition in vivo. Abbreviations Used: AQ: Amodiaquine, AP: Andrographis paniculata, CYP: Cytochrome P450, DEAQ: Desethylamodiaquine, EP: Echinacea purpurea, ELJ: Eurycoma longifolia Jack, GB: Ginkgo biloba, Ki: Inhibition constant, LP: Labisia pumila, Vmax: Maximal velocity, Km: Michaelis-Menten constant. PMID:27695271
Specificity of neutral amino acid uptake at the basolateral side of the epithelium in the cat salivary gland in situ.

PubMed

Bustamante, J C; Mann, G E; Yudilevich, D L

1981-01-01

1. Amino acid uptake was measured in resting cat submandibular glands with either a natural blood supply or perfused at constant flow with a Krebs-albumin solution. Following a bolus arterial injection of a 3H-labelled amino acid and D-[14C]mannitol (extracellular reference tracer), the venous effluent was immediately sampled sequentially. The maximal uptake, Umax, from the blood or perfusate was determined from the paired-tracer dilution curves using the expression: uptake % = (1 -- (3H/14C) X 100). 2. In glands with a natural blood supply, Umax values up to 46% were measured for short-chain (serine and alanine) and long-chain (valine, methionine, leucine, isoleucine, 1-amino-cyclopentane cyclopentane carboxylic acid, phenylalanine, tryptophan, tyrosine, histidine and glutamine) neutral amino acids. In contrast, Umax was negligible for amino acids of the imino-glycine group (proline and glycine) and the nonmetabolized amino acids, 2-aminoisobutyric acid (AIB) and methylaminoisobutyric acid (MeAIB). 3. In glands with a natural blood supply addition of an unlabelled amino acid to the tracer injectate reduced Umax for the test acid by up to 80%. The pattern of these interactions suggested the presence of two transport systems for neutral amino acids, one preferring short-chain and the other long-chain amino acids. 4. In glands perfused at constant flow rates with an amino acid-free Krebs-albumin solution high Umax values were measured: L-serine (66%), L-alanine (54%), L-leucine (43%), L-phenylalanine (42%) and L-tyrosine (51%). Only a low uptake was observed for L-proline (8%) and glycine (14%). There was no uptake of methylaminoisobutyric acid which confirms the result obtained in glands with an intact circulation. 5. Saturation of L-phenylalanine influx was observed in perfused glands as the perfusate concentration of unlabelled L-phenylalanine was increased from 0.5 to 20 mmol . 1-1. A Michaelis--Menten analysis based on a single entry system indicated an apparent Km of 6.4 +/- 0.8 mmol . 1-1 and a Vmax of 1719 +/- 94 nmol . min-1g.-1 6. Since the fenestrated capillaries in the salivary gland are readily permeable to the test amino acid and D-mannitol, it is most probable that the amino acid carriers are located in the basolateral side of the epithelium. 7. The use of a paired-tracer dilution technique to measure uptake in a single circulatory passage has enabled a detailed characterization of neutral amino acid transport in the salivary gland and has overcome the limitation of previous studies based on solute transfer from blood to saliva.
Peroxidase-like activity of apoferritin paired gold clusters for glucose detection.

PubMed

Jiang, Xin; Sun, Cuiji; Guo, Yi; Nie, Guangjun; Xu, Li

2015-02-15

The discovery and application of noble metal nanoclusters have received considerable attention. In this paper, we reported that apoferritin paired gold clusters (Au-Ft) could efficiently catalyze oxidation of 3.3',5.5'-tetramethylbenzidine (TMB) by H2O2 to produce a blue color reaction. Compared with natural enzyme, Au-Ft exhibited higher activity near acidic pH and could be used over a wide range of temperatures. Apoferritin nanocage enhanced the reaction activity of substrate TMB by H2O2. The reaction catalyzed by Au-Ft was found to follow a typical Michaelis-Menten kinetics. The kinetic parameters exhibited a lower K(m) value (0.097 mM) and a higher K(cat) value (5.8 × 10(4) s(-1)) for TMB than that of horse radish peroxidase (HRP). Base on these findings, Au-Ft, acting as a peroxidase mimetic, performed enzymatic spectrophotometric analysis of glucose. This system exhibited acceptable reproducibility and high selectivity in biosening, suggesting that it could have promising applications in the future. Copyright © 2014 Elsevier B.V. All rights reserved.
Effect of mass transfer in a recirculation batch reactor system for immobilized penicillin amidase.

PubMed

Park, J M; Choi, C Y; Seong, B L; Han, M H

1982-10-01

The effect of external mass transfer resistance on the overall reaction rate of the immobilized whole cell penicillin amidase of E. coli in a recirculation batch reactor was investigated. The internal diffusional resistance was found negligible as indicated by the value of effectiveness factor, 0.95. The local environmental change in a column due to the pH drop was successfully overcome by employing buffer solution. The reaction rate was measured by pH-stat method and was found to follow the simple Michaelis-Menten law at the initial stage of the reaction. The values of the net reaction rate experimentally determined were used to calculate the substrate concentration at the external surface of the catalyst pellet and then to calculate the mass transfer coefficient, k(L), at various flow rates and substrate concentrations. The correlation proposed by Chilton and Colburn represented adequately the experimental data. The linear change of log j(D) at low log N(Re) with negative slope was ascribed to the fact that the external mass transfer approached the state of pure diffusion in the limit of zero superficial velocity.
Facile synthesis of glucoamylase embedded metal-organic frameworks (glucoamylase-MOF) with enhanced stability.

PubMed

Nadar, Shamraja S; Rathod, Virendra K

2017-02-01

The self-assembled glucoamylase metal-organic framework (glucoamylase-MOF) was synthesized by facile one-step method within 20min by simply mixing aqueous solution of 2-methylimidazole (160mM), glucoamylase (5mg/mL) and zinc acetate (40mM) at room temperature (28±2°C). The prepared glucoamylase-MOF was characterized by using FT-IR, confocal scanning laser microscopy, XRD and SEM. The robustness and thermal stability of glucoamylase embedded MOF was evaluated in terms of half-life (in the range of 60-80°C) which showed 6 folds increment as against free form. Further, in Michaelis-Menten kinetics studies, glucoamylase entrapped MOF exhibited higher K m value and lower V max value as compared to native enzyme. Moreover, the immobilized glucoamylase exhibited up to 57% of residual activity after six consecutive cycles of reuse, whereas it retained 91% of residual activity till 25days of storage. Finally, the conformational changes occurred after the encapsulation of glucoamylase in the interior of MOF, which was analyzed by using FT-IR data analysis tools. Copyright © 2016 Elsevier B.V. All rights reserved.
Determining the Optimal Values of Exponential Smoothing Constants--Does Solver Really Work?

ERIC Educational Resources Information Center

Ravinder, Handanhal V.

2013-01-01

A key issue in exponential smoothing is the choice of the values of the smoothing constants used. One approach that is becoming increasingly popular in introductory management science and operations management textbooks is the use of Solver, an Excel-based non-linear optimizer, to identify values of the smoothing constants that minimize a measure…
Investigation of two- and three-bond carbon-hydrogen coupling constants in cinnamic acid based compounds.

PubMed

Pierens, Gregory K; Venkatachalam, Taracad K; Reutens, David C

2016-12-01

Two- and three-bond coupling constants ( 2 J HC and 3 J HC ) were determined for a series of 12 substituted cinnamic acids using a selective 2D inphase/antiphase (IPAP)-single quantum multiple bond correlation (HSQMBC) and 1D proton coupled 13 C NMR experiments. The coupling constants from two methods were compared and found to give very similar values. The results showed coupling constant values ranging from 1.7 to 9.7 Hz and 1.0 to 9.6 Hz for the IPAP-HSQMBC and the direct 13 C NMR experiments, respectively. The experimental values of the coupling constants were compared with discrete density functional theory (DFT) calculated values and were found to be in good agreement for the 3 J HC . However, the DFT method under estimated the 2 J HC coupling constants. Knowing the limitations of the measurement and calculation of these multibond coupling constants will add confidence to the assignment of conformation or stereochemical aspects of complex molecules like natural products. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.
Ferrocenyl-substituted dinuclear Cu(II) complex: Synthesis, spectroscopy, electrochemistry, DFT calculations and catecholase activity

NASA Astrophysics Data System (ADS)

Emirik, Mustafa; Karaoğlu, Kaan; Serbest, Kerim; Menteşe, Emre; Yilmaz, Ismail

2016-02-01

A new ferrocenyl-substituted heterocyclic hydrazide ligand and its Cu(II) complex were prepared. The DFT calculations were performed to determine the electronic and molecular structures of the title compounds. The electronic spectra were calculated by using time-dependent DFT method, and the transitions were correlated with the molecular orbitals of the compounds. The bands assignments of IR spectra were achieved in the light of the theoretical vibrational spectral data and total energy distribution values calculated at DFT/B3LYP/6-311++G(d,p) level. The redox behaviors of the ferrocene derivatives were investigated by cyclic voltammetry. The compounds show reversible redox couple assignable to Fc+/Fc couple. The copper(II) complex behaves as an effective catalyst towards oxidation of 3,5-di-tert-butylcatechol to its corresponding quinone derivative in DMF saturated with O2. The reaction follows Michaelis-Menten enzymatic reaction kinetics with turnover numbers 2.32 × 103.
Automatic network coupling analysis for dynamical systems based on detailed kinetic models.

PubMed

Lebiedz, Dirk; Kammerer, Julia; Brandt-Pollmann, Ulrich

2005-10-01

We introduce a numerical complexity reduction method for the automatic identification and analysis of dynamic network decompositions in (bio)chemical kinetics based on error-controlled computation of a minimal model dimension represented by the number of (locally) active dynamical modes. Our algorithm exploits a generalized sensitivity analysis along state trajectories and subsequent singular value decomposition of sensitivity matrices for the identification of these dominant dynamical modes. It allows for a dynamic coupling analysis of (bio)chemical species in kinetic models that can be exploited for the piecewise computation of a minimal model on small time intervals and offers valuable functional insight into highly nonlinear reaction mechanisms and network dynamics. We present results for the identification of network decompositions in a simple oscillatory chemical reaction, time scale separation based model reduction in a Michaelis-Menten enzyme system and network decomposition of a detailed model for the oscillatory peroxidase-oxidase enzyme system.
Phenytoin intoxication during concurrent diazepam therapy

PubMed Central

Rogers, Howard J.; Haslam, Robert A.; Longstreth, James; Lietman, Paul S.

1977-01-01

Phenytoin elimination is a saturable process obeying Michaelis-Menten kinetics. Plasma phenytoin levels are not related linearly to dose, and small changes in enzyme activity produced by concurrent drug therapy could alter plasma levels. Two cases of phenytoin intoxication associated with simultaneous administration of diazepam are reported. Intravenous phenytoin infusions were given and the apparent Km and Vmax computed from the resulting plasma phenytoin levels. In one case `Km' and `Vmax' were 0.8 μmol/1 and 1.3 μmol/1/hour respectively during concurrent diazepam administration, and 50.3 μmol/1 and 4.4 μmol/1/hour after discontinuation of diazepam. In the second case phenytoin infusion with diazepam gave `Km' and `Vmax' values of 0.012 μmol/1 and 0.95 μmol/1/hour. Without diazepam these were 28.8 μmol/1 and 0.92 μmol/1/hour respectively. PMID:599366
Yeast ribonuclease III uses a network of multiple hydrogen bonds for RNA binding and cleavage.

PubMed

Lavoie, Mathieu; Abou Elela, Sherif

2008-08-19

Members of the bacterial RNase III family recognize a variety of short structured RNAs with few common features. It is not clear how this group of enzymes supports high cleavage fidelity while maintaining a broad base of substrates. Here we show that the yeast orthologue of RNase III (Rnt1p) uses a network of 2'-OH-dependent interactions to recognize substrates with different structures. We designed a series of bipartite substrates permitting the distinction between binding and cleavage defects. Each substrate was engineered to carry a single or multiple 2'- O-methyl or 2'-fluoro ribonucleotide substitutions to prevent the formation of hydrogen bonds with a specific nucleotide or group of nucleotides. Interestingly, introduction of 2'- O-methyl ribonucleotides near the cleavage site increased the rate of catalysis, indicating that 2'-OH are not required for cleavage. Substitution of nucleotides in known Rnt1p binding site with 2'- O-methyl ribonucleotides inhibited cleavage while single 2'-fluoro ribonucleotide substitutions did not. This indicates that while no single 2'-OH is essential for Rnt1p cleavage, small changes in the substrate structure are not tolerated. Strikingly, several nucleotide substitutions greatly increased the substrate dissociation constant with little or no effect on the Michaelis-Menten constant or rate of catalysis. Together, the results indicate that Rnt1p uses a network of nucleotide interactions to identify its substrate and support two distinct modes of binding. One mode is primarily mediated by the dsRNA binding domain and leads to the formation of stable RNA/protein complex, while the other requires the presence of the nuclease and N-terminal domains and leads to RNA cleavage.
Inhibitory effects of nitrite on the reactions of bovine carbonic anhydrase II with CO2 and bicarbonate consistent with zinc-bound nitrite.

PubMed

Nielsen, Per M; Fago, Angela

2015-08-01

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

PubMed

Astola, Laura; Stigter, Hans; Gomez Roldan, Maria Victoria; van Eeuwijk, Fred; Hall, Robert D; Groenenboom, Marian; Molenaar, Jaap J

2016-01-01

We study the glycosylation processes that convert initially toxic substrates to nutritionally valuable metabolites in the flavonoid biosynthesis pathway of tomato (Solanum lycopersicum) seedlings. To estimate the reaction rates we use ordinary differential equations (ODEs) to model the enzyme kinetics. A popular choice is to use a system of linear ODEs with constant kinetic rates or to use Michaelis-Menten kinetics. In reality, the catalytic rates, which are affected among other factors by kinetic constants and enzyme concentrations, are changing in time and with the approaches just mentioned, this phenomenon cannot be described. Another problem is that, in general these kinetic coefficients are not always identifiable. A third problem is that, it is not precisely known which enzymes are catalyzing the observed glycosylation processes. With several hundred potential gene candidates, experimental validation using purified target proteins is expensive and time consuming. We aim at reducing this task via mathematical modeling to allow for the pre-selection of most potential gene candidates. In this article we discuss a fast and relatively simple approach to estimate time varying kinetic rates, with three favorable properties: firstly, it allows for identifiable estimation of time dependent parameters in networks with a tree-like structure. Secondly, it is relatively fast compared to usually applied methods that estimate the model derivatives together with the network parameters. Thirdly, by combining the metabolite concentration data with a corresponding microarray data, it can help in detecting the genes related to the enzymatic processes. By comparing the estimated time dynamics of the catalytic rates with time series gene expression data we may assess potential candidate genes behind enzymatic reactions. As an example, we show how to apply this method to select prominent glycosyltransferase genes in tomato seedlings.
Pharmacokinetic and pharmacodynamic interaction between nifedipine and metformin in rats: competitive inhibition for metabolism of nifedipine and metformin by each other via CYP isozymes.

PubMed

Choi, Young H; Lee, Myung G

2012-05-01

It has been reported that hypertension exponentially increases in the patients with type 2 diabetes mellitus. Thus, this study was performed to investigate the pharmacokinetic and pharmacodynamic interactions between nifedipine and metformin, since both drugs were commonly metabolized via hepatic CYP2C and 3A subfamilies in rats. Nifedipine (3 mg/kg) and metformin (100 mg/kg) were simultaneously administered intravenously or orally to rats. Concentrations (I) of each drug in the liver and intestine, maximum velocity (V(max)), Michaelis-Menten constant (K(m)), and intrinsic clearance (CL(int)) for the disappearance of each drug, apparent inhibition constant (K(i)) and [I]/K(i) ratios of each drug in liver and intestine were determined. Also the metabolism of each drug in rat and human CYPs and blood pressure were also measured. After the simultaneous single intravenous administration of both drugs together, the AUCs of each drug were significantly greater than that in each drug alone due to the competitive inhibition for the metabolism of nifedipine by metformin via hepatic CYP3A1/2 and of metformin by nifedipine via hepatic CYP2C6 and 3A1/2. After the simultaneous single oral administration of both drugs, the significantly greater AUCs of each drug than that in each drug alone could have mainly been due to the competitive inhibition for the metabolism of nifedipine and metformin by each other via intestinal CYP3A1/2 in addition to competitive inhibition for the hepatic metabolism of each drug as same as the intravenous study.
Characterization of a Naphthalene Dioxygenase Endowed with an Exceptionally Broad Substrate Specificity Toward Polycyclic Aromatic Hydrocarbons

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

Jouanneau,Y.; Meyer, C.; Jakoncic, J.

In Sphingomonas CHY-1, a single ring-hydroxylating dioxygenase is responsible for the initial attack of a range of polycyclic aromatic hydrocarbons (PAHs) composed of up to five rings. The components of this enzyme were separately purified and characterized. The oxygenase component (ht-PhnI) was shown to contain one Rieske-type [2Fe-2S] cluster and one mononuclear Fe center per {alpha} subunit, based on EPR measurements and iron assay. Steady-state kinetic measurements revealed that the enzyme had a relatively low apparent Michaelis constant for naphthalene (K{sub m} = 0.92 {+-} 0.15 {mu}M) and an apparent specificity constant of 2.0 {+-} 0.3 M{sup -1} s{sup -1}.more » Naphthalene was converted to the corresponding 1,2-dihydrodiol with stoichiometric oxidation of NADH. On the other hand, the oxidation of eight other PAHs occurred at slower rates and with coupling efficiencies that decreased with the enzyme reaction rate. Uncoupling was associated with hydrogen peroxide formation, which is potentially deleterious to cells and might inhibit PAH degradation. In single turnover reactions, ht-PhnI alone catalyzed PAH hydroxylation at a faster rate in the presence of organic solvent, suggesting that the transfer of substrate to the active site is a limiting factor. The four-ring PAHs chrysene and benz[a]anthracene were subjected to a double ring-dihydroxylation, giving rise to the formation of a significant proportion of bis-cis-dihydrodiols. In addition, the dihydroxylation of benz[a]anthracene yielded three dihydrodiols, the enzyme showing a preference for carbons in positions 1,2 and 10,11. This is the first characterization of a dioxygenase able to dihydroxylate PAHs made up of four and five rings.« less
Patchy gold coated Fe3O4 nanospheres with enhanced catalytic activity applied for paper-based bipolar electrode-electrochemiluminescence aptasensors.

PubMed

Zhang, Xin; Bao, Ning; Luo, Xiliang; Ding, Shou-Nian

2018-05-10

In this work, novel multifunctional patchy gold coated Fe 3 O 4 hybrid nanoparticles (PG-Fe 3 O 4 NPs) have been successfully synthesized in aqueous medium via a facile adsorption-reduction method. A rational formation mechanism has been proposed by monitoring the morphological evolution. The PG-Fe 3 O 4 NPs retained the good magnetic property and exhibited excellent catalytical effeciency towards the electrochemical reduction of hydrogen peroxide. Chronoamperometric and amperometric experiments indicated a relatively high catalytic rate constant of 3.13 × 10 5 M -1 s -1 , a high sensitivity of 578.87 µA mM -1 cm -2 and a low Michaelis-Menten constant of 462 µM. Meanwhile, the introduction of patchy gold could help biofunctionalization via Au-S bond for different biodetection and biosensing purposes. Here, as an example, thiol-terminated aptamers were immobilized onto the patchy gold part as a signal probe to detect carcinoembryonic antigen (CEA). A related paper-based bipolar electrode-electrochemiluminescence (pBPE-ECL) aptasensor was fabricated as the low-cost, disposable and miniature platform. To improve the sensitivity, Au nanodendrites were electrodeposited at the BPE cathode as the matrix for Apt1 immobilization. This aptasensor showed a wide linear range of 0.1 pg mL -1 -15 ng mL -1 with a low detection limit of 0.03 pg mL -1 , remaining competitive against other ones, and also demonstrating the PG-Fe 3 O 4 NPs have promising potential for catalysis and bioassays. Copyright © 2018 Elsevier B.V. All rights reserved.
The Hydrogenase Activity of the Molybdenum/Copper-containing Carbon Monoxide Dehydrogenase of Oligotropha carboxidovorans*

PubMed Central

Wilcoxen, Jarett; Hille, Russ

2013-01-01

The reaction of the air-tolerant CO dehydrogenase from Oligotropha carboxidovorans with H2 has been examined. Like the Ni-Fe CO dehydrogenase, the enzyme can be reduced by H2 with a limiting rate constant of 5.3 s−1 and a dissociation constant Kd of 525 μm; both kred and kred/Kd, reflecting the breakdown of the Michaelis complex and the reaction of free enzyme with free substrate in the low [S] regime, respectively, are largely pH-independent. During the reaction with H2, a new EPR signal arising from the Mo/Cu-containing active site of the enzyme is observed which is distinct from the signal seen when the enzyme is reduced by CO, with greater g anisotropy and larger hyperfine coupling to the active site 63,65Cu. The signal also exhibits hyperfine coupling to at least two solvent-exchangeable protons of bound substrate that are rapidly exchanged with solvent. Proton coupling is also evident in the EPR signal seen with the dithionite-reduced native enzyme, and this coupling is lost in the presence of bicarbonate. We attribute the coupled protons in the dithionite-reduced enzyme to coordinated water at the copper site in the native enzyme and conclude that bicarbonate is able to displace this water from the copper coordination sphere. On the basis of our results, a mechanism for H2 oxidation is proposed which involves initial binding of H2 to the copper of the binuclear center, displacing the bound water, followed by sequential deprotonation through a copper-hydride intermediate to reduce the binuclear center. PMID:24165123
Imidacloprid is hydroxylated by Laodelphax striatellus CYP6AY3v2.

PubMed

Wang, R; Zhu, Y; Deng, L; Zhang, H; Wang, Q; Yin, M; Song, P; Elzaki, M E A; Han, Z; Wu, M

2017-10-01

Laodelphax striatellus (Fallén) is one of the most destructive pests of rice, and has developed high resistance to imidacloprid. Our previous work indicated a strong association between imidacloprid resistance and the overexpression of a cytochrome P450 gene CYP6AY3v2 in a L. striatellus imidacloprid resistant strain (Imid-R). In this study, a transgenic Drosophila melanogaster line that overexpressed the L. striatellus CYP6AY3v2 gene was established and was found to confer increased levels of imidacloprid resistance. Furthermore, CYP6AY3v2 was co-expressed with D. melanogaster cytochrome P450 reductase (CPR) in Spodoptera frugiperda 9 (SF9) cells. A carbon monoxide difference spectra analysis indicated that CYP6AY3v2 was expressed predominately in its cytochrome P450 (P450) form, which is indicative of a good-quality functional enzyme. The recombinant CYP6AY3v2 protein efficiently catalysed the model substrate P-nitroanisole to p-nitrophenol with a maximum velocity (V max ) of 60.78 ± 3.93 optical density (mOD)/min/mg protein. In addition, imidacloprid itself was metabolized by the recombinant CYP6AY3v2/nicotinamide adenine dinucleotide 2'-phosphate reduced tetrasodium salt (NADPH) CPR microsomes in in vitro assays (catalytic constant (K cat ) = 0.34 pmol/min/pmol P450, michaelis constant (K m ) = 41.98 μM), and imidacloprid depletion and metabolite peak formation were with a time dependence. The data provided direct evidence that CYP6AY3v2 is capable of hydroxylation of imidacloprid and conferring metabolic resistance in L. striatellus. © 2017 The Royal Entomological Society.

Combined pressure and cosolvent effects on enzyme activity - a high-pressure stopped-flow kinetic study on α-chymotrypsin.

PubMed

Luong, Trung Quan; Winter, Roland

2015-09-21

We investigated the combined effects of cosolvents and pressure on the hydrolysis of a model peptide catalysed by α-chymotrypsin. The enzymatic activity was measured in the pressure range from 0.1 to 200 MPa using a high-pressure stopped-flow systems with 10 ms time resolution. A kosmotropic (trimethalymine-N-oxide, TMAO) and chaotropic (urea) cosolvent and mixtures thereof were used as cosolvents. High pressure enhances the hydrolysis rate as a consequence of a negative activation volume, ΔV(#), which, depending on the cosolvent system, amounts to -2 to -4 mL mol(-1). A more negative activation volume can be explained by a smaller compression of the ES complex relative to the transition state. Kinetic constants, such as kcat and the Michaelis constant KM, were determined for all solution conditions as a function of pressure. With increasing pressure, kcat increases by about 35% and its pressure dependence by a factor of 1.9 upon addition of 2 M urea, whereas 1 M TMAO has no significant effect on kcat and its pressure dependence. Similarly, KM increases upon addition of urea 6-fold. Addition of TMAO compensates the urea-effect on kcat and KM to some extent. The maximum rate of the enzymatic reaction increases with increasing pressure in all solutions except in the TMAO : urea 1 : 2 mixture, where, remarkably, pressure is found to have no effect on the rate of the enzymatic reaction anymore. Our data clearly show that compatible solutes can easily override deleterious effects of harsh environmental conditions, such as high hydrostatic pressures in the 100 MPa range, which is the maximum pressure encountered in the deep biosphere on Earth.
Mechanism of product inhibition for cellobiohydrolase Cel7A during hydrolysis of insoluble cellulose.

PubMed

Olsen, Johan P; Alasepp, Kadri; Kari, Jeppe; Cruys-Bagger, Nicolaj; Borch, Kim; Westh, Peter

2016-06-01

The cellobiohydrolase cellulase Cel7A is extensively utilized in industrial treatment of lignocellulosic biomass under conditions of high product concentrations, and better understanding of inhibition mechanisms appears central in attempts to improve the efficiency of this process. We have implemented an electrochemical biosensor assay for product inhibition studies of cellulases acting on their natural substrate, cellulose. Using this method we measured the hydrolytic rate of Cel7A as a function of both product (inhibitor) concentration and substrate load. This data enabled analyses along the lines of conventional enzyme kinetic theory. We found that the product cellobiose lowered the maximal rate without affecting the Michaelis constant, and this kinetic pattern could be rationalized by two fundamentally distinct molecular mechanisms. One was simple reversibility, that is, an increasing rate of the reverse reaction, lowering the net hydrolytic velocity as product concentrations increase. Strictly this is not a case of inhibition, as no catalytically inactive is formed. The other mechanism that matched the kinetic data was noncompetitive inhibition with an inhibition constant of 490 ± 40 μM. Noncompetitive inhibition implies that the inhibitor binds with comparable strength to either free enzyme or an enzymesubstrate complex, that is, that association between enzyme and substrate has no effect on the binding of the inhibitor. This mechanism is rarely observed, but we argue, that the special architecture of Cel7A with numerous subsites for binding of both substrate and product could give rise to a true noncompetitive inhibition mechanism. Biotechnol. Bioeng. 2016;113: 1178-1186. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.
Data Analysis and Its Impact on Predicting Schedule & Cost Risk

DTIC Science & Technology

2006-03-01

variance of the error term by performing a Breusch - Pagan test for constant variance (Neter et al., 1996:239). In order to test the normality of...is constant variance. Using Microsoft Excel®, we calculate a p- 68 value of 0.225678 for the Breusch - Pagan test . We again compare this p-value to...calculate a p-value of 0.121211092 Breusch - Pagan test . We again compare this p-value to an alpha of 0.05 indicating our assumption of constant variance
Crystal cryocooling distorts conformational heterogeneity in a model Michaelis complex of DHFR

PubMed Central

Keedy, Daniel A.; van den Bedem, Henry; Sivak, David A.; Petsko, Gregory A.; Ringe, Dagmar; Wilson, Mark A.; Fraser, James S.

2014-01-01

Summary Most macromolecular X-ray structures are determined from cryocooled crystals, but it is unclear whether cryocooling distorts functionally relevant flexibility. Here we compare independently acquired pairs of high-resolution datasets of a model Michaelis complex of dihydrofolate reductase (DHFR), collected by separate groups at both room and cryogenic temperatures. These datasets allow us to isolate the differences between experimental procedures and between temperatures. Our analyses of multiconformer models and time-averaged ensembles suggest that cryocooling suppresses and otherwise modifies sidechain and mainchain conformational heterogeneity, quenching dynamic contact networks. Despite some idiosyncratic differences, most changes from room temperature to cryogenic temperature are conserved, and likely reflect temperature-dependent solvent remodeling. Both cryogenic datasets point to additional conformations not evident in the corresponding room-temperature datasets, suggesting that cryocooling does not merely trap pre-existing conformational heterogeneity. Our results demonstrate that crystal cryocooling consistently distorts the energy landscape of DHFR, a paragon for understanding functional protein dynamics. PMID:24882744
A path-independent integral for the characterization of solute concentration and flux at biofilm detachments

USGS Publications Warehouse

Moran, B.; Kulkarni, S.S.; Reeves, H.W.

2007-01-01

A path-independent (conservation) integral is developed for the characterization of solute concentration and flux in a biofilm in the vicinity of a detachment or other flux limiting boundary condition. Steady state conditions of solute diffusion are considered and biofilm kinetics are described by an uptake term which can be expressed in terms of a potential (Michaelis-Menten kinetics). An asymptotic solution for solute concentration at the tip of the detachment is obtained and shown to be analogous to that of antiplane crack problems in linear elasticity. It is shown that the amplitude of the asymptotic solution can be calculated by evaluating a path-independent integral. The special case of a semi-infinite detachment in an infinite strip is considered and the amplitude of the asymptotic field is related to the boundary conditions and problem parameters in closed form for zeroth and first order kinetics and numerically for Michaelis-Menten kinetics. ?? Springer Science+Business Media, Inc. 2007.
Kinetics study of palm oil hydrolysis using immobilized lipase Candida rugosa in packed bed reactor.

PubMed

Min, C S; Bhatia, S; Kamaruddin, A H

1999-01-01

Continuous hydrolysis of palm oil triglyceride in organic solvent using immobilized Candida rugosa on the Amberlite MB-1 as a source of immobilized lipase was studied in packed bed reactor. The enzymatic kinetics of hydrolysis reaction was studied by changing the substrate concentration, reaction temperature and residence time(tau) in the reactor. At 55 degrees C, the optimum water concentration was found to be 15 % weight per volume of solution (%w/v). The Michaelis-Menten kinetic model was used to obtain the reaction parameters, Km(app) and V max(app). The activation energies were found to be quite low indicating that the lipase-catalyzed process is controlled by diffusion of substrates. The Michaelis-Menten kinetic model was found to be suitable at low water concentration 10-15 %w/v of solution. At higher water concentration, substrate inhibition model was used for data analysis. Reactor operation was found to play an important role in the palm oil hydrolysis kinetic.
A novel electrochemical biosensor based on Fe3O4 nanoparticles-polyvinyl alcohol composite for sensitive detection of glucose.

PubMed

Sanaeifar, Niuosha; Rabiee, Mohammad; Abdolrahim, Mojgan; Tahriri, Mohammadreza; Vashaee, Daryoosh; Tayebi, Lobat

2017-02-15

In this research, a new electrochemical biosensor was constructed for the glucose detection. Iron oxide nanoparticles (Fe 3 O 4 ) were synthesized through co-precipitation method. Polyvinyl alcohol-Fe 3 O 4 nanocomposite was prepared by dispersing synthesized nanoparticles in the polyvinyl alcohol (PVA) solution. Glucose oxidase (GOx) was immobilized on the PVA-Fe 3 O 4 nanocomposite via physical adsorption. The mixture of PVA, Fe 3 O 4 nanoparticles and GOx was drop cast on a tin (Sn) electrode surface (GOx/PVA-Fe 3 O 4 /Sn). The Fe 3 O 4 nanoparticles were characterized by X-ray diffraction (XRD). Also, Fourier transform infrared (FTIR) spectroscopy and field emission scanning electron microscopy (FE-SEM) techniques were utilized to evaluate the PVA-Fe 3 O 4 and GOx/PVA-Fe 3 O 4 nanocomposites. The electrochemical performance of the modified biosensor was investigated using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Presence of Fe 3 O 4 nanoparticles in the PVA matrix enhanced the electron transfer between enzyme and electrode surface and the immobilized GOx showed excellent catalytic characteristic toward glucose. The GOx/PVA-Fe 3 O 4 /Sn bioelectrode could measure glucose in the range from 5 × 10 -3 to 30 mM with a sensitivity of 9.36 μA mM -1 and exhibited a lower detection limit of 8 μM at a signal-to-noise ratio of 3. The value of Michaelis-Menten constant (K M ) was calculated as 1.42 mM. The modified biosensor also has good anti-interfering ability during the glucose detection, fast response (10 s), good reproducibility and satisfactory stability. Finally, the results demonstrated that the GOx/PVA-Fe 3 O 4 /Sn bioelectrode is promising in biosensor construction. Copyright © 2016 Elsevier Inc. All rights reserved.
Development of a phosphorylated Momordica charantia protein system for inhibiting susceptible dose-dependent C. albicans to available antimycotics: An allosteric regulation of protein.

PubMed

Qiao, Yuanbiao; Song, Li; Zhu, Chenchen; Wang, Qian; Guo, Tianyan; Yan, Yanhua; Li, Qingshan

2017-11-15

A regulatory Momordica charantia protein system was constructed allosterically by in vitro protein phosphorylation, in an attempt to evaluate antimycological pluripotency against dose-dependent susceptibilities in C. albicans. Fungal strain lineages susceptible to ketoconazole, econazole, miconazole, 5-flucytosine, nystatin and amphotericin B were prepared in laboratory, followed by identification via antifungal susceptibility testing. Protein phosphorylation was carried out in reactions with 5'-adenylic, guanidylic, cytidylic and uridylic acids and cyclic adenosine triphosphate, through catalysis of cyclin-dependent kinase 1, protein kinase A and protein kinase C respectively. Biochemical analysis of enzymatic reactions indicated the apparent Michaelis-Menten constants and maximal velocity values of 16.57-91.97mM and 55.56-208.33μM·min -1 , together with an approximate 1:1 reactant stoichiometric ratio. Three major protein phosphorylation sites were theoretically predicted at Thr255, Thr102 and Thr24 by a KinasePhos tool. Additionally, circular dichroism spectroscopy demonstrated that upon phosphorylation, protein folding structures were decreased in random coil, β6-sheet and α1-helix partial regions. McFarland equivalence standard testing yielded the concentration-dependent inhibition patterns, while fungus was grown in Sabouraud's dextrose agar. The minimal inhibitory concentrations of 0.16-0.51μM (at 50% response) were obtained for free protein and phosphorylated counterparts. With respect to the 3-cycling susceptibility testing regimen, individuals of total protein forms were administrated in-turn at 0.14μM/cycle. Relative inhibition ratios were retained to 66.13-81.04% of initial ones regarding the ketoconazole-susceptible C. albicans growth. An inhibitory protein system, with an advantage of decreasing antifungal susceptibilities to diverse antimycotics, was proposed because of regulatory pluripotency whereas little contribution to susceptibility in itself. Copyright © 2017 Elsevier B.V. All rights reserved.
Performance comparison between multienzymes loaded single and dual electrodes for the simultaneous electrochemical detection of adenosine and metabolites in cancerous cells.

PubMed

Hussain, Khalil K; Akhtar, Mahmood H; Kim, Moo-Hyun; Jung, Dong-Keun; Shim, Yoon-Bo

2018-06-30

The analytical performance of the multi enzymes loaded single electrode sensor (SES) and dual electrode sensor (DES) was compared for the detection of adenosine and metabolites. The SES was fabricated by covalent binding of tri-enzymes, adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), and xanthine oxidase (XO) along with hydrazine (Hyd) onto a functionalized conducting polymer [2,2:5,2-terthiophene-3-(p-benzoic acid)] (pTTBA). The enzyme reaction electrode in DES was fabricated by covalent binding of ADA and PNP onto pTTBA coated on Au nanoparticles. The detection electrode in DES was constructed by covalent binding of XO and Hyd onto pTTBA coated on porous Au. Due to the higher amount (3.5 folds) of the immobilized enzymes and Hyd onto the DES than SES, and the lower Michaelis constant (Km) value for DES (28.7 µM) compared to SES (36.1 µM), the sensitivity was significantly enhanced for the DES (8.2 folds). The dynamic range obtained using DES was from 0.5 nM to 120.0 µM with a detection limit of 1.43 nM ± 0.02, 0.76 nM ± 0.02, and 0.48 nM ± 0.01, for adenosine (AD), inosine (IN), and hypoxanthine (Hypo) respectively. Further, the DES was coupled with an electrochemical potential modulated microchannel for the separation and simultaneous detection of AD, IN, and Hypo in an extracellular matrix of cancerous (A549) and non-cancerous (Vero) cells. The sensor probe confirms a higher basal level of extracellular AD and its metabolites in cancer cells compared to normal cells. In addition, the effect of dipyridamole on released adenosine in A549 cells was investigated. Copyright © 2018 Elsevier B.V. All rights reserved.
In vitro metabolism of benzo[a]pyrene-7,8-dihydrodiol and dibenzo[def,p]chrysene-11,12 diol in rodent and human hepatic microsomes

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

Smith, Jordan N.; Mehinagic, Denis; Nag, Subhasree

Polycyclic aromatic hydrocarbons (PAHs) are contaminants that are ubiquitously found in the environment, produced through combustion of organic matter or petrochemicals, and many of which are procarcinogens. The prototypic PAH, benzo[a]pyrene (B[a]P) and the highly carcinogenic dibenzo[def,p]chrysene (DBC) are metabolically activated by isoforms of the P450 enzyme superfamily producing benzo[a]pyrene-7,8-dihydrodiol (B[a]P diol), dibenzo[def,p]chrysene-11,12 diol (DBC diol). Each of these diols can be further metabolized by cytochrome P450 enzymes to highly reactive diol-epoxide metabolites that readily react with DNA or by phase II conjugation facilitating excretion. To complement prior in vitro metabolism studies with parent B[a]P and DBC, both phase Imore » metabolism and phase II glucuronidation of B[a]P diol and DBC diol were measured in hepatic microsomes from female B6129SF1/J mice, male Sprague-Dawley rats, and female humans. Metabolic parameters, including intrinsic clearance and Michaelis-Menten kinetics were calculated from substrate depletion data. Mice and rats demonstrated similar B[a]P diol phase I metabolic rates. Compared to rodents, human phase I metabolism of B[a]P diol demonstrated lower overall metabolic capacity, lower intrinsic clearance at higher substrate concentrations (>0.14 µM), and higher intrinsic clearance at lower substrate concentrations (<0.07 µM). Rates of DBC diol metabolism did not saturate in mice or humans and were highest overall in mice. Higher affinity constants and lower capacities were observed for DBC diol glucuronidation compared to B[a]P diol glucuronidation; however, intrinsic clearance values for these compounds were consistent within each species. Kinetic parameters reported here will be used to extend physiologically based pharmacokinetic (PBPK) models to include the disposition of B[a]P and DBC metabolites in animal models and humans to support future human health risk assessments.« less
Model Parameter Variability for Enhanced Anaerobic Bioremediation of DNAPL Source Zones

NASA Astrophysics Data System (ADS)

Mao, X.; Gerhard, J. I.; Barry, D. A.

2005-12-01

The objective of the Source Area Bioremediation (SABRE) project, an international collaboration of twelve companies, two government agencies and three research institutions, is to evaluate the performance of enhanced anaerobic bioremediation for the treatment of chlorinated ethene source areas containing dense, non-aqueous phase liquids (DNAPL). This 4-year, 5.7 million dollars research effort focuses on a pilot-scale demonstration of enhanced bioremediation at a trichloroethene (TCE) DNAPL field site in the United Kingdom, and includes a significant program of laboratory and modelling studies. Prior to field implementation, a large-scale, multi-laboratory microcosm study was performed to determine the optimal system properties to support dehalogenation of TCE in site soil and groundwater. This statistically-based suite of experiments measured the influence of key variables (electron donor, nutrient addition, bioaugmentation, TCE concentration and sulphate concentration) in promoting the reductive dechlorination of TCE to ethene. As well, a comprehensive biogeochemical numerical model was developed for simulating the anaerobic dehalogenation of chlorinated ethenes. An appropriate (reduced) version of this model was combined with a parameter estimation method based on fitting of the experimental results. Each of over 150 individual microcosm calibrations involved matching predicted and observed time-varying concentrations of all chlorinated compounds. This study focuses on an analysis of this suite of fitted model parameter values. This includes determining the statistical correlation between parameters typically employed in standard Michaelis-Menten type rate descriptions (e.g., maximum dechlorination rates, half-saturation constants) and the key experimental variables. The analysis provides insight into the degree to which aqueous phase TCE and cis-DCE inhibit dechlorination of less-chlorinated compounds. Overall, this work provides a database of the numerical modelling parameters typically employed for simulating TCE dechlorination relevant for a range of system conditions (e.g, bioaugmented, high TCE concentrations, etc.). The significance of the obtained variability of parameters is illustrated with one-dimensional simulations of enhanced anaerobic bioremediation of residual TCE DNAPL.
Cation-dependent nutrient transport in shrimp digestive tract.

PubMed

Simmons, Tamla; Mozo, Julie; Wilson, Jennifer; Ahearn, Gregory A

2012-02-01

Purified epithelial brush border membrane vesicles (BBMV) were produced from the hepatopancreas of the Atlantic White shrimp, Litopeneaus setiferus, using standard methods originally developed for mammalian tissues and previously applied to other crustacean and echinoderm epithelia. These vesicles were used to study the cation dependency of sugar and amino acid transport across luminal membranes of hepatopancreatic epithelial cells. (3)H-D: -glucose uptake by BBMV against transient sugar concentration gradients occurred when either transmembrane sodium or potassium gradients were the only driving forces for sugar accumulation, suggesting the presence of a possible coupled transport system capable of using either cation. (3)H-L: -histidine transport was only stimulated by a transmembrane potassium gradient, while (3)H-L: -leucine uptake was enhanced by either a sodium or potassium gradient. These responses suggest the possible presence of a potassium-dependent transporter that accommodates either amino acid and a sodium-dependent system restricted only to L: -leucine. Uptake of (3)H-L: -leucine was significantly stimulated (P < 0.05) by several metallic cations (e.g., Zn(2+), Cu(2+), Mn(2+), Cd(2+), or Co(2+)) at external pH values of 7.0 or 5.0 (internal pH 7.0), suggesting a potential synergistic role of the cations in the transmembrane transfer of amino acids. (3)H-L: -histidine influxes (15 suptakes) were hyperbolic functions of external [zinc] or [manganese], following Michaelis-Menten kinetics. The apparent affinity constant (e.g., K (m)) for manganese was an order of magnitude smaller (K (m) = 0.22 μM Mn) than that for zinc (K (m) = 1.80 μM Zn), while no significant difference (P > 0.05) occurred between their maximal transport velocities (e.g., J (max)). These results suggest that a number of cation-dependent nutrient transport systems occur on the shrimp brush border membrane and aid in the absorption of these important dietary elements.
The Chemical Chaperone Phenylbutyrate Rescues MCT8 Mutations Associated With Milder Phenotypes in Patients With Allan-Herndon-Dudley Syndrome.

PubMed

Braun, Doreen; Schweizer, Ulrich

2017-03-01

Mutations in the thyroid hormone transporter monocarboxylate transporter 8 (MCT8) prevent appropriate entry of thyroid hormones into brain cells during development and cause severe mental retardation in affected patients. The current treatment options are thyromimetic compounds that enter the brain independently of MCT8. Some MCT8-deficient patients (e.g., those carrying MCT8delF501) will not be as severely affected as most others. We have shown that the MCT8delF501 protein has decreased protein stability but important residual function once it reaches the plasma membrane. We were able to rescue protein expression and the function of MCT8delF501 in a Madin-Darby canine kidney cell model by application of the chemical chaperone sodium phenylbutyrate (NaPB), a drug that has been used to treat patients with cystic fibrosis and urea cycle defects for extended periods of time. In the present study, we have extended our previous study and report on the NaPB-dependent rescue of a series of other pathogenic MCT8 mutants associated with milder patient phenotypes. We show that NaPB can functionally rescue the expression and activities of Ser194Phe, Ser290Phe, Leu434Trp, Arg445Cys, Leu492Pro, and Leu568Pro mutations in MCT8 in a dose-dependent manner. The soy isoflavone genistein, a dietary supplement, which was effective in MCT8delF501, was also effective in increasing the expression and transport of these MCT8 mutants; however, the effect size differed among mutants. Kinetic analyses revealed that the Michaelis constants of the mutants toward the primary substrate 3,3',5-triiodothyronine were not much different from the wild-type value, suggesting that these mutants are not impaired in their interaction with substrate but rather destabilized by the mutation and degraded. Copyright © 2017 by the Endocrine Society.
Semisupervised Gaussian Process for Automated Enzyme Search.

PubMed

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

2016-06-17

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

NASA Technical Reports Server (NTRS)

Hahne, G. E.

2003-01-01

The argument follows from the viewpoint that quantum mechanics is taken not in the usual form involving vectors and linear operators in Hilbert spaces, but as a boundary value problem for a special class of partial differential equations-in the present work, the nonrelativistic Schrodinger equation for motion of a structureless particle in four- dimensional space-time in the presence of a potential energy distribution that can be time-as well as space-dependent. The domain of interest is taken to be one of two semi-infinite boxes, one bounded by two t=constant planes and the other by two t=constant planes. Each gives rise to a characteristic boundary value problem: one in which the initial, input values on one t=constant wall are given, with zero asymptotic wavefunction values in all spatial directions, the output being the values on the second t=constant wall; the second with certain input values given on both z=constant walls, with zero asymptotic values in all directions involving time and the other spatial coordinates, the output being the complementary values on the z=constant walls. The first problem corresponds to ordinary quantum mechanics; the second, to a fully time-dependent version of a problem normally considered only for the steady state (time-independent Schrodinger equation). The second problem is formulated in detail. A conserved indefinite metric is associated with space-like propagation, where the sign of the norm of a unidirectional state corresponds to its spatial direction of travel.
[Nursing perspective on psychiatric care in Ivory Coast].

PubMed

Lecocq, Dan

2017-05-01

Michaël Bilson is a nurse at the psychiatric hospital of Bingerville, in Ivory Coast. Here, he describes his mission supporting the National Health Worker Training Institute. It is the only nurse training school in Ivory Coast. Copyright © 2017 Elsevier Masson SAS. All rights reserved.
The Kinetics and Inhibition of the Enzyme Methemoglobin Reductase

ERIC Educational Resources Information Center

Splittgerber, A. G.; And Others

1975-01-01

Describes an undergraduate biochemistry experiment which involves the preparation and kinetics of an oxidation-reduction enzyme system, methemoglobin reductase. A crude enzyme extract is prepared and assayed spectrophotometrically. The enzyme system obeys Michaelis-Menton kinetics with respect to both substrate and the NADH cofactor. (MLH)
Microbial respiration and kinetics of extracellular enzymes activities through rhizosphere and detritusphere at agricultural site

NASA Astrophysics Data System (ADS)

Löppmann, Sebastian; Blagodatskaya, Evgenia; Kuzyakov, Yakov

2014-05-01

Rhizosphere and detritusphere are soil microsites with very high resource availability for microorganisms affecting their biomass, composition and functions. In the rhizosphere low molecular compounds occur with root exudates and low available polymeric compounds, as belowground plant senescence. In detritusphere the substrate for decomposition is mainly a polymeric material of low availability. We hypothesized that microorganisms adapted to contrasting quality and availability of substrates in the rhizosphere and detritusphere are strongly different in affinity of hydrolytic enzymes responsible for decomposition of organic compounds. According to common ecological principles easily available substrates are quickly consumed by microorganisms with enzymes of low substrate affinity (i.e. r-strategists). The slow-growing K-strategists with enzymes of high substrate affinity are better adapted for growth on substrates of low availability. Estimation of affinity of enzyme systems to the substrate is based on Michaelis-Menten kinetics, reflecting the dependency of decomposition rates on substrate amount. As enzymes-mediated reactions are substrate-dependent, we further hypothesized that the largest differences in hydrolytic activity between the rhizosphere and detritusphere occur at substrate saturation and that these differences are smoothed with increasing limitation of substrate. Affected by substrate limitation, microbial species follow a certain adaptation strategy. To achieve different depth gradients of substrate availability 12 plots on an agricultural field were established in the north-west of Göttingen, Germany: 1) 4 plots planted with maize, reflecting lower substrate availability with depth; 2) 4 unplanted plots with maize litter input (0.8 kg m-2 dry maize residues), corresponding to detritusphere; 3) 4 bare fallow plots as control. Maize litter was grubbed homogenously into the soil at the first 5 cm to ensure comparable conditions for the herbivore and detritivore communities in the soil. The kinetics (Km and Vmax) of four extracellular hydrolytic enzymes responsible for C- and phosphorous-cycle (β-glucosidase, β-xylosidase, β-cellobiohydrolase and acid phosphatase), microbial biomass, basal respiration (BR) and substrate-induced respiration (SIR) were measured in rhizosphere, detritusphere and control from 0 - 10 and 10 - 20 cm. The metabolic quotient (qCO2) was calculated as specific indicator for efficiency of microbial substrate utilization. We observed clear differences in enzymes activities at low and high concentrations of substrate. At substrate saturation enzyme activity rates of were significantly higher in rooted plots compared to litter amended plots, whereas at lower concentration no treatment effect could be found. The BR, SIR and qCO2 values were significantly higher at 0 - 10 cm of the planted treatment compared to litter and control plots, revealing a significantly higher respiration at lower efficiency of microbial substrate utilization in the rhizosphere. The Michaelis-Menten constant (Km) decreased with depth, especially for β-glucosidase, acid phosphatase and β-xylosidase, indicating higher substrate affinity of microorganisms in deeper soil and therefore different enzyme systems functioning. The substrate affinity factor (Vmax/Km) increased 2-fold with depth for various enzymes, reflecting a switch of predominantly occurring microbial strategies. Vmax/Km ratio indicated relative domination of zymogenous microbial communities (r-strategists) in 0 - 10 cm depth as compared with 10 - 20 cm depth where the K-strategists dominated.
Maximum Mass of Hybrid Stars in the Quark Bag Model

NASA Astrophysics Data System (ADS)

Alaverdyan, G. B.; Vartanyan, Yu. L.

2017-12-01

The effect of model parameters in the equation of state for quark matter on the magnitude of the maximum mass of hybrid stars is examined. Quark matter is described in terms of the extended MIT bag model including corrections for one-gluon exchange. For nucleon matter in the range of densities corresponding to the phase transition, a relativistic equation of state is used that is calculated with two-particle correlations taken into account based on using the Bonn meson-exchange potential. The Maxwell construction is used to calculate the characteristics of the first order phase transition and it is shown that for a fixed value of the strong interaction constant αs, the baryon concentrations of the coexisting phases grow monotonically as the bag constant B increases. It is shown that for a fixed value of the strong interaction constant αs, the maximum mass of a hybrid star increases as the bag constant B decreases. For a given value of the bag parameter B, the maximum mass rises as the strong interaction constant αs increases. It is shown that the configurations of hybrid stars with maximum masses equal to or exceeding the mass of the currently known most massive pulsar are possible for values of the strong interaction constant αs > 0.6 and sufficiently low values of the bag constant.
Kinetics of Alcohol Dehydrogenase-Catalyzed Oxidation of Ethanol Followed by Visible Spectroscopy

ERIC Educational Resources Information Center

Bendinskas, Kestutis; DiJiacomo, Christopher; Krill, Allison; Vitz, Ed

2005-01-01

The effect of substrate concentration on the rate of enzymatic reaction was investigated and typical Michaelis-Mentin kinetics was observed during the first week. The first order reaction at relatively low concentrations of ethanol and the pseudo zero-order reaction at high concentrations of ethanol were emphasized.

Diagnosis of Enzyme Inhibition Using Excel Solver: A Combined Dry and Wet Laboratory Exercise

ERIC Educational Resources Information Center

Dias, Albino A.; Pinto, Paula A.; Fraga, Irene; Bezerra, Rui M. F.

2014-01-01

In enzyme kinetic studies, linear transformations of the Michaelis-Menten equation, such as the Lineweaver-Burk double-reciprocal transformation, present some constraints. The linear transformation distorts the experimental error and the relationship between "x" and "y" axes; consequently, linear regression of transformed data…
Making Enzyme Kinetics Dynamic via Simulation Software

ERIC Educational Resources Information Center

Potratz, Jeffrey P.

2017-01-01

An interactive classroom demonstration that enhances students' knowledge of steady-state and Michaelis-Menten enzyme kinetics is described. The instructor uses a free version of professional-quality KinTek Explorer simulation software and student input to construct dynamic versions of three static hallmark images commonly used to introduce enzyme…
A Qualitative Approach to Enzyme Inhibition

ERIC Educational Resources Information Center

Waldrop, Grover L.

2009-01-01

Most general biochemistry textbooks present enzyme inhibition by showing how the basic Michaelis-Menten parameters K[subscript m] and V[subscript max] are affected mathematically by a particular type of inhibitor. This approach, while mathematically rigorous, does not lend itself to understanding how inhibition patterns are used to determine the…
Enzyme Kinetics and the Michaelis-Menten Equation

ERIC Educational Resources Information Center

Biaglow, Andrew; Erickson, Keith; McMurran, Shawnee

2010-01-01

The concepts presented in this article represent the cornerstone of classical mathematical biology. The central problem of the article relates to enzyme kinetics, which is a biochemical system. However, the theoretical underpinnings that lead to the formation of systems of time-dependent ordinary differential equations have been applied widely to…
Calibration of the k- ɛ model constants for use in CFD applications

NASA Astrophysics Data System (ADS)

Glover, Nina; Guillias, Serge; Malki-Epshtein, Liora

2011-11-01

The k- ɛ turbulence model is a popular choice in CFD modelling due to its robust nature and the fact that it has been well validated. However it has been noted in previous research that the k- ɛ model has problems predicting flow separation as well as unconfined and transient flows. The model contains five empirical model constants whose values were found through data fitting for a wide range of flows (Launder 1972) but ad-hoc adjustments are often made to these values depending on the situation being modeled. Here we use the example of flow within a regular street canyon to perform a Bayesian calibration of the model constants against wind tunnel data. This allows us to assess the sensitivity of the CFD model to changes in these constants, find the most suitable values for the constants as well as quantifying the uncertainty related to the constants and the CFD model as a whole.
A sensitive glucose biosensor based on Ag@C core-shell matrix.

PubMed

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

2015-04-01

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

PubMed

Miao, Ming; Jiang, Bo; Jiang, Huan; Zhang, Tao; Li, Xingfeng

2015-11-01

This study evaluated the inhibitory effects of the green tea extract on human pancreatic α-amylase activity and its molecular mechanism. The green tea extract was composed of epicatechin (59.2%), epigallocatechin gallate (14.6%) and epicatechin gallate (26.2%) as determined by HPLC analysis. Enzyme activity measurement showed that % inhibition and IC50 of the green tea extract (10%, based on starch) were 63.5% and 2.07 mg/ml, respectively. The Michaelis-Menten constant remained unchanged but the maximal velocity decreased from 0.43 (control) to 0.07 mg/(ml × min) (4 mg/ml of the green tea extract), indicating that the green tea extract was an effective inhibitor against α-amylase with a non-competitive mode. The fluorescence data revealed that the green tea extract bound with α-amylase to form a new complex with static quenching mechanism. Docking study showed the epicatechin gallate in the green tea extract presented stronger affinity than epigallocatechin gallate, with more number of amino acid residues involved in amylase binding with hydrogen bonds and Van der Waals forces. Thus, the green tea extract could be used to manipulate starch digestion for potential health benefits. Copyright © 2015 Elsevier Ltd. All rights reserved.
[Hepatotoxicity of emodin based on UGT1A1 enzyme-mediated bilirubin in liver microsomes].

PubMed

Wang, Qi; Dai, Zhong; Zhang, Yu-Jie; Ma, Shuang-Cheng

2016-12-01

To study the hepatotoxicity of emodin based on bilirubin metabolism mediated by glucuronidation of UGT1A1 enzyme. In this study, three different incubation systems were established by using RLM, HLM, and rUGT1A1, with bilirubin as the substrate. Different concentrations of bilirubin and emodin were added in the incubation systems. The double reciprocal Michaelis equation was drawn based on the total amount of bilirubin glucuronidation. The apparent inhibition constant Ki was then calculated with the slope curve to predict the hepatotoxicity. The results indicated that emodin had a significant inhibition to the UGT1A1 enzyme in all of the three systems, with Ki=5.400±0.956(P<0.05) in HLM system, Ki =10.020±0.611(P<0.05) in RLM system, Ki=4.850±0.528(P<0.05) in rUGT1A1 system. Meanwhile, emodin had no significant difference between rat and human in terms of inhibition of UGT1A1 enzyme. Emodin had a potential risk of the hepatotoxicity by inhibiting the UGT1A1 enzyme activity. And the method established in this study provides a new thought and new method to evaluate hepatotoxicity and safety of traditional Chinese medicines. Copyright© by the Chinese Pharmaceutical Association.
Hexagonal tungsten oxide nanoflowers as enzymatic mimetics and electrocatalysts.

PubMed

Park, Chan Yeong; Seo, Ji Min; Jo, Hongil; Park, Juhyun; Ok, Kang Min; Park, Tae Jung

2017-01-27

Tungsten oxide (WO x ) has been widely studied for versatile applications based on its photocatalytic, intrinsic catalytic, and electrocatalytic properties. Among the several nanostructures, we focused on the flower-like structures to increase the catalytic efficiency on the interface with both increased substrate interaction capacities due to their large surface area and efficient electron transportation. Therefore, improved WO x nanoflowers (WONFs) with large surface areas were developed through a simple hydrothermal method using sodium tungstate and hydrogen chloride solution at low temperature, without any additional surfactant, capping agent, or reducing agent. Structural determination and electrochemical analyses revealed that the WONFs have hexagonal Na 0.17 WO 3.085 ·0.17H 2 O structure and exhibit peroxidase-like activity, turning from colorless to blue by catalyzing the oxidation of a peroxidase substrate, such as 3,3',5,5'-tetramethylbenzidine, in the presence of H 2 O 2 . Additionally, a WONF-modified glassy carbon electrode was adopted to monitor the electrocatalytic reduction of H 2 O 2 . To verify the catalytic efficiency enhancement by the unique shape and structure of the WONFs, they were compared with calcinated WONFs, cesium WO x nanoparticles, and other peroxidase-like nanomaterials. The results indicated that the WONFs showed a low Michaelis-Menten constant (k m ), high maximal reaction velocity (v max ), and large surface area.
Novel epoxy activated hydrogels for solving lactose intolerance.

PubMed

Elnashar, Magdy M M; Hassan, Mohamed E

2014-01-01

"Lactose intolerance" is a medical problem for almost 70% of the world population. Milk and dairy products contain 5-10% w/v lactose. Hydrolysis of lactose by immobilized lactase is an industrial solution. In this work, we succeeded to increase the lactase loading capacity to more than 3-fold to 36.3 U/g gel using epoxy activated hydrogels compared to 11 U/g gel using aldehyde activated carrageenan. The hydrogel's mode of interaction was proven by FTIR, DSC, and TGA. The high activity of the epoxy group was regarded to its ability to attach to the enzyme's -SH, -NH, and -OH groups, whereas the aldehyde group could only bind to the enzyme's -NH2 group. The optimum conditions for immobilization such as epoxy chain length and enzyme concentration have been studied. Furthermore, the optimum enzyme conditions were also deliberated and showed better stability for the immobilized enzyme and the Michaelis constants, K m and V max, were doubled. Results revealed also that both free and immobilized enzymes reached their maximum rate of lactose conversion after 2 h, albeit, the aldehyde activated hydrogel could only reach 63% of the free enzyme. In brief, the epoxy activated hydrogels are more efficient in immobilizing more enzymes than the aldehyde activated hydrogel.
Purification and characterization of chondroitinase ABC from Acinetobacter sp. C26.

PubMed

Zhu, Changliang; Zhang, Jingliang; Zhang, Jing; Jiang, Yanhui; Shen, Zhaopeng; Guan, Huashi; Jiang, Xiaolu

2017-02-01

An extracellular chondroitinase ABC (ChSase ABC, EC 4.2.2.4) produced by cultivating Acinetobacter sp. C26, was purified to homogeneity from the supernatant by ammonium sulfate fractionation, Q-Sepharose Fast Flow and Sephadex G-100 chromatography. The 76kDa enzyme was purified 48.09-fold to homogeneity with specific activity of 348.64U/mg, Using the chondroitin sulfate A (CS-A) as substrate, the maximal reaction rate (Vmax) and Michaelis-Menten constant (Km) of ChSase ABC were found to be 10.471μmol/min/ml and 0.105mg/ml, respectively. The enzyme showed the highest activity at the optimal conditions of pH 6.0 and 42 ∘C, respectively. This enzyme was stable at pH 5-10, 5-9 and 5-7 at 4°C, 37°C and 42°C, respectively. Investigation about thermal stability of ChSase ABC displayed that it was stable at 37°C. ChSase ABC activity was increased in presence of Na + , K + , Mn 2+ , 1,10-phenanthrolin and strongly inhibited by Cu 2+ , Hg 2+ , Al 3+ and SDS. These properties suggested that ChSase ABC from Acinetobacter sp. C26 bring promising prospects in medical and industry applications. Copyright © 2016 Elsevier B.V. All rights reserved.
Screening of neuraminidase inhibitors from traditional Chinese medicines by integrating capillary electrophoresis with immobilized enzyme microreactor.

PubMed

Zhao, Haiyan; Chen, Zilin

2014-05-02

A simple and effective neuraminidase-immobilized capillary microreactor was fabricated by glutaraldehyde cross-linking technology for screening the neuraminidase inhibitors from traditional Chinese medicines. The substrate and product were separated by CE in short-end injection mode within 2 min. Dual-wavelength ultraviolet detection was employed to eliminate the interference from the screened compounds. The parameters relating to the separation efficiency and the activity of immobilized neuraminidase were systematically evaluated. The activity of the immobilized neuraminidase remained 90% after 30 days storage at 4°C. The immobilized NA microreactor could be continuously used for more than 200 runs. The Michaelis-Menten constant of neuraminidase was determined by the microreactor as 136.6 ± 10.8 μM. In addition, six in eighteen natural products were found as potent inhibitors and the inhibition potentials were ranked in the following order: bavachinin>bavachin>baicalein>baicalin>chrysin and vitexin. The half-maximal inhibitory concentrations were 59.52 ± 4.12, 65.28 ± 1.07, 44.79 ± 1.21 and 31.62 ± 2.04 for baicalein, baicalin, bavachin and bavachinin, respectively. The results demonstrated that the neuraminidase-immobilized capillary microreactor was a very effective tool for screening neuraminidase inhibitors from traditional Chinese medicines. Copyright © 2014 Elsevier B.V. All rights reserved.
Screening of tyrosinase inhibitors by capillary electrophoresis with immobilized enzyme microreactor and molecular docking.

PubMed

Cheng, Mengxia; Chen, Zilin

2017-02-01

A new method for screening tyrosinase inhibitors from traditional Chinese medicines (TCMs) was successfully developed by capillary electrophoresis with reliable online immobilized enzyme microreactor (IMER). In addition, molecular docking study has been used for supporting inhibition interaction between enzyme and inhibitors. The IMER of tyrosinase was constructed at the outlet of the capillary by using glutaraldehyde as cross-linker. The parameters including enzyme reaction, separation of the substrate and product, and the performance of immobilized tyrosinase were investigated systematically. Because of using short-end injection procedure, the product and substrate were effectively separated within 2 min. The immobilized tyrosinase could remain 80% active for 30 days at 4°C. The Michaelis-Menten constant of tyrosinase was determined as 1.78 mM. Kojic acid, a known tyrosinase inhibitor, was used as a model compound for the validation of the inhibitors screening method. The half-maximal inhibitory concentration of kojic acid was 5.55 μM. The method was successfully applied for screening tyrosinase inhibitors from 15 compounds of TCM. Four compounds including quercetin, kaempferol, bavachinin, and bakuchiol were found having inhibitory potentials. The results obtained in this work were supported by molecular docking study. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Blood-brain barrier transport of an essential amino acid after cerebral ischemia reperfusion injury.

PubMed

Suzuki, Toyofumi; Miyazaki, Yumiko; Ohmuro, Aya; Watanabe, Masaki; Furuishi, Takayuki; Fukami, Toshiro; Tomono, Kazuo

2013-01-01

Under pathophysiological conditions such as -cerebral ischemia-reperfusion (IR), damage to cerebrovascular endothelial cells causes alterations in the blood-brain barrier (BBB) function that can exacerbate neuronal cell injury and death. Clarifying changes in BBB transport in the early period of IR is important for understanding BBB function during therapy after cerebral ischemia. The present study was aimed at clarifying changes during IR in the BBB transport of L-phenylalanine (Phe) as a substrate of L-type amino acid transporter 1. An IR model was produced in mice by blood recirculation following occlusion of the middle cerebral artery. Permeability of the BBB to [(3)H]Phe was measured after IR injury using the brain perfusion method. Confocal microscopy of the IR injury showed no brain penetration of fluorescent tracer, thus confirming BBB integrity during 45 min of ischemia. Tight junction opening was not observed at 30 min after reperfusion following ischemia for 45 min. At the time of IR, [(3)H]Phe uptake into the brain appeared saturated. The Michaelis constant and maximum transport velocity in the IR group was reduced by 22 % compared with those in controls. These results suggest that the intrinsic transport clearance of Phe is slightly decreased in the early phase of IR.
Colorimetric Glucose Assay Based on Magnetic Particles Having Pseudo-peroxidase Activity and Immobilized Glucose Oxidase.

PubMed

Martinkova, Pavla; Opatrilova, Radka; Kruzliak, Peter; Styriak, Igor; Pohanka, Miroslav

2016-05-01

Magnetic particles (MPs) are currently used as a suitable alternative for peroxidase in the construction of novel biosensors, analytic and diagnostic methods. Their better chemical and thermal stabilities predestine them as appropriate pseudo-enzymatic catalysts. In this point of view, our research was focused on preparation of simply and fast method for immobilization of glucose oxidase onto surface of MPs with peroxidase-like activity. Spectrophotometric method (wavelength 450 nm) optimized for glucose determination using modified MPs has been successfully developed. Concentration curve for optimization of method was assayed, and Michaelis-Menten constant (K m) calculated, maximum reaction rate (V max), limit of detection, and correlation coefficient were determined to be 0.13 mmol/l (2.34 mg/dl), 1.79 pkat, 3.74 µmol/l (0.067 mg/dl), and 0.996, respectively. Interferences of other sugars such as sucrose, sorbitol, deoxyribose, maltose, and fructose were determined as well as effect of substances presenting in plasma (ascorbic acid, reduced glutathione, trolox, and urea). Results in comparison with positive and negative controls showed no interferences of the other sugars and no influence of plasma substances to measuring of glucose. The constructed method showed corresponding results with linear dependence and a correlation coefficient of 0.997. Possibility of repeated use of modified MPs was successfully proved.
Glucose biosensor based on functionalized ZnO nanowire/graphite films dispersed on a Pt electrode

NASA Astrophysics Data System (ADS)

Gallay, P.; Tosi, E.; Madrid, R.; Tirado, M.; Comedi, D.

2016-10-01

We present a glucose biosensor based on ZnO nanowire self-sustained films grown on compacted graphite flakes by the vapor transport method. Nanowire/graphite films were fragmented in water, filtered to form a colloidal suspension, subsequently functionalized with glucose oxidase and finally transferred to a metal electrode (Pt). The obtained devices were evaluated using scanning electron microscopy, energy-dispersive x-ray spectroscopy, cyclic voltammetry and chronoamperometry. The electrochemical responses of the devices were determined in buffer solutions with successive glucose aggregates using a tripolar electrode system. The nanostructured biosensors showed excellent analytical performance, with linear response to glucose concentrations, high sensitivity of up to ≈17 μA cm-2 mM-1 in the 0.03-1.52 mM glucose concentration range, relatively low Michaelis-Menten constant, excellent reproducibility and a fast response. The detection limits are more than an order of magnitude lower than those achievable in commercial biosensors for glucose control, which is promising for the development of glucose monitoring methods that do not require blood extraction from potentially diabetic patients. The strong detection enhancements provided by the functionalized nanostructures are much larger than the electrode surface-area increase and are discussed in terms of the physical and chemical mechanisms involved in the detection and transduction processes.
Facile and controllable preparation of glucose biosensor based on Prussian blue nanoparticles hybrid composites.

PubMed

Li, Lei; Sheng, Qinglin; Zheng, Jianbin; Zhang, Hongfang

2008-11-01

A glucose biosensor based on polyvinylpyrrolidone (PVP) protected Prussian blue nanoparticles (PBNPs)-polyaniline/multi-walled carbon nanotubes hybrid composites was fabricated by electrochemical method. A novel route for PBNPs preparation was applied in the fabrication with the help of PVP, and from scanning electron microscope images, Prussian blue particles on the electrode were found nanoscaled. The biosensor exhibits fast current response (<6 s) and a linearity in the range from 6.7x10(-6) to 1.9x10(-3) M with a high sensitivity of 6.28 microA mM(-1) and a detection limit of 6x10(-7) M (S/N=3) for the detection of glucose. The apparent activation energy of enzyme-catalyzed reaction and the apparent Michaelis-Menten constant are 23.9 kJ mol(-1) and 1.9 mM respectively, which suggests a high affinity of the enzyme-substrate. This easy and controllable construction method of glucose biosensor combines the characteristics of the components of the hybrid composites, which favors the fast and sensitive detection of glucose with improved analytical capabilities. In addition, the biosensor was examined in human serum samples for glucose determination with a recovery between 95.0 and 104.5%.
Enzymatic conversion of D-galactose to D-tagatose: heterologous expression and characterisation of a thermostable L-arabinose isomerase from Thermoanaerobacter mathranii.

PubMed

Jørgensen, F; Hansen, O C; Stougaard, P

2004-06-01

The ability to convert D-galactose into D-tagatose was compared among a number of bacterial L-arabinose isomerases ( araA). One of the most efficient enzymes, from the anaerobic thermophilic bacterium Thermoanaerobacter mathranii, was produced heterologously in Escherichia coli and characterised. Amino acid sequence comparisons indicated that this enzyme is only distantly related to the group of previously known araA sequences in which the sequence similarity is evident. The substrate specificity and the Michaelis-Menten constants of the enzyme determined with L-arabinose, D-galactose and D-fucose also indicated that this enzyme is an unusual, versatile L-arabinose isomerase which is able to isomerise structurally related sugars. The enzyme was immobilised and used for production of D-tagatose at 65 degrees C. Starting from a 30% solution of D-galactose, the yield of D-tagatose was 42% and no sugars other than D-tagatose and D-galactose were detected. Direct conversion of lactose to D-tagatose in a single reactor was demonstrated using a thermostable beta-galactosidase together with the thermostable L-arabinose isomerase. The two enzymes were also successfully combined with a commercially available glucose isomerase for conversion of lactose into a sweetening mixture comprising lactose, glucose, galactose, fructose and tagatose.
A new acylamidase from Rhodococcus erythropolis TA37 can hydrolyze N-substituted amides.

PubMed

Lavrov, K V; Zalunin, I A; Kotlova, E K; Yanenko, A S

2010-08-01

A new acylamidase was isolated from Rhodococcus erythropolis TA37 and characterized. N-Substituted acrylamides (isopropyl acrylamide, N,N-dimethyl-aminopropyl acrylamide, and methylene-bis-acrylamide), acid para-nitroanilides (4'-nitroacetanilide, Gly-pNA, Ala-pNA, Leu-pNA), and N-acetyl derivatives of glycine, alanine, and leucine are good substrates for this enzyme. Aliphatic amides (acetamide, acrylamide, isobutyramide, n-butyramide, and valeramide) are also used as substrates but with less efficiency. The enzyme subunit mass by SDS-PAGE is 55 kDa. Maximal activity is exhibited at pH 7-8 and 55°C. The enzyme is stable for 15 h at 22°C and for 0.5 h at 45°C. The Michaelis constant (K(m)) is 0.25 mM with Gly-pNA and 0.55 mM with Ala-pNA. The acylamidase activity is suppressed by inhibitors of serine proteases (phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate) but is not suppressed by inhibitors of aliphatic amidases (acetaldehyde and nitrophenyl disulfides). The N-terminal amino acid sequence of the acylamidase is highly homologous to those of two putative amidases detected from sequenced R. erythropolis genomes. It is suggested that the acylamidase together with the detected homologs forms a new class within the amidase signature family.
Biochemical and structural characterization of a novel arginine kinase from the spider Polybetes pythagoricus

DOE PAGES

Laino, Aldana; Lopez-Zavala, Alonso A.; Garcia-Orozco, Karina D.; ...

2017-09-11

Energy buffering systems are key for homeostasis during variations in energy supply. Spiders are the most important predators for insects and therefore key in terrestrial ecosystems. From biomedical interest, spiders are important for their venoms and as a source of potent allergens, such as arginine kinase (AK, EC 2.7.3.3). AK is an enzyme crucial for energy metabolism, keeping the pool of phosphagens in invertebrates, and also an allergen for humans. In this work, we studied AK from the Argentininan spider Polybetes pythagoricus ( PpAK), from its complementary DNA to the crystal structure. The PpAK cDNA from muscle was cloned, andmore » it is comprised of 1068 nucleotides that encode a 384-amino acids protein, similar to other invertebrate AKs. The apparent Michaelis-Menten kinetic constant ( K m) was 1.7 mM with a k cat of 75 s –1. Two crystal structures are presented, the apo PvAK and PpAK bound to arginine, both in the open conformation with the active site lid (residues 310–320) completely disordered. The guanidino group binding site in the apo structure appears to be organized to accept the arginine substrate. Lastly, these results contribute to knowledge of mechanistic details of the function of arginine kinase.« less

Biochemical and structural characterization of a novel arginine kinase from the spider Polybetes pythagoricus

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

Laino, Aldana; Lopez-Zavala, Alonso A.; Garcia-Orozco, Karina D.

Energy buffering systems are key for homeostasis during variations in energy supply. Spiders are the most important predators for insects and therefore key in terrestrial ecosystems. From biomedical interest, spiders are important for their venoms and as a source of potent allergens, such as arginine kinase (AK, EC 2.7.3.3). AK is an enzyme crucial for energy metabolism, keeping the pool of phosphagens in invertebrates, and also an allergen for humans. In this work, we studied AK from the Argentininan spider Polybetes pythagoricus ( PpAK), from its complementary DNA to the crystal structure. The PpAK cDNA from muscle was cloned, andmore » it is comprised of 1068 nucleotides that encode a 384-amino acids protein, similar to other invertebrate AKs. The apparent Michaelis-Menten kinetic constant ( K m) was 1.7 mM with a k cat of 75 s –1. Two crystal structures are presented, the apo PvAK and PpAK bound to arginine, both in the open conformation with the active site lid (residues 310–320) completely disordered. The guanidino group binding site in the apo structure appears to be organized to accept the arginine substrate. Lastly, these results contribute to knowledge of mechanistic details of the function of arginine kinase.« less
Construction of a D-amino acid oxidase reactor based on magnetic nanoparticles modified by a reactive polymer and its application in screening enzyme inhibitors.

PubMed

Mu, Xiaoyu; Qiao, Juan; Qi, Li; Liu, Ying; Ma, Huimin

2014-08-13

Developing facile and high-throughput methods for exploring pharmacological inhibitors of D-amino acid oxidase (DAAO) has triggered increasing interest. In this work, DAAO was immobilized on the magnetic nanoparticles, which were modified by a biocompatible reactive polymer, poly(glycidyl methacrylate) (PGMA) via an atom transfer radical polymerization technique. Interestingly, the enzyme immobilization process was greatly promoted with the assistance of a lithium perchlorate catalyst. Meanwhile, a new amino acid ionic liquid (AAIL) was successfully synthesized and employed as the efficient chiral ligand in a chiral ligand exchange capillary electrophoresis (CLE-CE) system for chiral separation of amino acids (AAs) and quantitation of methionine, which was selected as the substrate of DAAO. Then, the apparent Michaelis-Menten constants in the enzyme system were determined with the proposed CLE-CE method. The prepared DAAO-PGMA-Fe3O4 nanoparticles exhibited excellent reusability and good stability. Moreover, the enzyme reactor was successfully applied in screening DAAO inhibitors. These results demonstrated that the enzyme could be efficiently immobilized on the polymer-grafted magnetic nanoparticles and that the obtained enzyme reactor has great potential in screening enzyme inhibitors, further offering new insight into monitoring the relevant diseases.
Stochastic simulation of biological reactions, and its applications for studying actin polymerization.

PubMed

Ichikawa, Kazuhisa; Suzuki, Takashi; Murata, Noboru

2010-11-30

Molecular events in biological cells occur in local subregions, where the molecules tend to be small in number. The cytoskeleton, which is important for both the structural changes of cells and their functions, is also a countable entity because of its long fibrous shape. To simulate the local environment using a computer, stochastic simulations should be run. We herein report a new method of stochastic simulation based on random walk and reaction by the collision of all molecules. The microscopic reaction rate P(r) is calculated from the macroscopic rate constant k. The formula involves only local parameters embedded for each molecule. The results of the stochastic simulations of simple second-order, polymerization, Michaelis-Menten-type and other reactions agreed quite well with those of deterministic simulations when the number of molecules was sufficiently large. An analysis of the theory indicated a relationship between variance and the number of molecules in the system, and results of multiple stochastic simulation runs confirmed this relationship. We simulated Ca²(+) dynamics in a cell by inward flow from a point on the cell surface and the polymerization of G-actin forming F-actin. Our results showed that this theory and method can be used to simulate spatially inhomogeneous events.
Incorporating Human Interindividual Biotransformation ...

EPA Pesticide Factsheets

The protection of sensitive individuals within a population dictates that measures other than central tendencies be employed to estimate risk. The refinement of human health risk assessments for chemicals metabolized by the liver to reflect data on human variability can be accomplished through (1) the characterization of enzyme expression in large banks of human liver samples, (2) the employment of appropriate techniques for the quantification and extrapolation of metabolic rates derived in vitro, and (3) the judicious application of physiologically based pharmacokinetic (PBPK) modeling. While in vitro measurements of specific biochemical reactions from multiple human samples can yield qualitatively valuable data on human variance, such measures must be put into the perspective of the intact human to yield the most valuable predictions of metabolic differences among humans. For quantitative metabolism data to be the most valuable in risk assessment, they must be tied to human anatomy and physiology, and the impact of their variance evaluated under real exposure scenarios. For chemicals metabolized in the liver, the concentration of parent chemical in the liver represents the substrate concentration in the MichaelisMenten description of metabolism. Metabolic constants derived in vitro may be extrapolated to the intact liver, when appropriate conditions are met. Metabolic capacity Vmax; the maximal rate of the reaction) can be scaled directly to the concentration
Acetate transport and utilization in the rat brain.

PubMed

Deelchand, Dinesh K; Shestov, Alexander A; Koski, Dee M; Uğurbil, Kâmil; Henry, Pierre-Gilles

2009-05-01

Acetate, a glial-specific substrate, is an attractive alternative to glucose for the study of neuronal-glial interactions. The present study investigates the kinetics of acetate uptake and utilization in the rat brain in vivo during infusion of [2-13C]acetate using NMR spectroscopy. When plasma acetate concentration was increased, the rate of brain acetate utilization (CMR(ace)) increased progressively and reached close to saturation for plasma acetate concentration > 2-3 mM, whereas brain acetate concentration continued to increase. The Michaelis-Menten constant for brain acetate utilization (K(M)(util) = 0.01 +/- 0.14 mM) was much smaller than for acetate transport through the blood-brain barrier (BBB) (K(M)(t) = 4.18 +/- 0.83 mM). The maximum transport capacity of acetate through the BBB (V(max)(t) = 0.96 +/- 0.18 micromol/g/min) was nearly twofold higher than the maximum rate of brain acetate utilization (V(max)(util) = 0.50 +/- 0.08 micromol/g/min). We conclude that, under our experimental conditions, brain acetate utilization is saturated when plasma acetate concentrations increase above 2-3 mM. At such high plasma acetate concentration, the rate-limiting step for glial acetate metabolism is not the BBB, but occurs after entry of acetate into the brain.
In vitro activation of dibromoacetonitrile to cyanide by myeloperoxidase.

PubMed

Al-Abbasi, Fahad A

2016-08-01

Dibromoacetonitrile (DBAN) is a disinfection by-product classified as a potential human and animal carcinogen. This study aimed at investigating the ability of myeloperoxidase (MPO) to oxidize DBAN to cyanide (CN - ) in vitro Detection of CN - served as a marker for the possible generation of free radical intermediates implicated in DBAN-induced toxicity. Optimum conditions for the oxidation of DBAN to CN - were characterized with respect to pH, temperature, and time of incubation as well as DBAN, MPO, potassium chloride, and hydrogen peroxide (H 2 O 2 ) concentrations in incubation mixtures. Maximum reaction velocity and Michaelis-Menten constant were assessed. Addition of sodium hypochlorite to the reaction mixtures significantly enhanced the rate of the reaction. Addition of the MPO inhibitors, sodium azide, 4-amino benzoic acid hydrazine, or indomethacin to the reaction mixtures significantly decreased the rate of DBAN oxidation. Inclusion of the antioxidant enzyme superoxide dismutase in the incubation mixtures significantly decreased the rate of reaction. Inclusion of the sulfhydryl compounds as reduced glutathione, N-acetylcysteine, d-penicillamine, or l-cysteine enhanced the rate of DBAN oxidation. These results demonstrate the ability of MPO/H 2 O 2 /chloride ion system to oxidize DBAN to CN - and provide insight for the elucidation of DBAN chronic toxicity. © The Author(s) 2015.
Hydrogen peroxide biosensor based on a myoglobin/hydrophilic room temperature ionic liquid film.

PubMed

Safavi, Afsaneh; Farjami, Fatemeh

2010-07-01

The composite film based on Nafion and hydrophilic room temperature ionic liquid (RTIL) 1-butyl-3-methyl-imidazolium chloride ([bmim]Cl) was used as an immobilization matrix to entrap myoglobin (Mb). The study of ionic liquid (IL)-Mb interaction by ultraviolet-visible (UV-vis) spectroscopy showed that Mb retains its native conformation in the presence of IL. The immobilized Mb displayed a pair of well-defined cyclic voltammetric peaks with a formal potential (E(o)(')) of -0.35 V in a 0.1 M phosphate buffer solution (PBS) of pH 7.0. The immobilized Mb exhibited excellent electrocatalytic response to the reduction of hydrogen peroxide, based on which a mediator-free amperometric biosensor for hydrogen peroxide was designed. The linear range for the determination of hydrogen peroxide was from 1.0 to 180 microM with a detection limit of 0.14 microM at a signal/noise ratio of 3. The apparent Michaelis constant (K(m)(app)) for the electrocatalytic reaction was 22.6 microM. The stability, repeatability, and selectivity of the sensor were evaluated. The proposed biosensor has a lower detection limit than many other IL-heme protein-based biosensors and is free from common interference in hydrogen peroxide biosensors. 2010 Elsevier Inc. All rights reserved.
Amperometric cholesterol biosensor based on the direct electrochemistry of cholesterol oxidase and catalase on a graphene/ionic liquid-modified glassy carbon electrode.

PubMed

Gholivand, Mohammad Bagher; Khodadadian, Mehdi

2014-03-15

Cholesterol oxidase (ChOx) and catalase (CAT) were co-immobilized on a graphene/ionic liquid-modified glassy carbon electrode (GR-IL/GCE) to develop a highly sensitive amperometric cholesterol biosensor. The H2O2 generated during the enzymatic reaction of ChOx with cholesterol could be reduced electrocatalytically by immobilized CAT to obtain a sensitive amperometric response to cholesterol. The direct electron transfer between enzymes and electrode surface was investigated by cyclic voltammetry. Both enzymes showed well-defined redox peaks with quasi-reversible behaviors. An excellent sensitivity of 4.163 mA mM(-1)cm(-2), a response time less than 6s, and a linear range of 0.25-215 μM (R(2)>0.99) have been observed for cholesterol determination using the proposed biosensor. The apparent Michaelis-Menten constant (KM(app)) was calculated to be 2.32 mM. The bienzymatic cholesterol biosensor showed good reproducibility (RSDs<5%) with minimal interference from the coexisting electroactive compounds such as ascorbic acid and uric acid. The CAT/ChOx/GR-IL/GCE showed excellent analytical performance for the determination of free cholesterol in human serum samples. © 2013 Elsevier B.V. All rights reserved.
17Beta-hydroxysteroid dehydrogenase (17beta-HSD) in scleractinian corals and zooxanthellae.

PubMed

Blomquist, Charles H; Lima, P H; Tarrant, A M; Atkinson, M J; Atkinson, S

2006-04-01

Steroid metabolism studies have yielded evidence of 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity in corals. This project was undertaken to clarify whether there are multiple isoforms of 17beta-HSD, whether activity levels vary seasonally, and if zooxanthellae contribute to activity. 17Beta-HSD activity was characterized in zooxanthellate and azooxanthellate coral fragments collected in summer and winter and in zooxanthellae cultured from Montipora capitata. More specifically, 17beta-HSD activity was characterized with regard to steroid substrate and inhibitor specificity, coenzyme specificity, and Michaelis constants for estradiol (E2) and NADP+. Six samples each of M. capitata and Tubastrea coccinea (three summers, three winters) were assayed with E2 and NADP+. Specific activity levels (pmol/mg protein) varied 10-fold among M. capitata samples and 6-fold among T. coccinea samples. There was overlap of activity levels between summer and winter samples. NADP+/NAD+ activity ratios varied from 1.6 to 22.2 for M. capatita, 2.3 to 3.8 for T. coccinea and 0.7 to 1.1 for zooxanthellae. Coumestrol was the most inhibitory of the steroids and phytoestrogens tested. Our data confirm that corals and zooxanthellae contain 17beta-HSD and are consistent with the presence of more than one isoform of the enzyme.
Theoretical value of Newtonian constant G confirmed by the International Bureau of Weights & Measures in Paris, France

NASA Astrophysics Data System (ADS)

Omerbashich, Mensur

2015-01-01

World oldest authority for scientific constants and the keeper of the original metre standard, the International Bureau of Weights and Measures (BIPM) in Paris, France has accomplished a historic confirmation of the Omerbashich's (first-ever) scientific prediction of value of the Newtonian gravitational constant G.
Soil-moisture constants and their variation

Treesearch

Walter M. Broadfoot; Hubert D. Burke

1958-01-01

"Constants" like field capacity, liquid limit, moisture equivalent, and wilting point are used by most students and workers in soil moisture. These constants may be equilibrium points or other values that describe soil moisture. Their values under specific soil and cover conditions have been discussed at length in the literature, but few general analyses and...
Distribution Development for STORM Ingestion Input Parameters

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

Fulton, John

The Sandia-developed Transport of Radioactive Materials (STORM) code suite is used as part of the Radioisotope Power System Launch Safety (RPSLS) program to perform statistical modeling of the consequences due to release of radioactive material given a launch accident. As part of this modeling, STORM samples input parameters from probability distributions with some parameters treated as constants. This report described the work done to convert four of these constant inputs (Consumption Rate, Average Crop Yield, Cropland to Landuse Database Ratio, and Crop Uptake Factor) to sampled values. Consumption rate changed from a constant value of 557.68 kg / yr tomore » a normal distribution with a mean of 102.96 kg / yr and a standard deviation of 2.65 kg / yr. Meanwhile, Average Crop Yield changed from a constant value of 3.783 kg edible / m 2 to a normal distribution with a mean of 3.23 kg edible / m 2 and a standard deviation of 0.442 kg edible / m 2 . The Cropland to Landuse Database ratio changed from a constant value of 0.0996 (9.96%) to a normal distribution with a mean value of 0.0312 (3.12%) and a standard deviation of 0.00292 (0.29%). Finally the crop uptake factor changed from a constant value of 6.37e -4 (Bq crop /kg)/(Bq soil /kg) to a lognormal distribution with a geometric mean value of 3.38e -4 (Bq crop /kg)/(Bq soil /kg) and a standard deviation value of 3.33 (Bq crop /kg)/(Bq soil /kg)« less
Study of the charge dependence of the pion–nucleon coupling constant on the basis of data on low-energy nucleon–nucleon interactions

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

Babenko, V. A.; Petrov, N. M., E-mail: pet2@ukr.net

2016-01-15

The relation between quantities that characterize the pion–nucleon and nucleon–nucleon interactions is studied with allowance for the fact that, at low energies, nuclear forces in nucleon–nucleon systems are mediated predominantly by one-pion exchange. On the basis of the values currently recommended for the low-energy parameters of the proton–proton interaction, the charged pion–nucleon coupling constant is evaluated at g{sub π}{sup 2}±/4π = 14.55(13). This value is in perfect agreement with the experimental value of g{sub π}{sup 2}±/4π = 14.52(26) found by the Uppsala Neutron Research Group. At the same time, the value obtained for the charged pion–nucleon coupling constant differs sizablymore » from the value of the pion–nucleon coupling constant for neutral pions, which is g{sub π}{sup 2} 0/4π = 13.55(13). This is indicative of a substantial charge dependence of the coupling constant.« less
Trends in electron-ion dissociative recombination of benzene analogs with functional group substitutions: Negative Hammett σpara values

NASA Astrophysics Data System (ADS)

Osborne, David; Lawson, Patrick Andrew; Adams, Nigel; Dotan, Itzhak

2014-06-01

An in-depth study of the effects of functional group substitution on benzene's electron-ion dissociative recombination (e-IDR) rate constant has been conducted. The e-IDR rate constants for benzene, biphenyl, toluene, ethylbenzene, anisole, phenol, and aniline have been measured using a Flowing Afterglow equipped with an electrostatic Langmuir probe (FALP). These measurements have been made over a series of temperatures from 300 to 550 K. A relationship between the Hammett σpara values for each compound and rate constant has indicated a trend in the e-IDR rate constants and possibly in their temperature dependence data. The Hammett σpara value is a method to describe the effect a functional group substituted to a benzene ring has upon the reaction rate constant.
Investigation of dynamic characteristics of a turbine-propeller engine

NASA Technical Reports Server (NTRS)

Oppenheimer, Frank L; Jacques, James R

1951-01-01

Time constants that characterize engine speed response of a turbine-propeller engine over the cruising speed range for various values of constant fuel flow and constant blade angle were obtained both from steady-state characteristics and from transient operation. Magnitude of speed response to changes in fuel flow and blade angle was investigated and is presented in the form of gain factors. Results indicate that at any given value of speed in the engine cruising speed range, time constants obtained both from steady-state characteristics and from transient operation agree satisfactorily for any given constant fuel flow, whereas time constants obtained from transient operation exceed time constants obtained from steady-state characteristics by approximately 14 percent for any given blade angle.
Contributions of residues of pancreatic phospholipase A2 to interfacial binding, catalysis, and activation.

PubMed

Yu, B Z; Rogers, J; Tsai, M D; Pidgeon, C; Jain, M K

1999-04-13

Primary rate and equilibrium parameters for 60 site-directed mutants of bovine pancreatic phospholipase A2 (PLA2) are analyzed so incremental contributions of the substitution of specific residues can be evaluated. The magnitude of the change is evaluated so a functional role in the context of the N- and C-domains of PLA2 can be assigned, and their relationship to the catalytic residues and to the i-face that makes contact with the interface. The effect of substitutions and interfacial charge is characterized by the equilibrium dissociation constant for dissociation of the bound enzyme from the interface (Kd), the dissociation constant for dissociation of a substrate mimic from the active site of the bound enzyme (KL), and the interfacial Michaelis constants, KM and kcat. Activity is lost (>99.9%) on the substitution of H48 and D49, the catalytic residues. A more than 95% decrease in kcat is seen with the substitution of F5, I9, D99, A102, or F106, which form the substrate binding pocket. Certain residues, which are not part of the catalytic site or the substrate binding pocket, also modulate kcat. Interfacial anionic charge lowers Kd, and induces kcat activation through K56, K53, K119, or K120. Significant changes in KL are seen by the substitution of N6, I9, F22, Y52, K53, N71, Y73, A102, or A103. Changes in KM [=(k2+k-1)/k1] are attributed to kcat (=k2) and KL (=k-1/k1). Some substitutions change more than one parameter, implying an allosteric effect of the binding to the interface on KS, and the effect of the interfacial anionic charge on kcat. Interpreted in the context of the overall structure, results provide insights into the role of segments and domains in the microscopic events of catalytic turnover and processivity, and their allosteric regulation. We suggest that the interfacial recognition region (i-face) of PLA2, due to the plasticity of certain segments and domains, exercises an allosteric control on the substrate binding and chemical step.
Purification and Biochemical and Kinetic Properties of an Endo-Polygalacturonase from the Industrial Fungus Aspergillus sojae.

PubMed

Fratebianchi, Dante; Cavello, Ivana Alejandra; Cavalitto, Sebastián Fernando

2017-01-01

An endo-polygalacturonase secreted by Aspergillus sojae was characterized after being purified to homogeneity from submerged cultures with orange peel as the sole carbon source by gel filtration and ion-exchange chromatographies. According to SDS-PAGE and analytical isoelectric focusing analyses, the enzyme presents a molecular weight of 47 kDa and pI value of 4.2. This enzyme exhibits considerable stability under highly acidic to neutral conditions (pH 1.5-6.5) and presents a half-life of 2 h at 50°C. Besides its activity towards pectin and polygalacturonic acid, the enzyme displays pectin-releasing activity, acting best in a pH range of 3.3-5.0. Thin-layer chromatographic analysis revealed that tri-galacturonate is the main enzymatic end product of polygalacturonic acid hydrolysis, indicating that it is an endo-polygalacturonase. The enzyme exhibits Michaelis-Menten kinetics, with KM and VMAX values of 0.134 mg/mL and 9.6 µmol/mg/min, respectively, and remained stable and active in the presence of SO2, ethanol, and various cations assayed except Hg2+. © 2017 S. Karger AG, Basel.
Application of the compensated Arrhenius formalism to explain the dielectric constant dependence of rates for Menschutkin reactions.

PubMed

Petrowsky, Matt; Glatzhofer, Daniel T; Frech, Roger

2013-11-21

The dependence of the reaction rate on solvent dielectric constant is examined for the reaction of trihexylamine with 1-bromohexane in a series of 2-ketones over the temperature range 25-80 °C. The rate constant data are analyzed using the compensated Arrhenius formalism (CAF), where the rate constant assumes an Arrhenius-like equation that also contains a dielectric constant dependence in the exponential prefactor. The CAF activation energies are substantially higher than those obtained using the simple Arrhenius equation. A master curve of the data is observed by plotting the prefactors against the solvent dielectric constant. The master curve shows that the reaction rate has a weak dependence on dielectric constant for values approximately less than 10 and increases more rapidly for dielectric constant values greater than 10.
Use of Mushroom Tyrosinase to Introduce Michaelis-Menten Enzyme Kinetics to Biochemistry Students

ERIC Educational Resources Information Center

Flurkey, William H.; Inlow, Jennifer K.

2017-01-01

An inexpensive enzyme kinetics laboratory exercise for undergraduate biochemistry students is described utilizing tyrosinase from white button mushrooms. The exercise can be completed in one or two three-hour lab sessions. The optimal amounts of enzyme, substrate (catechol), and inhibitor (kojic acid) are first determined, and then kinetic data is…
Bionomic Exploitation of a Ratio-Dependent Predator-Prey System

ERIC Educational Resources Information Center

Maiti, Alakes; Patra, Bibek; Samanta, G. P.

2008-01-01

The present article deals with the problem of combined harvesting of a Michaelis-Menten-type ratio-dependent predator-prey system. The problem of determining the optimal harvest policy is solved by invoking Pontryagin's Maximum Principle. Dynamic optimization of the harvest policy is studied by taking the combined harvest effort as a dynamic…

Some links on this page may take you to non-federal websites. Their policies may differ from this site.