Showing papers on "Michaelis–Menten kinetics published in 1981"
01 Jan 1981
TL;DR: In this paper, the activation of Gluplasminogen and Lys-minminogen in the presence of fibrinogen (f) was studied in purified systems, and the initial rate of activation (u) was calculated.
Abstract: The kinetics of the activation of Glu-plasminogen and Lys-plasminogen (P) by a two-chain form of human tissue plasminogen activator (A) were studied in purified systems, and in the presence of fibrinogen (f) and of fibrin films (F) of increasing size and surface density. The activation in the purified systems followed Michaelis-Menten kinetics with a Michaelis constant of 65 p~ and a catalytic rate constant of 0.06 s-' for Glu-plasminogen as compared to 19 p~ and 0.2 s" for Lysplasminogen. In the presence of fibrinogen plots of l/u uersus 1/[P] or l/u uersus l/[fl yielded straight lines with an apparent Michaelis constant at infinite [fl of 28 PM and a catalytic rate constant of 0.3 s-' for Gluplasminogen as compared to 1.8 p~ and 0.3 s-' for Lysplasminogen. In the systems with fibrin, plasmin was estimated from the rate of release of '"I from "'I-labeled fibrin films. The initial rate of activation (u) was calculated and Lineweaver-Burk plots of l/u uersus 1/ [PI or l/u uersus l/[F] yielded straight lines. Activation occurred with an intrinsic Michaelis constant of 0.16 PM and a catalytic rate constant of 0.1 s-' for Gluplasminogen as compared to 0.02 PM and 0.2 s" for Lysplasminogen. The kinetic analysis suggested that the activation in the presence of fibrin occurs through binding of an activator molecule to the clot surface and subsequent addition of plasminogen (sequential ordered mechanism) to form a cyclic ternary complex. The low Michaelis constant in the presence of fibrin allows efficient plasminogen activation on a fibrin clot, while its high value in the absence of fibrin prevents efficient activation in plasma.
1,149 citations
TL;DR: D-Fructose dehydrogenase was shown to be a satisfactory reagent for microdetermination of D-fructose and was stable at pH 4.5 to 6.0 Stability of the purified enzyme was much enhanced by the presence of detergent in the enzyme solution.
Abstract: D-Fructose dehydrogenase was solubilized and purified from the membrane fraction of glycerol-grown Gluconobacter industrius IFO 3260 by a procedure involving solubilization of the enzyme with Triton X-100 and subsequent fractionation on diethylaminoethyl-cellulose and hydroxylapatite columns. The purified enzyme was tightly bound to a c-type cytochrome and another peptide existing as a dehydrogenase-cytochrome complex. The purified enzyme was deemed pure by analytical ultracentrifugation as well as by gel filtration on a Sephadex G-200 column. The molecular weight of the enzyme complex was determined to be about 140,000, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of three components having molecular weights of 67,000 (dehydrogenase), 50,800 (cytochrome c), and 19,700 (unknown function). Only D-fructose was readily oxidized by the enzyme in the presence of dyes such as ferricyanide, 2,6-dichlorophenolindophenol, or phenazine methosulfate. Nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, and oxygen did not function as electron acceptors. The optimum pH of D-fructose oxidation was 4.0. The enzyme was stable at pH 4.5 to 6.0 Stability of the purified enzyme was much enhanced by the presence of detergent in the enzyme solution. Removal of detergent from the enzyme solution facilitated the aggregation of the enzyme and caused its inactivation. An apparent Michaelis constant for D-fructose was observed to be 10(-2) M with the purified enzyme. D-Fructose dehydrogenase was shown to be a satisfactory reagent for microdetermination of D-fructose.
193 citations
TL;DR: Two kinetically distinguishable sn-glycerol 3-phosphate ( glycerol-P) acryltransferase activities were detected in Escherichia coli inner membranes using acyl-acyl carrier protein (ACP) substrates.
80 citations
TL;DR: It is proposed that the interaction of the reductase with P-450LM2 is inefficient in neutral vesicles and thus rate determining for the overall hydroxylation activities.
Abstract: The phospholipid specificity of rabbit liver microsomal cytochrome P-450LM2 catalyzed hydroxylation reactions was examined in reconstituted phospholipid vesicles. An apparent linear relationship between the negative charge of the vesicles and the rate of P-450LM2-catalyzed O-dealkylation of p-nitroanisole or 7-ethoxycoumarin was obtained. The membrane charge-mediated increase in hydroxylation activities was found not to be due to (i) an altered lipid/water partition coefficient of the substrate, (ii) a change in the apparent Michaelis constant of P-450LM2 for the substrate, (iii) a different activation energy of the O-demethylation of p-nitroanisole, (iv) different spin states of P-450LM2 or (v) an altered secondary structure of this enzyme as monitored by circular dichroism. However, when the formation of the ferrous carbonyl complex of P-450LM2 was followed under aerobic or anaerobic conditions after the addition of NADPH to the vesicles, an increased negative charge of the membrane was accompanied by an increased reducibility of P-450LM2. A similar linear relationship between the reducibility of cytochrome b5 and the negative charge of the liposomes was also evident in membranes containing NADPH-cytochrome P-450 reductase and cytochrome b5. It is proposed that the interaction of the reductase with P-450LM2 is inefficient in neutral vesicles and thus rate determining for the overall hydroxylation activities.
56 citations
TL;DR: The ill-conditioning of the estimation of parameters of a differential equation that includes the so-called Michaelis-Menten output is discussed, where parameter values in an infinite region of the parameter space are shown to have solutions also lying within these two bounds.
49 citations
TL;DR: The immobilized d -glucose oxidase membrane was sufficiently stable to perform 1000 assays for the determination of d - glucose in human whole blood and retained 77% of its initial activity after storage at 4°C for 16 months.
45 citations
TL;DR: A 5000-fold purification of the enzyme responsible for the rapid inactivation of enkephalin in human blood has been achieved: this enzyme cleaves the N-terminal tyrosine from enkePHalin and from short peptides provided their first amino acid is aromatic.
34 citations
TL;DR: The observed effects of the protonmotive force on the apparent Michaelis constant for galactoside can be explained in terms of theProton being a substrate of the transport reaction.
Abstract: beta-Galactoside transport by Escherichia coli occurs with the concomitant uptake of a proton. The kinetics of beta-galactoside uptake at various values of external pH are interpreted in terms of a model in which both the galactoside and the proton are substrates of the transport reaction. The values of some of the kinetic constants for this two-substrate reaction were determined. The observed effects of the protonmotive force on the apparent Michaelis constant for galactoside can be explained in terms of the proton being a substrate of the transport reaction.
33 citations
TL;DR: Hexosaminidase C (2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase), partially purified from bovine brain tissue, is apparently not a glycoprotein since it did not bind to various lectins, nor did sialidase change its isoelectric point.
30 citations
TL;DR: A simple method is presented that establishes intrinsic rate parameters when slow pore diffusion of substrate limits immobilized enzyme reactions that obey Michaelis‐Menten kinetics, and the Aris‐Bischoff modulus is employed.
Abstract: A simple method is presented that establishes intrinsic rate parameters when slow pore diffusion of substrate limits immobilized enzyme reactions that obey Michaelis-Menten kinetics. The Aris-Bischoff modulus is employed. Data at high substrate concentrations, where the enzyme would be saturated in the absence of diffusion limitation, and at low substrate concentrations, where effectiveness factors are inversely proportional to reaction modulus, are used to determine maximum rate and Michaelis constant, respectively. Because Michaelis-Menten and Langmuir-Hinshelwood kinetics are formally identical, this method may be used to estimate intrinsic rate parameters of many heterogeneous catalysts. The technique is demonstrated using experimental data from the hydrolysis of maize dextrin with diffusion-limited immobilized glucoamylase. This system yields a Michaelis constant of 0.14%, compared to 0.11% for soluble glucoamylase and 0.24% for immobilized glucoamylase free of diffusional effects.
30 citations
TL;DR: Human liver enzyme exhibited normal Michaelis-Menten kinetics both in phosphate and Tris buffers, and the apparent Michaelis constant for L-argininosuccinate ws 0.1 mM, unlike bovine liver argininosuccinase, which failed to show any negative homotropic interactions with substrate.
TL;DR: In this paper, the kinetics of oxidation of dimethyl, dipropyl, and diphenyl sulfides by pyridinium chloro chromate in chlorobenzene-nitrobenzene mixtures are reported.
Abstract: The kinetics of oxidation of dimethyl, dipropyl, and diphenyl sulfides by pyridinium chloro chromate in chlorobenzene–nitrobenzene mixtures are reported. The rate data show Michaelis–Menten behavior. The oxidation process is catalyzed by the organic acids like dichloro and trichloro acetic acids. The rate-determining step appears to be a unimolecular decomposition of a complex of the reactants.
TL;DR: The L-α-aminoamidase of Brevibacterium A4 is a particle enzyme with a molecular weight of 135, 000; its optimum pH is 9.5 and its optimum temperature 60°; it is probably a metallo-enzyme with an SH group in the active site, and requiring the presence of Mg2+ or Mn2+.
Abstract: The L-α-aminoamidase of Brevibacterium A4 is a particle enzyme with a molecular weight of 135, 000; its optimum pH is 9.5 and its optimum temperature 60°. It is probably a metallo-enzyme with an SH group in the active site, and requiring the presence of Mg2+ or Mn2+. The purification tests showed that it hydrolyses a large number of α-aminoamides. The Michaelis constant Km varies little for the different substrates. On the other hand the Vm varies greatly, in particular according to the hydrocarbon chain length.The use of the α-amidoamidasic activity for the preparation of optically active, natural or non-natural α-aminoacids has been considered.
TL;DR: Taurodeoxycholate 7 alpha-monooxygenase was purified from rat liver microsomes of phenobarbital-treated rats and reconstituted from the purified enzyme, NADPH-cytochrome P-450 reductase, dilauroylglyceryl-3-phosphorylcholine, and NADPH.
TL;DR: The intrinsic nucleotide content of the dicyclohexylcarbodiimide-sensitive ATPase (DSA) from spinach chloroplasts and its interactions with ADP have been studied.
Abstract: The intrinsic nucleotide content of the dicyclohexylcarbodiimide-sensitive ATPase (DSA) from spinach chloroplasts and its interactions with ADP have been studied. Both partially purified and sucrose gradient purified DSA contain at least 1 mol of ADP/mol of enzyme and 1 mol of ATP/mol of enzyme, although considerable variation exists between different preparations. Radioactively labeled ADP is incorporated into DSA in the presence of 5 mM MgCl2 and 10 mM octyl glucoside with a half-life of approximately 30 min. Incorporation of ADP into DSA reconstituted in phospholipid vesicles occurs at about twice this rate, and a slightly slower rate of uptake is observed with [3H]ADP and [3H]ATP in the presence of 2 mM ethylenediaminetetraacetic acid. The [3H]ATP always appears as bound [3H]ADP on the enzyme. Nucleotide analyses indicate that this incorporation represents an exchange with tightly bound ADP. The nucleotide exchange requires binding at another nucleotide site or sites on the enzyme and is essentially a one-turnover process. Even during ATP synthesis less than 20% of incorporated 3H-labeled nucleotide is removed. Binding studies with forced dialysis indicate the presence of a reversible binding site for ADP distinct from the nucleotide exchange. Similar binding isotherms are obtained for the partially purified enzyme stabilized with 10 mM octyl glucoside, the gradient-purified enzyme stabilized with 0.4% sodium cholate, and the reconstituted, partially purified enzyme. The binding stoichiometry is approximately 0.5 mol of ADP/mol of DSA and the dissociation constant is approximately 2 microM, which is similar to the Michaelis constant for ADP estimated from kinetic studies of ATP synthesis.
TL;DR: In this paper, the existence of thermal rate maxima was shown to be conditional on the relative magnitudes of the three apparent activation energies associated with the Michaelis-Menten mechanism.
TL;DR: Rate expression for enzyme poisoning which are consistent with a Michaelis‐Menten main reaction are used to analyze the performance of a fixed bed reactor containing immobilized enzyme, showing that minimum enzyme activity can occur in the interior of the bed.
Abstract: Rate expression for enzyme poisoning which are consistent with a Michaelis-Menten main reaction are used to analyze the performance of a fixed bed reactor containing immobilized enzyme. When enzyme deactivation results from the irreversible bonding of a product molecule to an existing substrate-enzyme complex, it is shown that minimum enzyme activity can occur in the interior of the bed, well away from the ends. This suggests that bed sectioning techniques may enable direct evaluation of fundamental poisoning mechanisms.
TL;DR: In this paper, an approximate analytical technique employing a finite integral transform was developed to solve the reaction diffusion problem with Michaelis-Menten kinetics in a solid of general shape, and a simple infinite series solution for the substrate concentration was obtained as a function of the Thiele modulus, modified Sherwood number, and Michaelis constant.
Abstract: An approximate analytical technique employing a finite integral transform is developed to solve the reaction diffusion problem with Michaelis-Menten kinetics in a solid of general shape. A simple infinite series solution for the substrate concentration is obtained as a function of the Thiele modulus, modified Sherwood number, and Michaelis constant. An iteration scheme is developed to bring the approximate solution closer to the exact solution. Comparison with the known exact solutions for slab geometry (quadrature) and numerically exact solutions for spherical geometry (orthogonal collocation) shows excellent agreement for all values of the Thiele modulus and Michaelis constant.
TL;DR: Analysis of simulated experimental data suggests that this design of Michaelis-Menten parameters will probably give more precise estimates of Km and V than the conventional design in which v is measured at several different concentrations of substrate.
Abstract: Michaelis-Menten parameters can be estimated by measuring initial velocities (v) in replicate at 2 concentrations of substrate, one much lower than Km and the other much higher than it. Analysis of simulated experimental data suggests that this design will probably give more precise estimates of Km and V than the conventional design in which v is measured at several different concentrations of substrate.
TL;DR: Simulated Lineweaver-Burk plots are presented which show distortions due to product inhibition, reaction reversal, and the absence of initial velocity conditions, which can be properly handled by employing an integrated Michaelis-Menten equation.
TL;DR: The rate equation for trans inhibition shows that the velocity constant for recovery of the unloaded carrier may be determined with steady state experimental data, and it is suggested that the observed recovery constant is independent of the substrates and trans inhibitors chosen for an assay of a particular carrier system.
TL;DR: The irreversible Michaelis-Menten reaction is studied by the use of the method of multiple scales and three stages are identified, one of which is studied in detail.
TL;DR: The presented nonlinear equation of the first-pass effect is an explicit integration of the Michaelis-Menten equation and involves only general mathematics, however, the problem of evaluating the Michael is-menten constants Vm and Km is not resolved.
TL;DR: The pH-dependence of fumaryl acetoacetase (4-fumarylacetoacetate fum Daryl-hydrolase, EC 3.7.2) activity was studied and the Michealis constant was found to be pH independent in this range.
TL;DR: In this article, a detailed description of reactor performance from start-up to final catalyst death is obtained for parallel and series self-poisoning mechanisms for a CSTR with one-half and integer-order kinetics, as well as Michaelis-Menten kinetics are considered.
TL;DR: The validity of the above methods was proved by the model experiments with purified enzymes; and the radiometric model experiment offered a good example for a new enzymatic assay method of many substrates.
TL;DR: In this article, the irreversible Michaelis-Menten scheme was reduced to a pair of autonomous first-order differential equations and the phaseplane behavior of these was investigated. But the phase-plane behavior was not investigated.
01 Jan 1981
TL;DR: The discrepancies between the measured kinetics and real mechanism may be due to the existence of isoenzymes, the non-identical behaviour of subunits in oligomeric enzymes, the association-dissociation of oligomers, the instability of the enzyme under assay conditions, the effect of ligands, interaction with proteins, other macromolecules or membrances, etc.
Abstract: One can observe Michaelian kinetics in the case when the enzyme does not follow Michaelis-Menten mechanism and one can determine deviations from Michaelis-Menten kinetics with an enzyme that follows Michaelian mechanism. The discrepancies between the measured kinetics and real mechanism may be due to the existence of isoenzymes, the non-identical behaviour of subunits in oligomeric enzymes, the association-dissociation of oligomers, the instability of the enzyme under assay conditions, the effect of ligands, interaction with proteins, other macromolecules or membrances, etc.
TL;DR: In this paper, the rate of formation of peroxidatically active reaction intermediate(s) via oxidation of the iron(III)-porphyrin complex, deuteroferriheme, with hydrogen peroxide decreases with increasing borate content of mixed borate-carbonate buffer solutions.