scholarly journals Effect of thrombomodulin on the kinetics of the interaction of thrombin with substrates and inhibitors

1986 ◽  
Vol 237 (1) ◽  
pp. 243-251 ◽  
Author(s):  
J Hofsteenge ◽  
H Taguchi ◽  
S R Stone
Keyword(s):  
Dissociation Constant ◽  
Antithrombin Iii ◽  
Dissociation Constants ◽  
Competitive Inhibitor ◽  
Mean Value ◽  
Similar Amount ◽  
Michaelis Constant ◽  
Saturation Kinetics ◽  
Slow Binding Inhibitor ◽  
Kinetics Of

Thrombomodulin decreased by 20-30% the Michaelis constant of two tripeptidyl p-nitroanilide substrates of thrombin. Thrombomodulin increased the rate of inactivation of thrombin by two peptidyl chloromethane inhibitors by a similar amount. This effect appeared to be due to a decrease in the dissociation constants of the inhibitors. An improved method for the separation of fibrinopeptides A and B by h.p.l.c. was developed, and this method was used to study the effect of thrombomodulin on the thrombin-catalysed cleavage of fibrinogen. In this reaction, thrombomodulin was a competitive inhibitor with respect to the A alpha-chain of fibrinogen. The release of fibrinopeptide B was also inhibited by thrombomodulin. Analysis of the inhibition caused by thrombomodulin with respect to fibrinopeptides A and B yielded the same dissociation constant for the thrombin-thrombomodulin complex. In the presence of thrombomodulin, the rate of inactivation of thrombin by antithrombin III was stimulated 4-fold. This stimulation showed saturation kinetics with respect to thrombomodulin. Thrombomodulin was found to compete with hirudin for a binding site on thrombin. As a result of this competition, hirudin became a slow-binding inhibitor of thrombin at high thrombomodulin concentrations. Estimates of the dissociation constant for thrombomodulin were obtained in several of the above experiments, and the weighted mean value was 0.7 nM.

scholarly journals A Competitive Inhibitor of Tyrosinase

1951 ◽  
Vol 4 (4) ◽  
pp. 554 ◽  
Author(s):  
C Warner
Keyword(s):  
Enzyme Inhibitor ◽  
Dissociation Constants ◽  
Competitive Inhibitor ◽  
Enzyme Preparations ◽  
Enzyme Substrate ◽  
Kinetics Of

The kinetics of the activation of catechol by tyrosinase prepared from the potato and the mushroom, and of its inhibition by sodium m-hydroxybenzoate, have been studied. The enzyme-substrate dissociation constants differed markedly between the two enzyme sources (K. potato = 5.OmM, Kg mushroom = O.28mM), as did also the enzyme-inhibitor dissociation constants (K; potato = 2.5mM, Ki mushroom = O.BmM). For both enzyme preparations sodium m-hydroxybenzoate met the requirements of a competitive inhibitor.

scholarly journals Mechanism of regulation of actin polymerization by Physarum profilin.

1984 ◽  
Vol 98 (6) ◽  
pp. 1919-1925 ◽  
Author(s):  
K Ozaki ◽  
S Hatano
Keyword(s):  
Steady State ◽  
Dissociation Constant ◽  
Actin Polymerization ◽  
Dissociation Constants ◽  
Critical Concentration ◽  
Cross Linking ◽  
Kinetics Of Polymerization ◽  
Kinetics Of ◽  
Chemical Cross Linking

Physarum profilin reduces the rates of nucleation and elongation of F-actin and also reduces the extent of polymerization of actin at the steady state in a concentration-dependent fashion. The apparent critical concentration for polymerization of actin is increased by the addition of profilin. These results can be explained by the idea that Physarum profilin forms a 1:1 complex with G-actin and decreases the concentration of actin available for polymerization. The dissociation constant for binding of profilin to G-actin is estimated from the kinetics of polymerization of G-actin and elongation of F-actin nuclei and from the increase of apparent critical concentration in the presence of profilin. The dissociation constants for binding of Physarum profilin to Physarum and muscle actins under physiological ionic conditions are in the ranges of 1.4-3.7 microM and 11.3-28.5 microM, respectively. When profilin is added to an F-actin solution, profilin binds to G-actin which co-exists with F-actin, and then G-actin is dissociated from F-actin to compensate for the decrease of the concentration of free G-actin and to keep it constant at the critical concentration. At the steady state, free G-actin of the critical concentration is in equilibrium not only with F-actin but also with profilin-G-actin complex. The stoichiometry of 1:1 for the formation of complex between profilin and G-actin is directly shown by means of chemical cross-linking.

Michaelis-Menten kinetics of galactose elimination by the isolated perfused pig liver

1976 ◽  
Vol 230 (5) ◽  
pp. 1302-1313 ◽  
Author(s):  
S Keiding ◽  
S Johansen ◽  
K Winkler ◽  
K Tonnesen ◽  
N Tygstrup
Keyword(s):  
Irreversible Process ◽  
Experimental Uncertainty ◽  
Maximal Rate ◽  
Michaelis Constant ◽  
Pig Liver ◽  
Blood Concentrations ◽  
Saturation Kinetics ◽  
Rate Limiting ◽  
Kinetics Of ◽  
Galactose Elimination

The relation between galactose elimination rates and blood concentrations in the isolated perfused pig liver was analyzed by a mathematical kinetic model. It assumes that the substrate, under steady-state conditions, is removed from the blood that flows through the sinusoids by an irreversible process which follows Michaelis-Menten (i.e., saturation) kinetics. The experiments consisted of successive periods with constant infusions of galactose. The model fitted the data to within the experimental uncertainty. The estimated maximal rate (Vmax) ranged from 0.34 to 0.57 mmol-min(-1)-kg(-1) liver, and the Michaelis constant, Km, ranged from 0.12 to 0.30 mmol-liter(-1) plasma water in nine experiments. The ratio between the galactose concentration in hepatocyte water and plasma water was not significantly different from 1.0, indicating that membrane transport is not rate limiting for the elimination of galactose. In experiments with increasing concentrations of galactose in hepatocyte water and approximately saturated elimination rates, the concentrations of galactose 1-phosphate, UDPgalactose, and UDPglucose remained essentially constant. This indicates that the phosphorylation of galactose to galactose 1-phosphate is the rate-determining process.

Inhibition Kinetics of Polyphenol Oxidase from Purple Sweet Potato by Ascorbic Acid

2013 ◽  
Vol 333-335 ◽  
pp. 1921-1925
Author(s):  
Lu Gao ◽  
Ke Da Li ◽  
Ying Chang Li
Keyword(s):  
Ascorbic Acid ◽  
Sweet Potato ◽  
Polyphenol Oxidase ◽  
Substrate Inhibition ◽  
Competitive Inhibitor ◽  
Inhibitory Effects ◽  
Michaelis Constant ◽  
Maximum Reaction ◽  
Purple Sweet Potato ◽  
Kinetics Of

With catechol as a substrate, some kinetic parameters, including Michaelis constant (Km), maximum reaction velocity (Vmax) and substrate inhibition constant (KI) for the reaction catalyzed by polyphenol oxidase (PPO) from purple sweet potato (PSP) were mainly studied here by spectrophotometry. Kmand Vmaxwere determined depending on bi-reciprocal diagram of Lineweaver-Burk and Hanes-Woolf diagram respectively, with Kmof 12.06 mM and Vmaxof 43.66 mM·min. The effects of four various inhibitors on PPO activity were different. Ascorbic acid (AA) and phytic acid (PA) showed strong inhibitory effects, with AA of the highest effect and citric acid (CA) the lowest. Among these inhibitors, AA was a reversible competitive inhibitor with KIof 15.26 mM, which was significant and instructive to the quality and benefit improvement of processed PSP products.

scholarly journals A study of the kinetics of the muscarinic effect on phosphatidylinositol and phosphatidic acid metabolism in rat brain synaptosomes

1977 ◽  
Vol 168 (3) ◽  
pp. 549-555 ◽  
Author(s):  
J C Miller
Keyword(s):  
Phosphatidic Acid ◽  
Rat Brain ◽  
Acid Metabolism ◽  
Dissociation Constants ◽  
Competitive Inhibitor ◽  
Bicarbonate Buffer ◽  
Brain Synaptosomes ◽  
32P Uptake ◽  
Rate Limiting ◽  
Kinetics Of

The uptake of [32P]phosphate into phosphatidylinositol and phosphatidate was measured in synaptosomes incubated in Krebs-Ringer bicarbonate buffer, pH7.4. The apparent dissociation constants for acetylcholine and carbamoylcholine was estimated from the increase in 32P uptake caused by these agents. These apparent constants were similar for both phosphatidylinositol and phosphatidate and were 2.7 +/- 0.5 MICROmeter for acetylcholine and 12 +/- 2 micrometer for carbamoylcholine when Ca2+ concentration was 0.75 mM. Under the same conditions the inhibition of the carbamoylcholine-induced increase in 32P uptake, caused by atropine, is consistent with atropine being a competitive inhibitor, with an apparent inhibition constant of 0.35 +/- 0.05 micrometer. The apparent constants were dependent on the Ca2+ concentration, and were greater in 2.54 mM-Ca2+. The former values for the kinetic constants are similar to the muscarinic-receptor dissociation constant, which indicates that the binding of the agonist to the receptor may be rate-limiting in this series of reactions when the Ca2+ concentration is 0.75 mM.

Kinetic Aspects of the Interaction of Blood Clotting Enzymes

1968 ◽  
Vol 19 (03/04) ◽  
pp. 364-367 ◽  
Author(s):  
H. C Hemker ◽  
P. W Hemker
Keyword(s):  
Enzyme Kinetics ◽  
Blood Clotting ◽  
Competitive Inhibition ◽  
Competitive Inhibitor ◽  
Kinetics Of

SummaryThe enzyme kinetics of competitive inhibition under conditions prevailing in clotting tests are developed and a method is given to measure relative amounts of a competitive inhibitor by means of the t — D plot.

Antithrombin III Antigen in Human Platelets

1985 ◽  
Vol 54 (03) ◽  
pp. 599-602 ◽  
Author(s):  
M Léon Alhenc-Gelas ◽  
M Aiach ◽  
A Gorenflot ◽  
J P Andreux
Keyword(s):  
Arachidonic Acid ◽  
Enzyme Immunoassay ◽  
Antithrombin Iii ◽  
Human Platelets ◽  
Mean Value ◽  
Normal Blood ◽  
Freezing And Thawing ◽  
Healthy Donors ◽  
At Iii ◽  
Storage Granules

SummaryImmunoreactive AT III was found in human platelets. AT III antigen was quantified in platelets taken from each of 17 healthy donors by a specific competitive enzyme immunoassay using purified AT III and AT III antibodies. AT III antigen levels in extracts of washed platelets disrupted by freezing and thawing ranged from 32 to 140 ng per 109 platelets with a mean value of 70.3 ± 27.3. When stimulated by arachidonic acid, the platelets released AT III antigen together with immunoreactive fibrinogen. These results show that AT III is present in platelets at a level corresponding to approximately 0.01% of total antithrombin in normal blood, and suggest that platelet AT III, like fibrinogen, is contained in the storage granules.

scholarly journals Inhibition of Amino Acid Transport in Rabbit Intestine by p-Chloromercuriphenyl Sulfonic Acid

1973 ◽  
Vol 62 (2) ◽  
pp. 131-146 ◽  
Author(s):  
John F. Schaeffer ◽  
Robert L. Preston ◽  
Peter F. Curran
Keyword(s):  
Transport System ◽  
Conformational Changes ◽  
Marked Reduction ◽  
Sulfhydryl Group ◽  
Major Effect ◽  
Acid Transport ◽  
Michaelis Constant ◽  
Saturation Kinetics ◽  
Rabbit Intestine ◽  
Transport Site

Influx of phenylalanine across the brush border of rabbit intestine is markedly reduced by treatment with 5 mM p-chloromercuriphenyl sulfonate (PCMBS). The effect is rapidly and completely reversed by dithiothreitol. Phenylalanine influx into PCMBS-treated tissue can be competitively inhibited by other neutral amino acids and follows saturation kinetics. PCMBS causes an increase in the apparent Michaelis constant from the value observed in control tissue but does not alter the maximal influx significantly. Treatment of the tissue with PCMBS leads to a significant reduction in the Na-sensitivity of the transport, and a number of results indicate that the major effect of the reagent is to cause a marked reduction in the affinity of the transport system for Na. The transport system can be partially protected against reaction with PCMBS by phenylalanine and tryptophan but not by methionine or norleucine. The results suggest that PCMBS reacts with a sulfhydryl group in the region of the transport site and may alter conformational changes associated with the binding of substrates.

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