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.

Interaction of lactate dehydrogenase isoenzymes with ligands

1988 ◽  
Vol 53 (8) ◽  
pp. 1857-1861 ◽  
Author(s):  
Jana Barthová ◽  
Jana Kučerová ◽  
Sylva Leblová
Keyword(s):  
Lactate Dehydrogenase ◽  
Enzyme Inhibitor ◽  
Dissociation Constants ◽  
Physiological Role ◽  
Competitive Inhibitor ◽  
Kinetic Measurements ◽  
Cibacron Blue ◽  
Michaelis Constants ◽  
Sepharose 4B ◽  
Lactate Dehydrogenase Isoenzymes

Isoenzymes of bovine lactate dehydrogenase (H4, H3M, and H2M2) were prepared by affinity chromatography on a 5'-AMP-Sepharose 4B column. The interaction of isoenzymes with two ligands, coenzyme NADH and the competitive inhibitor Cibacron Blue F3GA was followed by means of kinetic measurements and by affinity electrophoresis. The Michaelis constants of NADH were compared with the inhibition constants of Cibacron Blue and dissociation constants of enzyme-inhibitor complexes. It was found that the M subunit of lactate dehydrogenase exhibits always higher affinity both to NADH and Cibacron Blue in comparison to the H subunit. This finding corresponds to the physiological role of lactate dehydrogenase isoenzymes.

scholarly journals Rate studies of polysaccharide biosynthesis from guanosine diphosphate α-d-glucose and guanosine diphosphate α-d-mannose

1971 ◽  
Vol 121 (1) ◽  
pp. 151-157 ◽  
Author(s):  
C. L. Villemez
Keyword(s):  
Reaction Rate ◽  
Initial Rate ◽  
Competitive Inhibitor ◽  
Enzyme Preparations ◽  
Polysaccharide Biosynthesis ◽  
Guanosine Diphosphate ◽  
Straight Line ◽  
Kinetics Of ◽  
Total Reaction Time ◽  
Stimulation Of

With enzyme preparations from Phaseolus aureus seedlings, the initial rate of 14C-labelled polysaccharide formation from GDP-α-d-[14C]glucose is not increased by additions of GDP-α-d-mannose. However, final incorporation is increased by addition of GDP-α-d-mannose, since the total reaction-time is extended. In contrast, the initial rate of 14C-labelled polysaccharide formation from GDP-α-d-[14C]mannose is increased by all concentrations of GDP-α-d-glucose that are less than that of the GDP-α-d-[14C]mannose. Maximum stimulation of the initial rate occurs at a GDP-α-d-[14C]mannose/GDP-α-d-glucose concentration ratio of about 4:1. However, eventual incorporation from GDP-α-d-[14C]mannose is decreased by the addition of GDP-α-d-glucose, since the reaction rate falls off sharply after about 2min. Reciprocal plots of 14C-labelled polysaccharide formation from GDP-α-d-[14C]mannose result in biphasic graphs. The two straight-line portions of the plot are joined by a curved line in the concentration range between 2–3 and 50μm. Extrapolated Km values for the two linear components are 0.4–1.0 and 700–1500μm. The effect of GDP-α-d-glucose on the kinetics of 14C-labelled polysaccharide formation from GDP-α-d-[14C]mannose is complex, and depends on relative concentrations of the two sugar nucleotides. 14C-labelled polysaccharide formation from GDP-α-d-[14C]glucose also results in biphasic reciprocal plots. One component appears to have Km about 2–3μm, the other about 200–400μm. In this reaction, GDP-α-d-mannose appears to be a competitive inhibitor with Ki 20–30μm. With particulate preparations of P. aureus, GDP-α-d-[14C]glucose appears to be a precursor for the synthesis of one polysaccharide, a glucomannan, the mannose moieties of which are derived from an intermediate existing in the particulate preparation. From the rate results, GDP-α-d-[14C]mannose appears to be a precursor for at least two polysaccharides, one of which is a glucomannan.

scholarly journals Partial purification and kinetics of oestriol 16α-glucuronyltransferase from the cytosol fraction of human liver

1970 ◽  
Vol 118 (4) ◽  
pp. 625-634 ◽  
Author(s):  
Govind S. Rao ◽  
Marie Luise Rao ◽  
Heinz Breuer
Keyword(s):  
Human Liver ◽  
Glucuronic Acid ◽  
Specific Radioactivity ◽  
Competitive Inhibitor ◽  
Enzymic Activity ◽  
Hydroxyl Group ◽  
Partial Purification ◽  
Cytosol Fraction ◽  
Enzyme Substrate ◽  
Kinetics Of

An enzyme that conjugates the 16α-hydroxyl group of oestriol with glucuronic acid was found in the cytosol fraction of human liver. The enzymic activity could not be sedimented when the cytosol fraction was centrifuged at 158000gav. for 120min. The oestriol 16α-glucuronyltransferase was purified 100-fold by 0–30% saturation of the cytosol fraction with ammonium sulphate followed by filtration of the precipitate through Sephadex G-200. The activity was eluted at the void volume. The product of the reaction, oestriol 16α-monoglucuronide, was identified by paper chromatography and by crystallization of radioactive product to constant specific radioactivity. The optimum temperature was 37°C, and the activation energy was calculated to be 11.1kcal/mol. The apparent Michaelis–Menten constants for oestriol and UDP-glucuronic acid were 13.3 and 100μm respectively. Cu2+, Zn2+ and Hg2+ inhibited, whereas Mg2+, Mn2+ and Fe2+ stimulated the enzyme. Substrate-specificity studies indicated that the amount of oestradiol-17β, oestradiol-17α and oestrone conjugated was not more than about 5% of that found for oestriol. Oestriol 16α-monoglucuronide, a product of the reaction, did not inhibit the 16α-oestriol glucuronyltransferase; in contrast, UDP, another product of the reaction, inhibited the enzyme competitively with respect to UDP-glucuronic acid as the substrate, and non-competitively with respect to oestriol as the substrate. ATP and UDP-N-acetylglucosamine did not affect the oestriol 16α-glucuronyltransferase. 17-Epioestriol acted as a competitive inhibitor and 16-epioestriol as a non-competitive inhibitor of the glucuronidation of oestriol. 5α-Pregnane-3α,20α-diol also inhibited the enzyme non-competitively. It is most likely that the oestriol 16α-glucuronyltransferase described here is bound to the membranes of the endoplasmic reticulum.

Kinetics of pulmonary angiotensin-converting enzyme and 5'-nucleotidase in vivo

1984 ◽  
Vol 57 (4) ◽  
pp. 1173-1181 ◽  
Author(s):  
J. D. Catravas ◽  
R. E. White
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Enzyme Inhibitor ◽  
Competitive Inhibitor ◽  
Converting Enzyme ◽  
Microvascular Endothelium ◽  
Vasoactive Substances ◽  
Alpha Beta ◽  
Kinetics Of

Angiotensin-converting enzyme and 5′-nucleotidase line the luminal surface of pulmonary microvascular endothelium and participate in the synthesis and/or degradation of potent vasoactive substances. We applied Michaelis-Menten kinetics in simultaneous estimations of apparent constants Km and Amax (product of Vmax and microvascular plasma volume) of these two enzymes for the substrates 3H-labeled benzoyl-Phe-Ala-Pro and 14C-labeled 5′AMP, respectively, in vivo. Values of angiotensin-converting enzyme for benzoyl-Phe-Ala-Pro (Km = 10–11 microM; Amax = 12–13 mumol X min-1) were somewhat higher than published estimates in vitro and changed predictably in response to the known enzyme inhibitor captopril. Kinetic values of 5′nucleotidase for 5′AMP (Km = 3–4 microM; Amax = 3–4 mumol/min) were substantially lower than those reported in vitro but also responded predictably to the competitive inhibitor of 5′nucleotidase, adenosine 5′[alpha, beta-methylene]diphosphate. These data offer in vivo estimates of enzyme kinetics that are useful in revealing enzyme behavior in their normal physiological environment and provide means of evaluating the action of pharmacological, physiological, and pathological modulators of enzyme activity, in vivo.

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.

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.

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.

m-[o-(2-Chloro-5-Fluorosulfonylphenylureido)Phenoxybutoxy]Benzamidine: Affinity Labeling Reagent Which Can Identify Secondary Binding Sites of Coagulation Complement and Fibrinolytic Serine Proteases

1979 ◽  
Author(s):  
D Bing ◽  
D Robison ◽  
J Andrews ◽  
R Laura
Keyword(s):  
Binding Sites ◽  
Serine Proteases ◽  
Enzyme Inhibitor ◽  
Molecular Model ◽  
Factor Xa ◽  
Affinity Labeling ◽  
Catalytic Center ◽  
Inhibitor Complex ◽  
Polypeptide Chains ◽  
Kinetics Of

We have determined that m-[o-(2-chloro-5-fluorosulfonylphenylureido)phenoxybutoxy]benza-midine [mCP(PBA)-F] is an affinity labeling reagent which labels both polypeptide chains of thrombin, factor Xa, complement component CIS and plasmin. As this means it is reacting outside of the catalytic center, we have called this reagent an exo-site affinity labeling reagent. Progressive irreversible inhibition of these enzymes by this reagent is rapid (k1st 2.5-4.6 x 10-3sec-1), the kinetics of inactivation are consistent with inhibition proceding via formation of a specific enzyme-inhibitor complex analogous to a Michaelis-Menton complex (KL - 115-26 μM), and diisopropylfluorophosphate or p-amidino-phenylmethanesulfonyfluoride Prevent labeling by [3H]mCP(PBA)-F. A molecular model of mCP(PBA)-F shows that the reactive SO2F group can be 17 A from the cationic amidine. The data are consistent with the hypothesis that both peptide chains are required for the specific proteolytic activity exhibited by these proteases and that the peptide chain which does not contain the active site serine is close to the catalytic center. (Supported by NIH and AHA grants

The role of secondary alcoholic groups of D-galacturonan in its degradation with exo-D-galacturonanase

1980 ◽  
Vol 45 (2) ◽  
pp. 427-434 ◽  
Author(s):  
Kveta Heinrichová ◽  
Rudolf Kohn
Keyword(s):  
Acetyl Derivative ◽  
Competitive Inhibitor ◽  
Hydroxyl Groups ◽  
Pectic Acid ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
The Individual ◽  
Degradation Degree ◽  
Derivatives Of

The effect of exo-D-galacturonanase from carrot on O-acetyl derivatives of pectic acid of variousacetylation degree was studied. Substitution of hydroxyl groups at C(2) and C(3) of D-galactopyranuronic acid units influences the initial rate of degradation, degree of degradation and its maximum rate, the differences being found also in the time of limit degradations of the individual O-acetyl derivatives. Value of the apparent Michaelis constant increases with increase of substitution and value of Vmax changes. O-Acetyl derivatives act as a competitive inhibitor of degradation of D-galacturonan. The extent of the inhibition effect depends on the degree of substitution. The only product of enzymic reaction is D-galactopyranuronic acid, what indicates that no degradation of the terminal substituted unit of O-acetyl derivative of pectic acid takes place. Substitution of hydroxyl groups influences the affinity of the enzyme towards the modified substrate. The results let us presume that hydroxyl groups at C(2) and C(3) of galacturonic unit of pectic acid are essential for formation of the enzyme-substrate complex.

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