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.

scholarly journals Studies on a testosterone glucuronyltransferase from the cytosol fraction of human liver

1970 ◽  
Vol 119 (4) ◽  
pp. 635-642 ◽  
Author(s):  
Govind S. Rao ◽  
Marie Luise Rao ◽  
Heinz Breuer
Keyword(s):  
Human Liver ◽  
Enzyme Preparation ◽  
Reaction Rate ◽  
Specific Radioactivity ◽  
Enzymic Activity ◽  
Hydroxyl Group ◽  
Freezing And Thawing ◽  
Cytosol Fraction ◽  
Tris Maleate ◽  
Conjugating Enzyme

An enzyme that conjugates the 17β-hydroxyl group of testosterone was found in the cytosol fraction of human liver. The same enzyme preparation also conjugates the 16α-hydroxyl group of oestriol. The enzymic activity could not be sedimented by centrifuging the cytosol fraction at 158000gav. for 120min. The testosterone-conjugating as well as the oestriol-conjugating activities were found in the precipitate obtained after 30% saturation of the cytosol fraction with ammonium sulphate. Filtration of the precipitate through Sephadex G-200 enriched the testosterone-conjugating enzyme 50-fold and the oestriol-conjugating enzyme 100-fold. No separation of the two activities was achieved. With labelled testosterone the product of the reaction, testosterone 17β-glucuronide, was identified by paper chromatography and by crystallization to constant specific radioactivity. Testosterone 17β-glucuronyltransferase was active between pH7.0 and 8.6 in tris–HCl and tris–maleate buffers. The apparent Km values for testosterone and UDP-glucuronic acid were 6.4 and 25μm respectively. The enzyme was active between 37 and 45°C; the activation energy was calculated to be 5kcal/mol. Oestriol did not influence the glucuronidation of testosterone. Controlled heating as well as alternate freezing and thawing of the purified enzyme preparation led to an inactivation of both testosterone-conjugating and oestriol-conjugating activities at similar rates. Testosterone and oestriol, when incubated together, gave a reaction rate that was approximately equal to the sum of the rates when the two substrates were incubated separately. The present findings suggest that testosterone and oestriol are conjugated by two separate enzymes.

On the molecular specificity of steroid-enzyme combinations. The kinetics of β -hydroxysteroid dehydrogenase

1955 ◽  
Vol 144 (914) ◽  
pp. 116-132 ◽  
Keyword(s):  
Hydroxysteroid Dehydrogenase ◽  
Hydroxyl Group ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Molecular Specificity ◽  
Planar Molecules ◽  
High Concentrations ◽  
Kinetics Of ◽  
Androstane Derivatives ◽  
Marked Tendency

β -Hydroxysteroid dehydrogenase is a purified enzymic protein of bacterial origin which catalyzes oxidations of 3 β - and 17 β -hydroxysteroids to their respective ketones with diphosphopyridine nucleotide as a hydrogen acceptor. The reaction kinetics of this enzyme with a variety of steroids are not in accordance with the predictions of the theory of Michaelis & Menten (1913), since the velocity of oxidation shows a marked tendency to decline at high concentrations of substrate. The behaviour of these compounds may be fully analyzed on the assumption of the formation of an enzyme-substrate complex involving two substrate molecules. The theory for bimolecular complex formation and its implications are examined. Affinity constants have been calculated for various steroids and conclusions drawn as to the structural requirements favouring attachment to the enzyme surface. Phenolic compounds of the oestra-1:3:5(10)-triene-3-ol family are most firmly bound. Planar molecules of the androst-4-ene, androst-5-ene or 5 α -androstane series show intermediate affinity, while testane (5 β -androstane) derivatives which deviate considerably from planarity are most poorly bound to the enzyme surface. The presence of oxygenated functions at positions 3 and 17 promotes high affinity, whereas an additional 11 α - or 11 β ?-hydroxyl group opposes this effect. Conclusions have been drawn as to the manner of attachment of substrates to the enzyme surface. Certain correlations between the molecular requirements for efficient binding of steroids to the enzyme surface and their physiological activities are demonstrated.

scholarly journals A kinetic study of irreversible enzyme inhibition by an inhibitor that is rendered unstable by enzymic catalysis. The inhibition of polyphenol oxidase by l-cysteine

1991 ◽  
Vol 277 (3) ◽  
pp. 869-874 ◽  
Author(s):  
E Valero ◽  
R Varón ◽  
F García-Carmona
Keyword(s):  
Kinetic Study ◽  
Polyphenol Oxidase ◽  
Dynamic Behaviour ◽  
Equation System ◽  
Enzymic Activity ◽  
Differential Equation System ◽  
Enzyme Substrate ◽  
Lag Period ◽  
Linear Differential ◽  
Kinetics Of

A kinetic study of the irreversible inhibition of an enzyme by an inhibitor that is depleted in the medium by its reaction with the product of enzymic analysis was made. The model is illustrated by the study of the inhibition of catecholase activity of polyphenol oxidase by L-cysteine. The inhibition is characterized by an initial lag period followed by a concomitant decrease in enzymic activity expressed when the steady state is reached, both kinetic parameters being modulated by enzyme, substrate and inhibitor concentrations. There is no analytical solution to the non-linear differential-equation system that describes the kinetics of the reaction, and so computer simulations of this dynamic behaviour are presented. The results obtained show that the system here studied presents kinetic co-operativity for a target enzyme that follows the simple Michaelis-Menten mechanism in its action on the substrate.

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 Inhibition of human alkaline phosphatases by vanadate

1979 ◽  
Vol 181 (1) ◽  
pp. 247-250 ◽  
Author(s):  
L E Seargeant ◽  
R A Stinson
Keyword(s):  
Alkaline Phosphatase ◽  
Binding Site ◽  
Human Liver ◽  
Competitive Inhibitor ◽  
Enzymic Activity ◽  
Alkaline Phosphatases

Orthovanadate was shown to be a potent competitive inhibitor (Ki less than 1 microM) of purified alkaline phosphatase from human liver, intestine of kidney. Inhibition was reversed and full enzymic activity restored in the presence of 1mM-adrenaline. Phosphate and vanadate competed for the same binding site on the enzyme.

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.

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.

Effect of Commonly Used Organic Solvents on the Kinetics of Cytochrome P450 2B6- and 2C8-Dependent Activity in Human Liver Microsomes

2007 ◽  
Vol 35 (11) ◽  
pp. 1990-1995 ◽  
Author(s):  
Ragini Vuppugalla ◽  
Shu-Ying Chang ◽  
Hongjian Zhang ◽  
Punit H. Marathe ◽  
David A. Rodrigues
Keyword(s):  
Cytochrome P450 ◽  
Organic Solvents ◽  
Human Liver ◽  
Liver Microsomes ◽  
Human Liver Microsomes ◽  
Cytochrome P450 2B6 ◽  
Dependent Activity ◽  
Kinetics Of
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