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Kinetics and mechanism of action of aldehyde reductase from pig kidney.

ABSTRACT: An improved procedure for purifying aldehyde reductase is described. Utilization of Blue Dextran--Sepharose 4B and elimination of hydroxyapatite chromatography greatly improves the yield and ease of purification. Starting with 340 g of kidney tissue (two pig kidneys) approx. 50 mg of purified reductase may be routinely and reproducibly obtained. The purified reductase was used to establish the kinetic reaction mechanism of the enzyme. Initial-velocity analysis and product-inhibition data revealed that pig kidney aldehyde reductase follows an Ordered Bi Bi reaction mechanism in which NADPH binds first before D-glyceraldehyde. The limiting Michaelis constants for D-glyceraldehyde and NADPH were 4.8 +/- 0.7 mM and 9.1 +/- 2.1 micrometer respectively. The mechanism is similar to that of another monomeric oxidoreductase, octopine dehydrogenase, towards which aldehyde reductase exhibits several similarities, but differs from that of other aldehyde reductases. Phenobarbital is a potent inhibitor of aldehyde reductase, inhibiting both substrate and cofactor non-competitively (Ki = 80.4 +/- 10.5 micrometer and 66.9 +/- 1.6 micrometer respectively). Barbiturate inhibition seems to be a common property of NADPH-dependent aldehyde reductases.

SUBMITTER: Davidson WS

PROVIDER: S-EPMC1186410 | biostudies-other | 1979 Feb

REPOSITORIES: biostudies-other

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Kinetic studies of the mechanism of pig kidney aldehyde reductase.

Project description:Initial-rate measurements were made of the oxidations of pyridine-3-methanol and glycerol by NADP+ and of the reduction of the corresponding aldehydes by NADPH catalysed by pig kidney aldehyde reductase. In addition, a brief survey of the specificity of the enzyme towards aldehyde substrates and its sensitivity to the inhibitors ethacrynic acid, sodium barbitone and warfarin was made. The detailed kinetic work indicates a compulsory mechanism for aldehyde reduction, with NADPH binding before aldehyde. For alcohol oxidation, however, it is necessary to postulate the formation of kinetically significant amounts of binary complexes of the type enzyme-alcohol to explain the results. Thus, for alcohol oxidation random-order addition of substrates may occur. Inhibition studies of the kinetics of aldehyde reduction in the presence of the corresponding alcohol product provide further evidence for the existence of enzyme-alcohol complexes. Finally, detailed kinetic studies were made of the inhibition of pyridine-3-aldehyde reduction by sodium barbitone. The mechanism of the inhibition is discussed.

| S-EPMC1162630 | biostudies-other

Some properties of pig kidney-cortex aldehyde reductase.

Project description:Aldehyde reductase was purified from pig kidney cortex to homogeneity by a new procedure. The molecular weight of the enzyme was estimated by sedimentation equilibrium to be 43 700 and by gel electrophoresis in the presence of sodium dodecyl sulphate to be 41 700. The enzyme is clearly a monomer. The enzyme preparation contained no significant quantities of zinc, manganese or copper and had no essential histidine or thiol groups. Changes in the absorption and fluorescence spectra of NADPH were observed on formation of the enzyme-NADPH complexes. Large changes in the fluorescence spectra were also observed in the presence of sodium barbitone or Warfarin. These changes were used as the basis of active-site titrations, which showed that the enzyme had one active site per molecule. The dissociation constants of NADPH and NADP+ from binary complexes with the enzyme were estimated in spectrophotometric titrations.

| S-EPMC1162254 | biostudies-other

Bovine lens aldehyde reductase (aldose reductase). Purification, kinetics and mechanism.

Project description:Aldehyde reductase (aldose reductase) was purified to homogeneity (as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis) from bovine lens by affinity chromatography on NADP+-Sepharose. The enzyme, a monomer of Mr about 40000, was active with a variety of alpha- hydroxyketones , including fructose. The minimum degree of the rate equation was 2:2 in the case of DL-glyceraldehyde, but linear kinetics were observed for glucose and NADPH over the concentration range studied. The enzyme largely followed a ternary-complex mechanism, with initial binding of NADPH before glucose and final release of NADP+.

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Purification and characterization of an enzymically active cleavage product of pig kidney aldehyde reductase.

Project description:During the purification of pig kidney aldehyde reductase by an established procedure [Flynn, Cromlish & Davidson (1982) Methods Enzymol. 89, 501-506] a second enzyme with aldehyde reductase activity may be purified. When the procedure was performed in the presence of 5 mM-EDTA, only traces of the second reductase, pig kidney aldehyde reductase (minor form), were present. By the criterion of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, pig kidney aldehyde reductase (minor form) had Mr 35 000, in comparison with Mr 40 200 found for pig kidney aldehyde reductase. Amino acid analysis of both enzymes and tryptic-peptide-map comparisons indicated differences in primary structure. The N-terminus of pig kidney aldehyde reductase (minor form) had the sequence Lys-Val-Leu, in contrast with the blocked (acetylated) N-terminus of pig kidney aldehyde reductase. The C-terminal sequence of both enzymes was the same. Both reductases were immunologically identical by double immunodiffusion and rocket immunoelectrophoresis. Pig kidney aldehyde reductase (minor form) had 50% of the specific activity of pig kidney aldehyde reductase when tested with a variety of aldehyde substrates. Michaelis constants of both enzymes for these substrates and for NADPH were similar, but values for kcat. and kcat./Km indicated that catalytically pig kidney aldehyde reductase was the more efficient enzyme. Typical aldehyde reductase inhibitors, such as phenobarbital and sodium valproate, had the same effect on both enzymes. It was concluded that pig kidney aldehyde reductase (minor form) is an enzymically active cleavage product of pig kidney aldehyde reductase which is formed when the latter is purified in the absence of the metalloproteinase inhibitor EDTA.

| S-EPMC1154136 | biostudies-other

Investigation of the arylnitroso reductase activity of pig liver aldehyde reductase.

Project description:The reduction of p-nitroso-N-dimethylaniline, p-nitroso-N-diethylaniline, p-nitrosophenol and p-nitroso-N-phenylaniline with NADPH in the presence of aldehyde reductases 1 and 2 is described. The reactivity of these nitroso substrates is increased by hydrophobic substituents and those promoting OH- elimination from the molecule of the reduced substrate. NN-Dimethylbenzoquinonedi-iminium cation was proved to be the reaction product formed from p-nitroso-N-dimethylaniline. The kinetics of the reduction of p-nitroso-N-dimethylaniline catalysed with aldehyde reductase 1 are rather complex at pH 7, and the preferred-pathway mechanism is probably involved. The reaction sequence approaches the ordered pattern at pH 8.5. It was shown that NADPH in equilibrium NADP+ recyclization proceeds in the presence of NADP+, p-nitroso-N-dimethylaniline, cyclohexanol and aldehyde reductase 1, the alcohol oxidation being the slowest step in this reaction. However, the rate of cyclohexanol oxidation surpasses that of the dissociation of NADPH from the enzyme.

| S-EPMC1146718 | biostudies-other

Bovine lens aldehyde dehydrogenase. Kinetics and mechanism.

Project description:Bovine lens cytoplasmic aldehyde dehydrogenase exhibits Michaelis-Menten kinetics with acetaldehyde, glyceraldehyde 3-phosphate, p-nitrobenzaldehyde, propionaldehyde, glycolaldehyde, glyceraldehyde, phenylacetylaldehyde and succinic semialdehyde as substrates. The enzyme was also active with malondialdehyde, and exhibited an esterase activity. Steady-state kinetic analyses show that the enzyme exhibits a compulsory-ordered ternary-complex mechanism with NAD+ binding before acetaldehyde. The enzyme was inhibited by disulfiram and by p-chloromercuribenzoate, and studies with with mercaptans indicated the involvement of thiol groups in catalysis.

| S-EPMC1152404 | biostudies-other

Kinetics of alkaline phosphatase from pig kidney. Mechanism of activation by magnesium ions.

Project description:The mechanism of activation of alkaline phosphatase (EC 3.1.3.1) from pig kidney by Mg(2+) ions was investigated with the aid of kinetic measurements. Mg(2+) ions are essential for enzyme activity. The following model (Scheme 1 of the text) for the reaction of enzyme, substrate and Mg(2+) ions was derived: [Formula: see text] The binding of the substrate to the enzyme is independent of the binding of the activator, and vice versa. Mg(2+) must therefore play a part in the substrate decomposition. It is not possible to determine whether the Mg(2+) ions are involved directly in the catalytic process, or whether they act as regulatory effectors. Because of the strong affinity existing between the alkaline phosphatase and Mg(2+), it is necessary to adjust the metal-ion concentration with the aid of a metal buffer. In the Appendix the necessary equations are derived for calculating the concentration of free metal ions in a system with several different metal ions. A FORTRAN IV program for solving these equations and for graphic presentation of the results has been deposited as Supplementary Publication SUP 50030 at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS 23 7 BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1973), 131, 5.

| S-EPMC1168072 | biostudies-other

The kinetic mechanism of the major form of ox kidney aldehyde reductase with D-glucuronic acid.

Project description:The steady-state kinetics of the major form of ox kidney aldehyde reductase with d-glucuronic acid have been determined at pH7. Initial rate and product inhibition studies performed in both directions are consistent with a Di-Iso Ordered Bi Bi mechanism. The mechanism of inhibition by sodium valproate and benzoic acid is shown to involve flux through an alternative pathway.

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Aldehyde reductase is a major protein associated with 3-deoxyglucosone reductase activity in rat, pig and human livers.

Project description:3-Deoxyglucosone reductase activity in the extracts of rat, pig and human livers was potently inhibited by aldehyde reductase inhibitors. The major species of 3-deoxyglucosone reductase purified from human and pig livers were biochemically and immunochemically identical with aldehyde reductase. The two enzymes and rat liver aldehyde reductase exhibited higher catalytic efficiency for 3-deoxyglucosone than for D-glucuronate, a representative substrate of aldehyde reductase.

| S-EPMC1151532 | biostudies-other

Isolation from pig lens of two proteins with dihydrodiol dehydrogenase and aldehyde reductase activities.

Project description:Dimeric and monomeric proteins containing dihydrodiol dehydrogenase and aldehyde reductase activities were purified from pig lens. The dimeric enzyme of Mr 65,000 specifically oxidized the trans-dihydrodiols of naphthalene and benzene with NADP+ as a strict cofactor, and reduced alpha-diketones, aromatic aldehydes and glyceraldehyde with NADPH as a cofactor. The monomeric enzyme of Mr 35,000, although identical with aldose reductase, oxidized the trans-dihydrodiol of naphthalene at a pH optimum of 7.6. These results suggest that the two enzymes are involved in the pathogenesis of naphthalene cataract.

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