Purification and comparative studies of alcohol dehydrogenases (original) (raw)
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Separation and partial characterization of multiple forms of rat liver alcohol dehydrogenase
Archives of Biochemistry and Biophysics, 1983
Rat liver alcohol dehydrogenase was purified and four isoenzyme forms, demonstrated by starch gel electrophoresis, were separated by 0-(carboxymethyl)-cellulose chromatography. Each of the isoenzymes had a distinct isoelectric point. All isoenzymes were active with both ethanol (or acetaldehyde) and steroid substrates, and had similar Michaelis-Menten constants for each of the substrates and coenzymes studied. The three isoenzymes with the lowest migration toward the cathode exhibited the same pH optimum of 10.7 for ethanol oxidation, a greater activity with 5@androstan-3@oll'l-one than with ethanol as a substrate, and an unchanged electrophoretic mobility following storage in the presence of 100 PM dithiothreitol. By contrast the isoenzyme with the highest mobility toward the cathode exhibited a pH optimum of 9.5 for ethanol oxidation, a low steroid/ethanol ratio of activity, and converted to the migrating pattern of the two isoenzymes with intermediate mobility when stored. The similarities between the isoenzymes of rat liver alcohol dehydrogenase differ considerably from differences in substrate specificity exhibited by isoenzymes of horse liver alcohol dehydrogenase.
Alcohol-oxidizing enzymes from various organisms
Comparative Biochemistry and Physiology Part B: Comparative Biochemistry, 1978
1. Enzymes that catalyse the oxidation of aliphatic alcohols to aldehydes are reviewed. 2. Special attention is given to phenazine methosulphate-linked alcohol dehydrogenases from bacteria and to flavin-containing alcohol oxidases from yeasts, moulds and higher plants.
Purification and molecular properties of mouse alcohol dehydrogenase isozymes
European Journal of Biochemistry, 1983
Alcohol dehydrogenase isozymes from mouse liver (A, and B,) and stomach (C,) tissues have been purified to homogeneity using triazine-dye affinity chromatography. The enzymes are dimers with similar but distinct subunit sizes, as determined by SDS/polyacrylamide gel electrophoresis: A, 43000; B, 39000, and C, 47000. Zinc analyses and 1,lO-phenanthroline inhibition studies indicated that the A and C subunits each contained two atoms of zinc, with at least one being involved catalytically, whereas the B subunit probably contained a single non-catalytic zinc atom. The isozymes exhibited widely divergent kinetic characteristics. A, exhibited a K,,, value for ethanol of 0.15 mM and a broad substrate specificity, with K, values decreasing dramatically with an increase in chain length; C, also exhibited this broad specificity for alcohols but showed a K , value of 232mM for ethanol. These isozymes also showed broad substrate specificities as aldehyde reductases. In contrast, B, showed no detectable activity as an aldehyde reductase for the aldehydes examined, and used ethanol as substrate only at very high concentrations (>0.5M). The isozyme exhibited low K, and high V,,, values, however, with medium-chain alcohols. Immunological studies showed that A, was immunologically distinct from the B, and C, isozymes. In vitro molecular hybridization studies gave no evidence for association between the alcohol dehydrogenase subunits. The results confirm genetic analyses [Holmes, Albanese, Whitehead and Duley (1981) J. Exp. Zool. 215, 151 -1 5 7 which are consistent with at least three structural genes encoding alcohol dehydrogenase in the mouse and confirm the role of the major liver isozyme (A,) in ethanol metabolism.
Mechanism of the Alcohol Dehydrogenases from Yeast and Horse Liver
European Journal of Biochemistry, 1971
Studies of the alcohol-acetaldehyde interchange, in the presence of analogues of NAD+ and brought about by yeast and horse liver alcohol dehydrogenases, have not provided any evidence in favour of the direct participation of the enzyme in the hydrogen transfer step. A new preparation of 1,4,5,6-tetrahydro-nicotinamide adenine dinucleotide is described. This analogue has been found to be a good competitive inhibitor for both enzymes, thus demonstrating the importance of fixation of the enzyme by a hydrogen bond to the group present in C-3 of the nicotinamide nucleus.
European Journal of Biochemistry, 1990
The major ethanol-active form of chicken liver alcohol dehydrogenase was characterized. The primary structure was determined by peptide analysis and, to a large part, was also deduced by cDNA analysis of a near full-length cDNA clone. The latter was detected by screening of a chicken liver cDNA library with antibodies raised against the purified dehydrogenase. The structure shows that the avian enzyme exhibits characteristics of the complex mammalian alcohol dehydrogenase system, tracing its origin and divergence, and allowing functional correlations. The chicken protein analyzed proves to be a class I alcohol dehydrogenase, with 74% residue identity to y chains of the human enzyme, a K, for ethanol of 0.5 mM and a Ki for 4-methyl pyrazole of 2.5 pM. Relationships to the other two classes are non-identical; residue exchanges towards the human classes increase in the order Correspondence to H. Jornvall,
Biochemistry, 1981
The alcohol dehydrogenase (ADH) of squirrel monkey liver can be purified and separated into pyrazole-insensitive and pyrazole-sensitive isoenzymes by affinity chromatography. This is the first demonstration of two functionally distinct classes of ADH in a species other than man. The inhibition of the two enzyme fractions by 4-methylpyrazole is analogous to that observed for the corresponding isoenzymes of human liver. Similarly, the substrate specificity of the pyrazole-insensitive form is more limited and its K, for ethanol
Purification of Alcohol Dehydrogenase Enzyme from Chicken Liver and Immobilization Onto Florisil
Asian Journal of Research in Biochemistry
The enzyme alcohol dehydrogenase (ADH) is a dimeric enzyme in which each of its subunits has a Zn2+ metal-containing catalytic domain and a cofactor binding domain. This enzyme converts alcohol into an aldehyde. In this article, the activity of the enzyme was investigated by applying the immobilization process directly to the alcohol dehydrogenase enzyme purified and activated florisil from the chicken liver. For this purpose, homogenization of chicken liver was achieved and its supernatants were separated by applying the ultracentrifugation process to the resulting homogenate. Then, % ammonium precipitation, dialysis, and ion exchange chromatography processes were performed, respectively. As a result of these processes, the hepatic alcohol dehydrogenase was purified 150.3 times compared to the coarse homogenate, and the specific activity of the enzyme was determined to be 0.631 U/mg protein. The activity of the enzyme directly immobilized was found to be 0.034 U/mg protein.
Biochemical Genetics, 1979
Chinese hamster cells were purified using gel filtration, ion-exchange, and affinity-column chromatography. Both enzymes exhibited the same isozyme band patterns on electrophoresis and isoelectric focusing. Physicochemical properties of the two enzymes such as pH and temperature optima, Km values, and temperature stability were found to be the same within the experimental errors. The genetic significance of these findings is discussed.
A convenient large scale preparation of the EE isozyme of horse liver alcohol dehydrogenase
Analytical Biochemistry, 1979
A modified method suitable for the preparation of large amounts of very pure EE isozyme of horse liver alcohol dehydrogenase is described. The enzyme was purified from fresh horse livers by ammonium sulfate precipitation, ion-exchange, and gel-filtration chromatography. The method has been used for large as well as small-scale purifications: a 5-g yield of pure EE isozyme is possible from 14 kg of fresh liver at 140% of the specific activity previously reported. Isoelectric focusing revealed a single band stained with Coomassie blue, and there was essentially no activity using an assay for steroid-active subunits. If stored under sterile conditions the purified enzyme is stable for 6 months and should be particularly suitable for investigations of potential site heterogeneity of the enzyme, an area of current controversy.