Rabbit liver alcohol dehydrogenase: purification and properties.
Alcohol dehydrogenase [EC 1.1.1.1] was purified to homogeneity from rabbit liver by water extraction, DEAE-cellulose treatment, affinity chromatography on 5'-AMP-Sepharose and gel filtration on Sephadex G-150 using dithiothreitol as a stabilizer. The purified enzyme has an estimated molecular weight of 72,000 and consists of two subunits with a molecular weight of about 36,000 each. The enzyme contains 4 g-atoms of zinc and 18 sulfhydryl groups per mol of protein and exhibits maximal activity at pH 10.8, with a second maximum at pH 7.5. The apparent Km values for ethanol and NAD+ are 0.45 mM and 53.19 microM, respectively, at pH 10.8 and 3.33 mM and 6.94 microM, respectively, at pH 7.5. The enzyme oxidizes ethanol most readily among the aliphatic alcohols studied and has very low substrate specificity for methanol. Among steroid alcohols, 5 beta-androstan-3 beta-ol-17-one serves as a substrate for the enzyme. Pyrazole and 4-methylpyrazole (which are well known alcohol dehydrogenase inhibitors), sulfhydryl reagents, heavy metal ions and metal-chelating agents inactivate the enzyme.
Hoshino
T
T
Ishiguro
I
I
Ohta
Y
Y
D000426:Alcohol Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002846:Chromatography, Affinity; D000077604:Fomepizole; D005978:Glutathione / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D011720:Pyrazoles / Q000494:pharmacology; D011817:Rabbits; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D008625:Tiopronin / Q000494:pharmacology; D015032:Zinc / Q000032:analysis
D000596:Amino Acids; D011720:Pyrazoles; D013439:Sulfhydryl Reagents; C031280:pyrazole; D000077604:Fomepizole; D008625:Tiopronin; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase; D005978:Glutathione; D015032:Zinc