1. Human liver alcohol dehydrogenase: purification and kinetic characterization of the beta 2 beta 2, beta 2 beta 1, alpha beta 2, and beta 2 gamma 1 "Oriental" isoenzymes.
- Author
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Yin SJ, Bosron WF, Magnes LJ, and Li TK
- Subjects
- Alcohol Dehydrogenase, Alcohol Oxidoreductases genetics, Alcohol Oxidoreductases metabolism, Animals, Chromatography, Cricetinae, Ethanol, Humans, Hydrogen-Ion Concentration, Isoelectric Focusing, Isoenzymes genetics, Isoenzymes metabolism, Japan, Kinetics, Macromolecular Substances, Oxidation-Reduction, Phenotype, Alcohol Oxidoreductases isolation & purification, Isoenzymes isolation & purification, Liver enzymology
- Abstract
Four alcohol dehydrogenase isoenzymes with "atypical" pH optima for ethanol oxidation at 8.8 were isolated from Japanese livers with the homozygous ADH2 2-2 and the heterozygous ADH2 2-1 phenotypes. Agarose gel isoelectric focusing patterns after dissociation--recombination of three isoenzymes purified from the homozygous livers indicate that they are beta 2 beta 2, alpha beta 2, and beta 2 gamma 1. A fourth isoenzyme, purified from livers with the heterozygous phenotype by agarose-hexane--AMP affinity chromatography, was identified as beta 2 beta 1 by dissociation-recombination studies. The kinetic properties of the three heterodimers, beta 2 beta 1, alpha beta 2, and beta 2 gamma 1, are intermediate between those of the respective homodimers, suggesting that the two subunits act independently. Product inhibition studies indicate that beta 2 beta 2 obeys an ordered sequential mechanism, as do the alpha alpha, beta 1 beta 1, gamma 1 gamma 1, and gamma 2 gamma 2 homodimers which have the "typical" pH optimum for ethanol oxidation at pH 10.0-10.5. The kinetic constants of beta 2 beta 2 differ substantially from those of the other homodimers. At pH 7.5, the Vmax for ethanol oxidation of beta 2 beta 2 is 5-40 times higher than that of alpha alpha, beta 1 beta 1, gamma 1 gamma 1, and gamma 2 gamma 2. The Km and Ki values of beta 2 beta 2 for NAD+ and NADH are also considerably higher than those of the other homodimers.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1984
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