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
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
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DOI：
10.1021/bi00319a026
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发表时间：
1984-01-01
期刊：
影响因子：
2.9
通讯作者：
LI, TK
中科院分区：
文献类型：
--
作者：
YIN, SJ;BOSRON, WF;LI, TK
从具有纯合ADH 2 2-2和杂合ADH 2 1-2表型的日本肝脏中分离出4种乙醇脱氢酶[ADH]同工酶，其乙醇氧化的“非典型”最适pH为8.8。从纯合肝脏纯化的3种同工酶解离-重组后的琼脂糖凝胶等电聚焦图表明它们是β 2 β 2，α。β 2和β 2 γ 1。通过琼脂糖-己烷-AMP亲和层析从具有杂合表型的肝中纯化的第4种同工酶通过解离-重组研究鉴定为β 2 β 1。3种异源二聚体β 2 β 1，α 2的动力学性质β 2和β 2 γ 1的亚基在各自同源二聚体的亚基之间是中间的，表明这2个亚基独立地起作用。产物抑制研究表明，β 2 β 2服从有序的顺序机制，α 2 β 2也是如此。α，β 1 β 1、γ 1 γ 1和γ 2 γ 2同二聚体，其具有在pH 10.0-10.5的乙醇氧化的“典型”最佳pH。β 2 β 2的动力学常数与其它同二聚体的动力学常数基本上不同。在pH7.5时，β 2 β 2的乙醇氧化的Vmax比α高5-40倍。α，β 1 β 1、γ 1 γ 1和γ 2 γ 2。NAD+和NADH的β 2 β 2的Km和Ki值也显著高于其它同二聚体的Km和Ki值。“东方”β 2 β 2同二聚体和其它人肝同工酶之间的动力学差异可以通过在β 2 β 2的辅酶结合位点中用较弱的碱组氨酸取代精氨酸-47残基来解释，所述精氨酸-47残基出现在迄今为止检测的所有其它哺乳动物醇脱氢酶中。
Four alcohol dehydrogenase [ADH] 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 1-2 phenotypes. Agarose gel isoelectric focusing patterns after dissociation-recombination of 3 isoenzymes purified from the homozygous livers indicate that they are .beta.2.beta.2, .alpha..beta.2 and .beta.2.gamma.1. A 4th 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 3 heterodimers, .beta.2.beta.1, .alpha..beta.2 and .beta.2.gamma.1, are intermediate between those of the respective homodimers, suggesting that the 2 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. The kinetic differences between the "Oriental" .beta.2.beta.2 homodimer and other human liver isoenymzes can be explained by the substitution of a weaker base, histidine, in the coenzyme binding site of .beta.2.beta.2 for the arginine-47 residue that appears in all other mammalian alcohol dehydrogenases thus far examined.