PURIFICATION AND MOLECULAR-PROPERTIES OF MOUSE ALCOHOL-DEHYDROGENASE ISOZYMES
PURIFICATION AND MOLECULAR-PROPERTIES OF MOUSE ALCOHOL-DEHYDROGENASE ISOZYMES
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DOI：
10.1111/j.1432-1033.1983.tb07807.x
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发表时间：
1983-01-01
期刊：
影响因子：
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通讯作者：
HOLMES, RS
中科院分区：
文献类型：
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作者：
ALGAR, EM;SEELEY, TL;HOLMES, RS
小鼠肝(A2、B2)和胃(C2)乙醇脱氢酶同工酶经三嗪-染料亲和层析纯化为均一酶。这些酶是具有相似但不同亚基大小的二聚体，通过十二烷基硫酸钠/聚丙烯酰胺凝胶电泳法测定：A，43,000，B，39,000和C，47,000。锌分析和1，10-二氮杂菲抑制研究表明，A和C亚基各含有2个锌原子，其中至少有1个参与催化作用；B亚基可能含有一个非催化锌原子。同工酶表现出明显不同的动力学特征。A2对乙醇的Km值为0.15 mm，具有广泛的底物专一性，Km值随着链长的增加而急剧下降；C2对酒精也表现出这种广泛的专一性，但对乙醇的Km值为232 mm。这些同工酶也表现出广泛的底物特异性，如乙醛还原酶。对于所考察的醛，B2没有检测到作为醛还原酶的活性，并且仅在高浓度(~gt；0.5M)下使用乙醇作为底物。该同工酶对中链醇表现出较低的Km和较高的Vmax。免疫学研究表明，A2在免疫学上不同于B2和C2同工酶。体外分子杂交研究没有证据表明酒精脱氢酶亚基之间存在关联。这一结果证实了与至少3个编码小鼠酒精脱氢酶结构基因一致的遗传分析，并证实了主要肝脏同工酶(A2)在乙醇代谢中的作用。
Alcohol dehydrogenase isozymes from mouse liver (A2 and B2) and stomach (C2) tissues were purified to homogeneity using triazine-dye affinity chromatography. The enzymes were dimers with similar but distinct subunit sizes, as determined by SDS[sodium dodecylsulfate]/polyacrylamide gel electrophoresis: A, 43,000, B, 39,000 and C, 47,000. Zn analyses and 1,10-phenanthroline inhibition studies indicated that the A and C subunits each contained 2 atoms of Zn, with at least 1 being involved catalytically; the B subunit probably contained a single noncatalytic Zn atom. The isozymes exhibited widely divergent kinetic characteristics. A2 exhibited a Km value for ethanol of 0.15 mM and a broad substrate specificity, with Km values decreasing dramatically with an increase in chain length; C2 also exhibited this broad specificity for alcohols but showed a Km value of 232 mM for ethanol. These isozymes also showed broad substrate specificities as aldehyde reductases. B2 showed no detectable activity as an aldehyde reductase for the aldehydes examined, and used ethanol as substrate only at high concentrations (> 0.5 M). The isozyme exhibited low Km and high Vmax values with medium-chain alcohols. Immunological studies showed that A2 was immunologically distinct from the B2 and C2 isozymes. In vitro molecular hybridization studies gave no evidence for association between the alcohol dehydrogenase subunits. The results confirmed genetic analyses which were consistent with at least 3 structural genes encoding alcohol dehydrogenase in the mouse and confirmed the role of the major liver isozyme (A2) in ethanol metabolism.