Kinetic and electrophoretic properties of native and recombined isoenzymes of human liver alcohol dehydrogenase.
Kinetic and electrophoretic properties of native and recombined isoenzymes of human liver alcohol dehydrogenase.
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DOI：
10.1021/bi00277a017
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发表时间：
1983-04
期刊：
影响因子：
2.9
通讯作者：
W. F. Bosron;L. Magnes;T. Li
中科院分区：
文献类型：
--
作者：
W. F. Bosron;L. Magnes;T. Li
通过亲和层析和离子交换层析，已经从单个人肝脏中分离出十种电泳上不同的乙醇脱氢酶分子形式。解离重组后的淀粉凝胶电泳模式与α、β 1、γ 1和γ 2亚基随机组合成6个异二聚体和4个同聚体同工酶的假设一致。同型二聚体同工酶，α α, β 1 β 1， γ 1 γ 1和γ 2 γ 2，在动力学性质上有很大的不同。在pH为7.5时，乙醇的β 1 β 1的Km值为0.049 mM， α α的Km值为4.2 mM。在pH为7.5时，形式γ 1 γ 1和γ 2 γ 2不服从Michaelis-Menten动力学，但与Hill系数分别为0.54和0.55和[S]0.5值分别为1.0和0.63 mM表现出负协同作用。然而，所有同功酶在pH为10.0时均表现出Michaelis-Menten氧化动力学，Km值在1.5 ~ 3.2 mM之间。在pH为7.5和10.0时，β 1 β 1的最大比活性明显低于其他三种同型二聚体。pH值为7.5时，四种同型二聚体对NAD+的Km值为7.4 ~ 13 μ m，对NADH的Km值为6.4 ~ 33 μ m。NADH的Ki值为0.19 ~ 1.6 μ m。在pH 7.5条件下，由α β 1分离和重组制备的α α和β 1 β 1的动力学性质与天然同型二聚体相似。分别由解离和重组的α γ 1和β 1 γ 2制备的形式γ 1 γ 1和γ 2 γ 2与希尔系数表现出负协同性，与各自的天然同型二聚体相似。
Ten, electrophoretically distinct, molecular forms of alcohol dehydrogenase have been isolated from a single human liver by affinity and ion-exchange chromatography. The starch gel electrophoresis patterns after the dissociation-recombination of the forms are consistent with the hypothesis that they arise from the random combination of alpha, beta 1, gamma 1, and gamma 2 subunits into six heterodimeric and four homodimeric isoenzymes. Large differences in kinetic properties are observed for the homodimeric isoenzymes, alpha alpha, beta 1 beta 1, gamma 1 gamma 1, and gamma 2 gamma 2. At pH 7.5, the Km value of beta 1 beta 1 for ethanol is 0.049 mM and that of alpha alpha is 4.2 mM. Forms gamma 1 gamma 1 and gamma 2 gamma 2 do not obey Michaelis-Menten kinetics at pH 7.5 but exhibit negative cooperativity with Hill coefficients of 0.54 and 0.55 and [S]0.5 values of 1.0 and 0.63 mM, respectively. However, all isoenzymes display Michaelis-Menten kinetics for ethanol oxidation at pH 10.0 with Km values ranging from 1.5 to 3.2 mM. The maximum specific activity of beta 1 beta 1 is considerably lower than that of the other three homodimers at both pH 7.5 and 10.0. The Km values of the four homodimers for NAD+ at pH 7.5 range from 7.4 to 13 microM and those for NADH, from 6.4 to 33 microM. Ki values for NADH range from 0.19 to 1.6 microM. At pH 7.5, the kinetic properties of alpha alpha and beta 1 beta 1, prepared in vitro from dissociated and recombined alpha beta 1, are similar to those of the native homodimers. The forms gamma 1 gamma 1 and gamma 2 gamma 2, prepared from dissociated and recombined alpha gamma 1 and beta 1 gamma 2, respectively, exhibit negative cooperativity with Hill coefficients that are similar to those seen with the respective native homodimers.