Purification and characterization of short-chain, medium-chain, and long-chain acyl-CoA dehydrogenases from rat liver mitochondria. Isolation of the holo- and apoenzymes and conversion of the apoenzyme to the holoenzyme.
Purification and characterization of short-chain, medium-chain, and long-chain acyl-CoA dehydrogenases from rat liver mitochondria. Isolation of the holo- and apoenzymes and conversion of the apoenzyme to the holoenzyme.
复制标题
DOI：
--
复制
发表时间：
1985-01
期刊：
影响因子：
--
通讯作者：
Y. Ikeda;K. Okamura-Ikeda;K. Tanaka
中科院分区：
文献类型：
--
作者：
Y. Ikeda;K. Okamura-Ikeda;K. Tanaka
采用DEAE-Sephadex A-50、羟基磷灰石、Matrex Gel Blue A、琼脂糖-己烷- coa和Bio-Gel A-0.5m层序层析，从大鼠肝脏线粒体中纯化出短链、中链和长链酰基辅酶A脱氢酶。研究了纯酰基辅酶a脱氢酶的分子、免疫学和催化性能。这三种酶的天然分子量分别为160,000、180,000和180,000。这三种酶的亚基分子量估计分别为41,000,45,000和45,000，这表明这些酶分别由四个大小相等的亚基组成。计算FAD含量为1 mol/mol亚基。短链酰基辅酶a脱氢酶与FAD的结合非常紧密，而中链酰基辅酶a和长链酰基辅酶a脱氢酶与FAD的结合不太紧密。中链和长链酰基辅酶a脱氢酶也被纯化为无fad的载脂蛋白酶。经FAD孵育，酶解转化为全酶。这三种酰基辅酶a脱氢酶在免疫学上彼此不同，即针对单个酶产生的抗体是单特异性的，不与任何其他酰基辅酶a脱氢酶交叉反应。我们的三种酶的制剂显示出底物特异性（如Vappmax和Kappmax所定义的），比以前从其他来源分离的制剂具有更高的特异性。底物特异性也通过测量线粒体声波中的活性来评估，在选择性地用单个单特异性抗体沉淀每种酶后。短链酰基辅酶a脱氢酶几乎完全脱氢了丁基辅酶a，而中链酰基辅酶a脱氢酶主要脱氢了C6-C10酰基辅酶a。用长链酰基辅酶a脱氢酶对C14-C22酰基辅酶a进行脱氢。C24酰基辅酶a不能被这种酶脱氢。月桂酰辅酶a似乎被后两种酶共同脱氢。短链酰基辅酶a脱氢酶不能使支链酰基辅酶a脱氢。在电子转移黄蛋白或苯那嗪甲硫代硫酸钠存在的情况下，2-烯基辅酶a被鉴定为相应酶/酰基辅酶a反应的产物。
Short-chain, medium-chain, and long-chain acyl-CoA dehydrogenases were purified to homogeneity from rat liver mitochondria by sequential chromatography on DEAE-Sephadex A-50, hydroxyapatite, Matrex Gel Blue A, agarose-hexane-CoA, and Bio-Gel A-0.5m. Molecular, immunological, and catalytic properties of the pure acyl-CoA dehydrogenases were investigated. The native molecular weights of these three enzymes were 160,000, 180,000, and 180,000, respectively. The subunit molecular weights of the three enzymes were estimated to be 41,000, 45,000, and 45,000, respectively, indicating that these enzymes are each composed of four subunits of equal size. The FAD content was calculated to be 1 mol/mol of subunit. While FAD binding by short-chain acyl-CoA dehydrogenase was very tight, that by medium-chain acyl-CoA and long-chain acyl-CoA dehydrogenases was less tight. The medium- and long-chain acyl-CoA dehydrogenases were also purified to homogeneity as FAD-free apoenzymes. The apoenzymes were converted to the fully active holoenzymes by incubation with FAD. The three acyl-CoA dehydrogenases were immunologically distinct from each other, i.e. the antibodies raised against the individual enzymes were monospecific and did not cross-react with any other acyl-CoA dehydrogenases. Our preparations of the three enzymes exhibited substrate specificities (as defined in Vappmax and Kappmax) significantly more specific than those of the previous preparations isolated from other sources. The substrate specificities were assessed also by measuring the activities in mitochondrial sonicates after selectively precipitating each enzyme with their individual monospecific antibodies. Butyryl-CoA was almost exclusively dehydrogenated by short-chain acyl-CoA dehydrogenase while C6-C10 acyl-CoAs were mainly dehydrogenated by medium-chain acyl-CoA dehydrogenase. C14-C22 acyl-CoAs were exclusively dehydrogenated by long-chain acyl-CoA dehydrogenase. C24 acyl-CoAs were not dehydrogenated by this enzyme. Lauroyl-CoA appeared to be jointly dehydrogenated by the latter two enzymes. Branched-chain acyl-CoAs were not dehydrogenated by short-chain acyl-CoA dehydrogenase. In the presence of electron-transfer flavoprotein or phenazine methosulfate, 2-enoyl-CoAs were identified as products from the corresponding enzyme/acyl-CoA reactions.