Resolution of mitochondrial NADH dehydrogenase and isolation of two iron-sulfur proteins.
Resolution of mitochondrial NADH dehydrogenase and isolation of two iron-sulfur proteins.
复制标题
线粒体 NADH 脱氢酶的解析和两种铁硫蛋白的分离。
DOI：
10.1021/bi00532a027
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发表时间：
1982
期刊：
影响因子：
2.9
通讯作者：
Ohnishi,T
中科院分区：
文献类型：
--
作者：
Ragan,CI;Galante,YM;Hatefi,Y;Ohnishi,T
方法NADH脱氢酶的拆分。NADH脱氢酶亚基分离的标准程序与Davis&Hatefi(1971)最初提出的琥珀酸脱氢酶亚基分离的标准程序基本相同。脱氢酶溶于50 mM Tris-HCl，pH 7.8，含2 mM二硫苏糖醇，最终蛋白浓度为2~4 mg/mL。然后加入4M的Cl3CCOONa溶液，最终浓度为0.2M。将溶液冷冻在液态氮气中，并立即在室温下解冻。冰冻和解冻过程重复进行。然后通过加入饱和和中和的(NH4)2S04进行分级，使其达到0.12、0.22和0.35的饱和度。每次加入(NH4)2S04后，溶液静置10分钟。以90000g离心5min，除去沉淀蛋白。除特别说明外，所有操作均在0-4℃下进行。所有的分析和测量都是对新鲜溶解的NADH脱氢酶和新制备的NADH脱氢酶亚单位进行的。分析方法。对铁(Doig&Ziegler，1962)、酸不稳定硫化物(Fogo&Popowski，1949)和黄素(Hatefi&Stempel，1969)进行了测定。用Bensadoun&Weinstein(1976)方法测定蛋白质，以避免二硫苏糖醇和
MethodsResolution of NADH Dehydrogenase. The standard pro-cedure for separation of NADH dehydrogenase subunits was basically the same as that originally developed by Davis & Hatefi (1971) for separating the subunits of succinate de-hydrogenase. The dehydrogenase was dissolved in 50 mM Tris-HCl, pH 7.8, containing 2 mM dithiothreitol, to give a final proteinconcentration of 2-4 mg/mL. A 4 M solution of Cl3CCOONa was then added to give a final concentration of 0.2 M. The solution was frozen in liquid N2 and immediately thawed at room temperature. The freezing and thawing was repeated. The solution was then fractionated by addition of saturated and neutralized (NH4) 2S04 to 0.12, 0.22, and 0.35 saturation. After each addition of (NH4) 2S04, the soltion was allowed to stand for 10 min. and precipitated protein was removed by centrifugation at 90000g for 5 min. Except where indicated, all operations were carried out at 0-4 C. All assays and measurements were carried out on freshly dissolved NADH dehydrogenase and on freshly preparedNADH dehydrogenase subunits. Analytical Methods. Iron (Doeg & Ziegler, 1962), acid-labile sulfide (Fogo & Popowski, 1949), and flavin (Hatefi & Stempel, 1969) were assayed as indicated in thereferences. The protein was measured by the method of Bensadoun & Weinstein (1976) to avoid interference by dithiothreitol and