Analysis of the subunit composition of complex I from bovine heart mitochondria
Analysis of the subunit composition of complex I from bovine heart mitochondria
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DOI：
10.1074/mcp.m300014-mcp200
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发表时间：
2003-02-01
影响因子：
7
通讯作者：
Walker, JE
中科院分区：
文献类型：
--
作者：
Carroll, J;Fearnley, IM;Walker, JE
从牛心脏线粒体中纯化的复合物 I 是一种多亚基膜结合组装体。过去，它的7个亚基被证明是线粒体基因组的产物，并鉴定出35个核编码亚基。该复合物呈 L 形，一只臂位于膜平面上，另一臂位于线粒体基质中与其正交。使用轻度离液去污剂，完整的复合物已分解成各种子复合物。子复合物 Ilambda 代表外源臂，子复合物 Ialpha 由子复合物 Ilambda 加上部分膜臂组成，子复合物 Ibeta 是膜臂的另一个重要部分。完整的复合体和这三个子复合体已经接受了广泛的重新分析。它们的亚基已通过三种独立的方法（一维 SDS-PAGE、二维等电聚焦/SDS-PAGE 和反相高压液相色谱 (HPLC)）分离，并通过胰蛋白酶肽质量指纹法和串联质谱法进行分析。许多完整亚基的质量也通过电喷雾电离质谱法进行了测量，并提供了有关翻译后修饰的有价值的信息。复合物 I 中已知的 35 个核编码亚基的存在已得到证实，并且还检测到了另外 4 个核编码亚基。亚基 B16.6、B14.7 和 ESSS 分别在子复合物 Ilambda 的 SDS-PAGE 分析、完整复合物的二维凝胶分析和子复合物 1,6 的 HPLC 分析中发现。尽管进行了多次尝试，但尚未获得在子复合物 Ibeta 的 HPLC 分析中检测到的第四个新亚基（质量 10,566+/-2 Da）的序列信息。不可能有更多的牛复合体亚基未被发现。因此，完整的酶是由 46 个亚基组成的复合体，假设复合体中每个亚基都有一个副本，则其质量为 980 kDa。
Complex I purified from bovine heart mitochondria is a multisubunit membrane-bound assembly. In the past, seven of its subunits were shown to be products of the mitochondrial genome, and 35 nuclear encoded subunits were identified. The complex is L-shaped with one arm in the plane of the membrane and the other lying orthogonal to it in the mitochondrial matrix. With mildly chaotropic detergents, the intact complex has been resolved into various subcomplexes. Subcomplex Ilambda represents the extrinsic arm, subcomplex Ialpha consists of subcomplex Ilambda plus part of the membrane arm, and subcomplex Ibeta is another substantial part of the membrane arm. The intact complex and these three subcomplexes have been subjected to extensive reanalysis. Their subunits have been separated by three independent methods (one-dimensional SDS-PAGE, two-dimensional isoelectric focusing/SDS-PAGE, and reverse phase high pressure liquid chromatography (HPLC)) and analyzed by tryptic peptide mass fingerprinting and tandem mass spectrometry. The masses of many of the intact subunits have also been measured by electrospray ionization mass spectrometry and have provided valuable information about post-translational modifications. The presence of the known 35 nuclear encoded subunits in complex I has been confirmed, and four additional nuclear encoded subunits have been detected. Subunits B16.6, B14.7, and ESSS were discovered in the SDS-PAGE analysis of subcomplex Ilambda, in the two-dimensional gel analysis of the intact complex, and in the HPLC analysis of subcomplex 1,6, respectively. Despite many attempts, no sequence information has been obtained yet on a fourth new subunit (mass 10,566+/-2 Da) also detected in the HPLC analysis of subcomplex Ibeta. It is unlikely that any more subunits of the bovine complex remain undiscovered. Therefore, the intact enzyme is a complex of 46 subunits, and, assuming there is one copy of each subunit in the complex, its mass is 980 kDa.