Carroll Joe, Fearnley Ian M, Shannon Richard J, Hirst Judy, Walker John E
Medical Research Council Dunn Human Nutrition Unit, The Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 2XY, United Kingdom.
Mol Cell Proteomics. 2003 Feb;2(2):117-26. doi: 10.1074/mcp.M300014-MCP200. Epub 2003 Feb 22.
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 Ibeta, 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.
从牛心线粒体中纯化得到的复合物I是一种多亚基膜结合装配体。过去，已证明其七个亚基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，一条臂位于膜平面内，另一条臂与线粒体基质中的膜平面正交。使用轻度离液剂，完整的复合物已被解析为各种亚复合物。亚复合物Ilambda代表外在臂，亚复合物Ialpha由亚复合物Ilambda加上膜臂的一部分组成，亚复合物Ibeta是膜臂的另一个主要部分。完整的复合物和这三个亚复合物已进行了广泛的重新分析。它们的亚基已通过三种独立方法（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图谱和串联质谱进行分析。许多完整亚基的质量也已通过电喷雾电离质谱测量，并提供了有关翻译后修饰的有价值信息。已确认复合物I中存在已知的35个核编码亚基，并检测到另外四个核编码亚基。亚基B16.6、B14.7和ESS分别在亚复合物Ilambda的SDS-PAGE分析、完整复合物的二维凝胶分析以及亚复合物Ibeta的HPLC分析中被发现。尽管进行了多次尝试，但在亚复合物Ibeta的HPLC分析中也检测到的第四个新亚基（质量为10,566±2 Da）仍未获得序列信息。牛复合物不太可能还有未被发现的亚基。因此，完整的酶是一个由46个亚基组成的复合物，假设复合物中每个亚基有一个拷贝，其质量为980 kDa。
