Szewczyk A, Nałecz M J, Broger C, Wojtczak L, Azzi A
Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw.
Biochim Biophys Acta. 1987 Nov 19;894(2):252-60. doi: 10.1016/0005-2728(87)90194-0.
Submitochondrial particles were prepared from bovine heart mitochondria, solubilized with Triton X-114 in the presence of lipids and submitted to hydroxylapatite chromatography. The eluate obtained, containing a mixture of mitochondrial carriers, was processed further by affinity chromatography using as ligand p-aminophenylsuccinate coupled via a diazo bond to aminohexyl-Sepharose 4B. The activity of the dicarboxylate exchanger was measured after reconstitution into asolectin vesicles at each step of the purification procedure. All samples studied were found to display substrate and inhibitor specificity similar to those described for the dicarboxylate carrier in mitochondria. The specific activity of the final material eluted from the affinity column was found to be about 1000-times higher than that of the Triton X-114 extract of submitochondrial particles. SDS-polyacrylamide gel electrophoresis analysis of the affinity chromatography eluate showed the presence of only two polypeptides.
从牛心线粒体中制备亚线粒体颗粒，在脂质存在的情况下用 Triton X - 114 溶解，并进行羟基磷灰石色谱分析。所得洗脱液含有线粒体载体混合物，通过使用对氨基苯基琥珀酸盐作为配体，经重氮键偶联到氨基己基 - Sepharose 4B 上的亲和色谱进一步处理。在纯化过程的每个步骤中，将其重构到大豆卵磷脂囊泡中后，测定二羧酸交换体的活性。研究发现，所有研究的样品都表现出与线粒体中二羧酸载体所描述的底物和抑制剂特异性相似。从亲和柱洗脱的最终物质的比活性比亚线粒体颗粒的 Triton X - 114 提取物高约 1000 倍。亲和色谱洗脱液的 SDS - 聚丙烯酰胺凝胶电泳分析表明仅存在两种多肽。
