脱辅基细胞色素c定向于线粒体的转运特异性研究

Study on the Specificity of Targeting of Apocytochrome c to Mitochondria

用分离纯化的完整线粒体和部分细胞器组分,初步研究了脱辅基细胞色素c在细胞内转运的特异性。完整线粒体用差速离心和密度梯度离心的方法,从幼龄鸡心肌组织中获得,对胞内几种细胞器标志酶比活力的测量表明,纯化的线粒体单胺氧化酶活力提高25.6倍,腺苷酸激酶活力提高3.59倍,细胞色素c氧化酶活力提高5.48倍,外膜完整性达90%以上,呼吸控制率大于20。以上数据表明该纯化的线粒体受胞内其它囊泡成分污染少,外膜完整并具有较高的氧化磷酸化偶联效率;在纯化线粒体的同时,得到另两种细胞器组分-内质网和溶酶体囊泡。体外转录翻译的apo.c与上述几个组分的结合实验表明,完整线粒体与apo.c的结合能力明显高于其它组分。

The specificity of translocation of apocytochrome c within a cell was studied by measurement of 35 S labeled apocytochrome c binding to isolated mitochondria and some other organelles.Chicken heart mitochondria were isolated and purified by a combination of differential and gradient centrifugation.The marker enzymes analysis showed that the mitochondria were highly pure,the specific activities of monoamine oxidase,adenylate kinase and cytochrome c oxidase increased 25.6,3.59 and 5.48 folds,respectively;its intactness of outer membrane was over 90%,and respiratory control ratio(RCR) over 20.The thin section electron micrograph showed the typical ultrastructure of the mitochondria,in which the features of the outer and inner membranes were distinguishable.In order to explore the specificity of targeting of apocytochrome c to mitochondria,the ability of apocytochrome c binding to different fractions obtained in the procedure of the isolation of mitochondria was examined.The mitochondria exhibited the highest binding to apocytochrome c.