Tong J, Margoliash E
Laboratory for Molecular Biology, Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois 60607, USA.
J Biol Chem. 1998 Oct 2;273(40):25695-702. doi: 10.1074/jbc.273.40.25695.
A highly efficient in vitro system was established for measuring by high performance liquid chromatography the formation of holocytochrome c by yeast mitochondria. Holocytochrome c formation required reducing agents, of which dithiothreitol was the most effective. With biosynthetically made, pure Drosophila melanogaster apocytochrome c and Saccharomyces cerevisiae mitochondria, the activity of cytochrome c heme lyase amounted to about 800 fmol min-1 mg-1 mitochondrial protein. The kinetics were typical Michaelis-Menten (Km approximately 1 nM), as were those of mitoplasts with broken outer membranes (Km approximately 3 nM). As tested with mitoplasts, holocytochromes c from a range of species were found to be competitive inhibitors of heme lyase at physiological concentrations, providing a mechanism for controlling this concentration in vivo. Apocytochrome c associated with yeast mitochondria in two phases of Kd approximately 2 x 10(-10) and 10(-8) M, respectively, whereas mitoplasts had lost the high affinity binding. A site-directed mutant of apocytochrome c (lysines 5, 7, and 8 replaced by glutamine, glutamic acid, and asparagine) was found to be converted to holocytochrome c (Km approximately 3.3 nM; maximal activity unchanged), even though the mutations completely eliminated the high affinity binding. Thus, the high affinity binding of apocytochrome c to mitochondria is not directly related to holocytochrome c formation.
建立了一种高效的体外系统，用于通过高效液相色谱法测量酵母线粒体中全细胞色素c的形成。全细胞色素c的形成需要还原剂，其中二硫苏糖醇最为有效。使用生物合成制备的纯黑腹果蝇脱辅基细胞色素c和酿酒酵母线粒体，细胞色素c血红素连接酶的活性约为800 fmol min-1 mg-1线粒体蛋白。动力学是典型的米氏动力学（Km约为1 nM），具有破损外膜的线粒体球状体的动力学也是如此（Km约为3 nM）。用线粒体球状体进行测试时，发现一系列物种的全细胞色素c在生理浓度下是血红素连接酶的竞争性抑制剂，这为体内控制该浓度提供了一种机制。脱辅基细胞色素c与酵母线粒体以两个阶段结合，解离常数分别约为2×10(-10)和10(-8) M，而线粒体球状体失去了高亲和力结合。发现脱辅基细胞色素c的一个定点突变体（赖氨酸5、7和8分别被谷氨酰胺、谷氨酸和天冬酰胺取代）可转化为全细胞色素c（Km约为3.3 nM；最大活性不变），尽管这些突变完全消除了高亲和力结合。因此，脱辅基细胞色素c与线粒体的高亲和力结合与全细胞色素c的形成没有直接关系。
