Basile G, Di Bello C, Taniuchi H
J Biol Chem. 1980 Aug 10;255(15):7181-91.
Incubation of the 125I-labeled apoprotein, prepared from 125I-labeled iso-1-cytochrome c, with a yeast mitochondrial fraction in the presence of hemin, NADPH, and an extract of the postmitochondrial fraction at 32 +/- 1 degree C for 30 min has resulted in formation of cytochrome c-like species in yields of up to 35%. This radioactive synthesized species contains a functional group which responds to reduction with ascorbate and oxidation with K3Fe(CN)6 in that it is resistant in the reduced form and susceptible in the oxidized form to trypsin action in a manner characteristic of native cytochrome c. The functional group cannot be removed from the protein by cold HCl-acetone or 8 M urea treatment. The reduced form of the synthesized species exhibits resistance against autoxidation and the oxidized form can be reduced also by cytochrome b2. The synthesized species exhibits the same compact hydrodynamic volume of native cytochrome c. Treatment with silver sulfate followed by incubation with dithiothreitol converts the synthesized species to the original apoprotein as judged by an increase in the hydrodynamic volume. Thus, the synthesized species is indistinguishable from the original labeled iso-1-cytochrome c by these measurements; i.e. the synthesized species consists of the apoprotein to which heme is covalently attached through the thioether bond(s). The active factor of the mitochondrial fraction is heat-labile. The synthetic activity is strongly dependent on pH with a maximum approximately at pH 7.0. Hemin (or heme) appears to be required for this synthesis. The postmitochondrial fraction is inactive by itself. However, its addition markedly increases the synthetic activity. This factor is heat-stable, soluble in 80% methanol (or 75% ethanol), and insoluble in ethyl ether or ethyl acetate. Addition of NADP(H) (or NAD(H)) also increases the synthetic activity, the reduced form being more effective than the oxidized form. The postmitochondrial factor and the pyridine nucleotides appear to enhance the effect of each other. Thus, it seems that cytochrome c or a cytochrome c-like species is formed from the apoprotein and heme (or hemin) by an enzyme, cytochrome c synthetase, present in mitochondria.
将由¹²⁵I标记的同工-1-细胞色素c制备的¹²⁵I标记载脂蛋白，与酵母线粒体组分在血红素、NADPH和线粒体后组分提取物存在的情况下，于32±1℃孵育30分钟，已导致形成产率高达35%的细胞色素c样物质。这种放射性合成物质含有一个官能团，该官能团对用抗坏血酸还原和用铁氰化钾氧化有反应，因为它在还原形式下对胰蛋白酶作用有抗性，而在氧化形式下以天然细胞色素c特有的方式对胰蛋白酶作用敏感。该官能团不能通过冷盐酸 - 丙酮或8M尿素处理从蛋白质中去除。合成物质的还原形式表现出抗自氧化能力，氧化形式也能被细胞色素b2还原。合成物质表现出与天然细胞色素c相同的紧密流体动力学体积。用硫酸银处理，然后与二硫苏糖醇孵育，根据流体动力学体积的增加判断，合成物质转化为原始载脂蛋白。因此，通过这些测量，合成物质与原始标记的同工-1-细胞色素c无法区分；即合成物质由通过硫醚键与血红素共价连接的载脂蛋白组成。线粒体组分的活性因子对热不稳定。合成活性强烈依赖于pH，最大值约在pH 7.0。血红素（或血色素）似乎是这种合成所必需的。线粒体后组分本身无活性。然而，它的加入显著增加了合成活性。这个因子对热稳定，可溶于80%甲醇（或75%乙醇），不溶于乙醚或乙酸乙酯。添加NADP(H)（或NAD(H)）也会增加合成活性，还原形式比氧化形式更有效。线粒体后因子和吡啶核苷酸似乎相互增强作用。因此，似乎细胞色素c或细胞色素c样物质是由线粒体中存在的一种酶——细胞色素c合成酶，从载脂蛋白和血红素（或血色素）形成的。
