Studies on cytochrome oxidase. Interactions of the cytochrome oxidase protein with phospholipids and cytochrome c.
Studies on cytochrome oxidase. Interactions of the cytochrome oxidase protein with phospholipids and cytochrome c.
复制标题
细胞色素氧化酶的研究。
DOI：
10.1016/s0021-9258(19)41825-5
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发表时间：
1975
期刊：
影响因子：
--
通讯作者：
T. King
中科院分区：
文献类型：
--
作者：
C. Yu;L. Yu;T. King
1.应用呼吸链顺序断裂原理，建立了一种分离去磷脂和富磷脂细胞色素氧化酶制剂的简便方法。2.富含磷脂的氧化物酶含有约20%的脂肪，主要包括磷脂酰乙醇胺、磷脂酰胆碱和心磷脂。它的酶活性不受外部脂类如asolectin的刺激。3.去磷脂氧化物酶的脂肪含量低于0.1%。它在催化还原的细胞色素c被分子氧氧化方面没有酶活性。这一活性可由asolectin完全恢复，并可通过单独或联合使用纯化的磷脂部分恢复(约75%)。从线粒体、酶本身和相关制剂中分离出的磷脂混合物也可以部分恢复这种活性。在测试的洗涤剂中，只有Emasol-1130和Tween80显示出一定的刺激活性。4.去磷脂的氧化物酶与细胞色素c明显通过“蛋白质-蛋白质”相互作用结合，富磷脂或补充磷脂的氧化物酶也是如此，形成细胞色素c与血红素a之比为1的复合体。由去磷脂的细胞色素氧化酶制备的络合物在一氧化碳还原形式与还原形式的差光谱中，在415 nm处有一个特征的Soret吸收峰。这个415 nm的最大值可通过用磷脂补充络合物或通过络合物在胆酸盐或在高离子强度的介质中解离而取消。当使用抗坏血酸作为电子供体时，由去磷脂的细胞色素氧化酶制备的络合物不会引起细胞色素A3的还原，这与由富含磷脂或补充磷脂的氧化酶制备的络合物形成鲜明的对比。然而，在所有细胞色素c-细胞色素氧化酶复合体的制备中，二亚硫酸盐都会减少细胞色素A3的含量。这些事实表明，磷脂对细胞色素氧化酶电子传递的作用可能位于细胞色素a和A3之间。初步的动力学结果证实了这一结论，即在去磷脂的酶中，从细胞色素a到A3的电子转移比在富含磷脂或补充磷脂的酶中慢得多。根据细胞色素c的含量，已发现在具有络合物的体系中(存在补充的外部介质，除非提供能量)，酶活性约高10倍，并且
1. By the application of the principle of the sequential fragmentation of the respiratory chain, a simple-method has been developed for the isolation of phospholipid-depleted and phospholipid-rich cytochrome oxidase preparations. 2. The phospholip-rich oxidase contains about 20% lipid, including mainly phosphatidylethanolamine, phosphatidylcholine, and cardiolipin. Its enzymic activity is not stimulated by an external lipid such as asolectin. 3. The phospholipid-depleted oxidase contains less than 0.1% lipid. It is enzymically inactive in catalyzing the oxidation of reduced cytochrome c by molecular oxygen. This activity can be fully restored by asolectin; and partially restored (approximately 75%) by purified phospholipids individually or in combination. The activity can be partially restored also by phospholipid mixtures isolated from mitochondria, from the oxidase itself, and from related preparations. Among the detergents tested only Emasol-1130 and Tween 80 show some stimulatory activity. 4. The phospholipid-depleted oxidase binds with cytochrome c evidently by "protein-protein" interactions as does the phospholipid-rich or the phospholipid-replenished oxidase to form a complex with the ratio of cytochrome c to heme a of unity. The complex prepared from phospholipid-depleted cytochrome oxidase exhibits a characteristic Soret absorption maximum at 415 nm in the difference spectrum of the carbon monoxide-reacted reduced form minus the reduced form. This 415-nm maximum is abolished by the replenishment of the complex with a phospholipid or by the dissociation of the complex in cholate or in a medium of high ionic strength. When ascorbate is used as an electron donor, the complex prepared from phospholipid-depleted cytochrome oxidase does not cause the reduction of cytochrome a3 which is in dramatic contrast to the complex from the phospholipid-rich or the phospholipid-replenished oxidase. However, dithionite reduces cytochrome a3 in all of the preparations of the cytochrome c-cytochrome oxidase complex. These facts suggest that the action of phospholipid on the electron transfer in cytochrome oxidase may be at the step between cytochromes a and a3. This conclusion is substantiated by preliminary kinetic results that the electron transfer from cytochrome a to a3 is much slower in the phospholipid-depleted than in phospholipid-rich or phospholipid-replenished oxidase. On the basis of the cytochrome c content, the enzymic activity has been found to be about 10 times higher in the system with the complex (in the presence of the replenishedhe external medium unless energy is provided, and that