Characterization of cytochrome P-450scc-containing liposomes.
Characterization of cytochrome P-450scc-containing liposomes.
复制标题
含细胞色素 P-450scc 的脂质体的表征。
DOI：
10.1016/0005-2736(81)90424-7
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发表时间：
1981
期刊：
影响因子：
--
通讯作者：
Kimura,T
中科院分区：
文献类型：
--
作者：
Yamakura,F;Kido,T;Kimura,T
从牛肾上腺皮质线粒体中纯化的细胞色素p450 SCC通过透析或Sephadex凝胶过滤的胆碱稀释法掺入脂质体。在所测试的合成磷脂中，二油酰甘油磷胆碱在p450 SCC掺入脂质体时表现出最好的稳定性。当磷脂与细胞色素的摩尔比约为时，血红素被掺入脂质体的最大量。200. 当p450 SCC通过胆碱滤过法掺入二油酰甘油磷脂胆碱脂质体时，含有p450 SCC的脂质体在Sepharose 4B凝胶滤过的洗脱模式上显示出两大种群，并且在直径200-600 Å及其聚集形式中可见。当细胞色素掺入二油酰甘油磷脂胆碱脂质体或无胆固醇肾上腺皮质线粒体脂质体时，p450 SCC在水溶液中的稳定性低于p450 SCC。胆固醇或肾上腺素可显著稳定p450 SCC脂质体。在CO存在的情况下，脂质体p450 SCC需要胆固醇才能通过肾上腺素还氧素、肾上腺素还氧素还原酶和NADPH进行最佳还原。在胆固醇存在的情况下，二油基甘油磷脂胆碱脂质体中约70%的总血红素被酶还原，这表明70%的总分子暴露在外单分子层的表面。为了观察血红素在膜中的位置，对二油基甘油磷胆碱脂质体p450 SCC进行了对氯苯磺酸处理。该试剂破坏脂质体p450 SCC。这些结果表明，血红素位于暴露在膜表面的对氯苯磺酸反应位点附近，或位于膜极头附近。
Purified cytochrome P 450 SCC from bovine adrenocortical mitochondria was incorporated into liposomes by the cholate-dilution method utilizing either dialysis or Sephadex gel filtration. Among synthetic phospholipids tested, dioleoylglycerophosphocholine showed the best stability during the incorporation of P 450 SCC into liposomes. A maximum amount of heme was incorporated into liposomes at a molar ratio of phospholipid to the cytochrome of approx. 200. When P 450 SCC was incorporated into the dioleoylglycerophosphocholine liposomes by the cholate-filtration method, the P 450 SCC-containing liposomes showed two major populations on the elution pattern of the Sepharose 4B gel filtration, and were seen at a diameter of 200–600 Å and its aggregated forms. When the cytochrome was incorporated into dioleoylglycerophosphocholine liposomes or cholesterol-free adrenocortical mitochondrial liposomes, P 450 SCC was less stable than P 450 SCC in aqueous solution. Cholesterol or adrenodoxin markedly stabilized the liposomal P 450 SCC. Liposomal P 450 SCC required cholesterol for its optimum reduction with adrenodoxin, adrenodoxin reductase, and NADPH in the presence of CO. About 70% of the total heme in the dioleoylglycerophosphocholine liposomes was reduced by the enzymatic reduction in the presence of cholesterol, indicating that 70% of the total molecules are exposed to the surface of the outer monolayer. In order to see the location of the heme in membrane, the dioleoylglycerophosphocholine-liposomal P 450 SCC was subjected to p-chloromercuriphenyl sulfonic acid treatment. This reagent destroyed the liposomal P 450 SCC. These results suggest that the heme is located in the proximity of the p-chloromercuriphenyl sulfonic acid reacting sites which are exposed to the surface, or located on the vincinity of polar heads of the membrane.