Participation of the membrane in the side chain cleavage of cholesterol. Reconstitution of cytochrome P-450scc into phospholipid vesicles.
Participation of the membrane in the side chain cleavage of cholesterol. Reconstitution of cytochrome P-450scc into phospholipid vesicles.
复制标题
膜参与胆固醇侧链裂解。
DOI：
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发表时间：
1979
影响因子：
4.8
通讯作者：
H. Kamin
中科院分区：
文献类型：
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作者：
D. Seybert;J. Lancaster;J. Lambeth;H. Kamin
通过将纯化的血红素蛋白与预先形成的磷脂酰胆碱囊泡一起孵育，可以在不添加去垢剂的情况下将细胞色素 P-450scc 重建为磷脂双层。盐效应表明细胞色素和磷脂囊泡之间的主要相互作用是疏水性的而不是离子性的；相反，肾上腺素还蛋白还原酶和肾上腺素还素都不会通过疏水相互作用与磷脂酰胆碱囊泡结合。将细胞色素 P-450scc 插入磷脂双层会导致光谱转换为低自旋型，但如果胆固醇掺入双层内，这种转变会明显减弱。囊泡重建的细胞色素 P-450scc 在双层内代谢胆固醇（周转 = 13 nmol/min/nmol 细胞色素 P-450scc）；事实上，囊泡内的所有胆固醇（超过 94%）都可以被酶接触。在胆固醇和细胞色素 P-450scc 含量恒定的情况下，通过增加磷脂/胆固醇比率来“稀释”双层内的胆固醇，导致侧链裂解率降低，并且掺入无胆固醇囊泡中的细胞色素 P-450scc 不能代谢单独囊泡内的胆固醇。此外，重构血红素蛋白的活性对磷脂的脂肪酸组成敏感。这些结果表明囊泡重建的细胞色素 P-450scc 上的胆固醇结合位点与膜的疏水双层连通。囊泡重建的细胞色素 P-450scc 的还原性以及分光光度和活性滴定实验表明，所有重建的细胞色素 P-450scc 分子都具有可从囊泡外部接近的肾上腺素结合位点。用肾上腺氧还蛋白还原酶进行的活性滴定还表明，催化不需要肾上腺氧还蛋白还原酶、肾上腺氧还蛋白和细胞色素 P-450scc 之间的三元或四元复合物，这一发现与我们提出的类固醇生成电子传递机制一致，其中肾上腺氧还蛋白充当肾上腺氧还蛋白还原酶和细胞色素 P-450 之间的移动电子穿梭（Lambeth，J.D.，Seybert， D.W. 和 Kamin, H. (1979) J. Biol. 254, 7255-7264。
Cytochrome P-450scc can be reconstituted into a phospholipid bilayer in the absence of added detergent by incubation of purified hemoprotein with preformed phosphatidylcholine vesicles. Salt effects demonstrate that the primary interaction between the cytochrome and phospholipid vesicles is hydrophobic rather than ionic; in contrast, neither adrenodoxin reductase nor adrenodoxin will bind to phosphatidylcholine vesicles by hydrophobic interactions. Insertion of cytochrome P-450scc into a phospholipid bilayer results in conversion of the optical spectrum to a low spin type, but this transition is markedly diminished if cholesterol is incorporated within the bilayer. Vesicle-reconstituted cytochrome P-450scc metabolizes cholesterol within the bilayer (turnover = 13 nmol/min/nmol of cytochrome P-450scc); virtually all (greater than 94%) of the cholesterol within the vesicle is accessible to the enzyme. "Dilution" of cholesterol within the bilayer by increasing the phospholipid/cholesterol ratio at a constant amount of cholesterol and cytochrome P-450scc results in a decreased rate of side chain cleavage, and cytochrome P-450scc incorporated into a cholesterol-free vesicle cannot metabolize cholesterol within a separate vesicle. In addition, activity of the reconstituted hemoprotein is sensitive to the fatty acid composition of the phospholipid. These results indicate that the cholesterol binding site on vesicle-reconstituted cytochrome P-450scc is in communication with the hydrophobic bilayer of the membrane. The reducibility of vesicle-reconstituted cytochrome P-450scc as well as spectrophotometric and activity titration experiments show that all of the reconstituted cytochrome P-450scc molecules possess an adrenodoxin binding site which is accessible from the exterior of the vesicle. Activity titrations with adrenodoxin reductase also demonstrate that a ternary or quaternary complex among adrenodoxin reductase, adrenodoxin, and cytochrome P-450scc is not required for catalysis, a finding consistent with our proposed mechanism of steroidogenic electron transport in which adrenodoxin acts as a mobile electron shuttle between adrenodoxin reductase and cytochrome P-450 (Lambeth, J.D., Seybert, D.W., and Kamin, H. (1979) J. Biol. Chem. 254, 7255-7264.