Kanaeva I P, Skotselyas E D, Turkina I F, Petrochenko E V, Davydov D R, Kondrashin S K, Dzuzenova Ch S, Bachmanova G I, Archakov A I
Institute of Physico-Chemical Medicine, Moscow, USSR.
Biochem Biophys Res Commun. 1987 Sep 30;147(3):1295-9. doi: 10.1016/s0006-291x(87)80211-5.
The membrane microsomal monooxygenase system can be reconstituted in solution from NADPH-specific flavoprotein and cytochrome P-450 which exist in the monomeric state in the presence of Emulgen 913 at molar ratio of the proteins and detergent of 1:1:300. Oxidized and dithionite-reduced monomers of cytochrome P-450 were much less thermostable than its initial aggregates, while thermal stability of NADPH-specific flavoprotein did not depend on its aggregation state. Binding spectra of cytochrome P-450 monomers with benzphetamine were atypical and had an absorbance minimum at 422 nm only. The addition of benzphetamine and/or flavoprotein to cytochrome P-450 monomers did not cause the spin equilibrium shift and the low-spin form content was higher than 85% in all cases. Investigation of the dependence of the initial rates of NADPH-dependent cytochrome P-450 reduction and benzphetamine oxidation on the stoichiometry of the flavoprotein and cytochrome P-450 at their constant total concentration showed that the molar ratio of 1:1 was required for maximal activity. Thus this system works in full accordance with the mass action law.
膜微粒体单加氧酶系统可在溶液中由NADPH特异性黄素蛋白和细胞色素P - 450重构而成，在存在乳化剂913的情况下，这些蛋白以单体状态存在，蛋白质与去污剂的摩尔比为1:1:300。细胞色素P - 450的氧化态和连二亚硫酸盐还原态单体的热稳定性远低于其初始聚集体，而NADPH特异性黄素蛋白的热稳定性并不取决于其聚集状态。细胞色素P - 450单体与苄非他明的结合光谱是非典型的，仅在422 nm处有一个吸光度最小值。向细胞色素P - 450单体中添加苄非他明和/或黄素蛋白不会导致自旋平衡移动，在所有情况下低自旋形式的含量均高于85%。在黄素蛋白和细胞色素P - 450总浓度恒定的情况下，研究NADPH依赖性细胞色素P - 450还原和苄非他明氧化的初始速率对黄素蛋白和细胞色素P - 450化学计量比的依赖性，结果表明最大活性需要1:1的摩尔比。因此，该系统完全符合质量作用定律。
