Miwa G T, Lu A Y
Arch Biochem Biophys. 1984 Oct;234(1):161-6. doi: 10.1016/0003-9861(84)90337-0.
NADPH-cytochrome P-450 reductase and two purified isozymes of cytochrome P-450 have been incorporated into phospholipid vesicles by a cholate dialysis technique. The enzyme system reconstituted in this manner was catalytically active. The observed kinetics for substrate oxidation indicated that both enzymes were associated with the liposomal membranes, and were not simply entrapped in the interior of the vesicle. The N-demethylation of benzphetamine was measured in order to determine the effect of variations in the mole ratio between the two enzymes and between the lipid and the total enzyme on the observed steady-state kinetics. In addition, the kinetic isotope effects for the O-deethylation of 7-ethoxycoumarin were measured in order to compare these parameters to those previously observed in a reconstituted system [G. T. Miwa, and A. Y. H. Lu (1981) Arch. Biochem. Biophys. 211, 454-458]. The results were all consistent with the association of the two proteins by lateral diffusion in the vesicle membrane. Moreover, the observed reduction in catalytic activity, as the enzymes were diluted in the vesicle membrane, can only be explained by the formation of a transient P-450-reductase complex, and not by the existence of a stable complex between the two proteins. These results provide compelling evidence for a mass action model for the interaction of these two enzymes in liposomal membranes.
通过胆酸盐透析技术，将NADPH-细胞色素P-450还原酶和两种纯化的细胞色素P-450同工酶掺入磷脂囊泡中。以这种方式重建的酶系统具有催化活性。观察到的底物氧化动力学表明，两种酶都与脂质体膜相关，而不仅仅是被困在囊泡内部。测定苄非他明的N-去甲基化，以确定两种酶之间以及脂质与总酶之间的摩尔比变化对观察到的稳态动力学的影响。此外，测量了7-乙氧基香豆素O-去乙基化的动力学同位素效应，以便将这些参数与先前在重建系统中观察到的参数进行比较[G. T. 三泽和A. Y. H. 卢（1981年）《生物化学与生物物理学报》211, 454 - 458]。结果都与这两种蛋白质通过囊泡膜中的侧向扩散缔合一致。此外，随着酶在囊泡膜中被稀释，观察到的催化活性降低只能用瞬时P-450-还原酶复合物的形成来解释，而不能用这两种蛋白质之间存在稳定复合物来解释。这些结果为脂质体膜中这两种酶相互作用的质量作用模型提供了令人信服的证据。
