Gut J, Richter C, Cherry R J, Winterhalter K H, Kawato S
J Biol Chem. 1982 Jun 25;257(12):7030-6.
Purified rat liver microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase were co-reconstituted in phosphatidylcholine-phosphatidylethanolamine-phosphatidylserine vesicles using a cholate dialysis technique. The co-reconstitution of the enzymes was demonstrated in proteoliposomes fractionated by centrifugation in a glycerol gradient. The proteoliposomes catalyzed the N-demethylation of a variety of substrates. Rotational diffusion of cytochrome P-450 was measured by detecting the decay of absorption anisotropy r(t), after photolysis of the heme.CO complex by a vertically polarized laser flash. The rotational mobility of cytochrome P-450, when reconstituted alone, was found to be dependent on the lipid to protein ratio by weight (L/P450) (Kawato, S., Gut, J., Cherry, R. J., Winterhalter, K. H., and Richter, C. (1982) J. Biol. Chem. 257, 7023-7029). About 35% of cytochrome P-450 was immobilized and the rest was rotating with a mean rotational relaxation time phi 1 of about 95 mus in L/P450 = 1 vesicle. In L/P450 = 10 vesicles, about 10% of P-450 was immobile and the rest was rotating with phi 1 congruent to 55 mus. Co-reconstitution of equimolar amounts of NADPH-cytochrome P-450 reductase into the above vesicles results in completely mobile cytochrome P-450 with a phi 1 congruent to 40 mus. Only a small decrease in the immobile fraction of cytochrome P-450 is observed when the molar ratio of cytochrome P-450 to the reductase is 5. The results suggest the formation of a monomolecular 1:1 complex between cytochrome P-450 and NADPH-cytochrome P-450 reductase in the liposomes.
使用胆酸盐透析技术，将纯化的大鼠肝脏微粒体细胞色素P - 450和NADPH - 细胞色素P - 450还原酶共重组于磷脂酰胆碱 - 磷脂酰乙醇胺 - 磷脂酰丝氨酸囊泡中。通过在甘油梯度中离心分级分离的蛋白脂质体证明了酶的共重组。该蛋白脂质体催化了多种底物的N - 去甲基化反应。通过垂直偏振激光闪光光解血红素.CO复合物后，检测吸收各向异性r(t)的衰减来测量细胞色素P - 450的旋转扩散。单独重组时，发现细胞色素P - 450的旋转流动性取决于重量上的脂质与蛋白质比例（L/P450）（川户，S.，古特，J.，切里，R. J.，温特哈尔特，K. H.，和里希特，C.（1982年）《生物化学杂志》257，7023 - 7029）。在L/P450 = 1的囊泡中，约35%的细胞色素P - 450被固定，其余部分以约95微秒的平均旋转弛豫时间phi 1旋转。在L/P450 = 10的囊泡中，约10%的P - 450不移动，其余部分以phi 1约等于55微秒旋转。将等摩尔量的NADPH - 细胞色素P - 450还原酶共重组到上述囊泡中，导致细胞色素P - 450完全可移动，phi 1约等于40微秒。当细胞色素P - 450与还原酶的摩尔比为5时，仅观察到细胞色素P - 450固定部分有小幅下降。结果表明在脂质体中细胞色素P - 450与NADPH - 细胞色素P - 450还原酶形成了单分子1:1复合物。
