Gut J, Richter C, Cherry R J, Winterhalter K H, Kawato S
J Biol Chem. 1983 Jul 25;258(14):8588-94.
Purified rat liver microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase were co-reconstituted in phosphatidylcholine-phosphatidylethanolamine-phosphatidylserine vesicles by a cholate dialysis technique. Rotational diffusion of cytochrome P-450 was measured by detecting the decay of absorption anisotropy r(t), after photolysis of the heme X CO complex by a vertically polarized laser flash. All cytochrome P-450 was found to be rotationally mobile when co-reconstituted with equimolar amounts of NADPH-cytochrome P-450 reductase in lipid to cytochrome P-450 ((L/P450)) = 1 (w/w] vesicles. Antibodies against NADPH-cytochrome P-450 reductase were raised. Their specificity was demonstrated by Ouchterlony double diffusion analysis. Antireductase Fab fragments were prepared from antireductase IgG by papain digestion. The N-demethylation of benzphetamine, catalyzed by the proteoliposomes, was significantly inhibited by antireductase IgG and by antireductase Fab fragments. Cross-linking of NADPH-cytochrome P-450 reductase by antireductase IgG resulted in complete immobilization of cytochrome P-450 in L/P450 = 1 vesicles. Antireductase IgG also immobilized cytochrome P-450 in L/P450 = 5 vesicles, although the degree of immobilization was slightly smaller. No immobilization of cytochrome P-450 in L/P450 = 1 vesicles was detected in the presence of antireductase Fab fragments or preimmune IgG. These results further support the proposal of the formation of monomolecular complexes between cytochrome P-450 and NADPH-cytochrome P-450 reductase in liposomal membranes (Gut, J., Richter, C., Cherry, R.J., Winterhalter, K.H., and Kawato, S. (1982) J. Biol. Chem. 257, 7030-7036).
通过胆酸盐透析技术，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。在通过垂直偏振激光闪光光解血红素X CO复合物后，通过检测吸收各向异性r(t)的衰减来测量细胞色素P-450的旋转扩散。当在脂质与细胞色素P-450的比例((L/P450)) = 1（w/w）的囊泡中与等摩尔量的NADPH-细胞色素P-450还原酶共重组时，发现所有细胞色素P-450都具有旋转流动性。制备了抗NADPH-细胞色素P-450还原酶的抗体。通过欧氏双扩散分析证明了它们的特异性。通过木瓜蛋白酶消化从抗还原酶IgG制备抗还原酶Fab片段。抗还原酶IgG和抗还原酶Fab片段显著抑制了由蛋白脂质体催化的苄非他明的N-去甲基化。抗还原酶IgG使NADPH-细胞色素P-450还原酶交联，导致细胞色素P-450在L/P450 = 1的囊泡中完全固定化。抗还原酶IgG也使细胞色素P-450在L/P450 = 5的囊泡中固定化，尽管固定化程度略小。在存在抗还原酶Fab片段或免疫前IgG的情况下，未检测到细胞色素P-450在L/P450 = 1的囊泡中的固定化。这些结果进一步支持了关于细胞色素P-450和NADPH-细胞色素P-450还原酶在脂质体膜中形成单分子复合物的提议（古特，J.，里希特，C.，彻里，R.J.，温特哈尔特，K.H.，和川户，S.（1982年）《生物化学杂志》257，7030 - 7036）。
