Kanaeva I P, Nikityuk O V, Davydov D R, Dedinskii I R, Koen Y M, Kuznetsova G P, Skotselyas E D, Bachmanova G I, Archakov A I
Institute of Biological and Medical Chemistry, Russian Academy of Medical Science, Moscow.
Arch Biochem Biophys. 1992 Nov 1;298(2):403-12. doi: 10.1016/0003-9861(92)90428-y.
The kinetic parameters of NADPH-dependent cytochrome P450 LM2 (2B4) reduction and substrate oxidation in the monomeric reconstituted system, consisting of purified NADPH-cytochrome P450 reductase and cytochrome P450 LM2 monomers, and in phenobarbital-induced rabbit liver microsomes were compared. In the absence of benzphetamine, NADPH-dependent reduction of cytochrome P450 LM2 was monophasic in the monomeric reconstituted system and biphasic in the microsomes. The presence of the substrate in the monomeric reconstituted system caused the appearance of the fast phase. In this system substrate-free cytochrome P450 LM2 was entirely low-spin, and the addition of benzphetamine shifted the spin equilibrium to a high state very weakly. No correlation between high-spin content and the proportion of the fast phase of NADPH-dependent LM2 reduction was found in the system. Vmax values for the oxidation of type I substrates (benzphetamine, dimethylaniline, aminopyrine) in the monomeric reconstituted system were higher or the same as in the microsomes, whereas Km values for the substrates and NADPH were lower in the microsomes. Maximal activity of the monomeric reconstituted system was observed at a 1:1 NADPH-cytochrome P450 reductase/cytochrome P450 LM2 ratio. Measurements of benzphetamine oxidation as a function of NADPH-cytochrome P450 reductase/cytochrome P450 LM2 ratio at a constant total protein concentration allowed the Kd of the NADPH-cytochrome P450 reductase/cytochrome P450 LM2 complex to be estimated as 6.4 +/- 0.5 microM. Complex formation between the NADPH-cytochrome P450 reductase and cytochrome P450 LM2 monomers was not detected by recording the difference binding spectra of the reductase monomers with LM2 monomers or by treatment the mixture of the monomers of the proteins with the crosslinking reagent, water-soluble carbodiimide.
比较了由纯化的NADPH-细胞色素P450还原酶和细胞色素P450 LM2单体组成的单体重组系统以及苯巴比妥诱导的兔肝微粒体中NADPH依赖性细胞色素P450 LM2（2B4）还原和底物氧化的动力学参数。在没有苄非他明的情况下，NADPH依赖性细胞色素P450 LM2的还原在单体重组系统中是单相的，而在微粒体中是双相的。单体重组系统中底物的存在导致快速相的出现。在该系统中，无底物的细胞色素P450 LM2完全是低自旋的，加入苄非他明后自旋平衡非常微弱地向高自旋状态转变。在该系统中未发现高自旋含量与NADPH依赖性LM2还原快速相比例之间的相关性。单体重组系统中I型底物（苄非他明、二甲基苯胺、氨基比林）氧化的Vmax值高于或与微粒体中的相同，而微粒体中底物和NADPH的Km值较低。在NADPH-细胞色素P450还原酶/细胞色素P450 LM2比例为1:1时观察到单体重组系统的最大活性。在恒定总蛋白浓度下测量苄非他明氧化作为NADPH-细胞色素P450还原酶/细胞色素P450 LM2比例的函数，使得NADPH-细胞色素P450还原酶/细胞色素P450 LM2复合物的Kd估计为6.4±0.5 microM。通过记录还原酶单体与LM2单体的差异结合光谱或用交联剂水溶性碳二亚胺处理蛋白质单体混合物，未检测到NADPH-细胞色素P450还原酶与细胞色素P450 LM2单体之间的复合物形成。
