Henry Wellcome Laboratories of Structural Biology, Department of Biochemistry, Henry Wellcome Building, University of Leicester, PO Box 138, Lancaster Road, Leicester LE1 9HN, UK.
Biochem J. 2010 Dec 15;432(3):485-93. doi: 10.1042/BJ20100744.
We have incorporated CYP3A4 (cytochrome P450 3A4) and CPR (NADPH-cytochrome P450 reductase) into liposomes with a high lipid/protein ratio by an improved method. In the purified proteoliposomes, CYP3A4 binds testosterone with Kd (app)=36±6 μM and Hill coefficient=1.5±0.3, and 75±4% of the CYP3A4 can be reduced by NADPH in the presence of testosterone. Transfer of the first electron from CPR to CYP3A4 was measured by stopped-flow, trapping the reduced CYP3A4 as its Fe(II)-CO complex and measuring the characteristic absorbance change. Rapid electron transfer is observed in the presence of testosterone, with the fast phase, representing 90% of the total absorbance change, having a rate of 14±2 s(-1). Measurements of the first electron transfer were performed at various molar ratios of CPR/CYP3A4 in proteoliposomes; the rate was unaffected, consistent with a model in which first electron transfer takes place within a relatively stable CPR-CYP3A4 complex. Steady-state rates of NADPH oxidation and of 6β-hydroxytestosterone formation were also measured as a function of the molar ratio of CPR/CYP3A4 in the proteoliposomes. These rates increased with increasing CPR/CYP3A4 ratio, showing a hyperbolic dependency indicating a Kd (app) of ~0.4 μM. This suggests that the CPR-CYP3A4 complex can dissociate and reform between the first and second electron transfers.
我们通过一种改良的方法，将细胞色素 P4503A4（CYP3A4）和细胞色素 P450 还原酶（CPR）与具有高脂质/蛋白比的脂质体结合。在纯化的蛋白脂质体中，CYP3A4 与睾酮的结合 Kd（app）为 36±6 μM，Hill 系数为 1.5±0.3，并且在存在睾酮的情况下，75±4%的 CYP3A4 可以被 NADPH 还原。通过停流技术测量 CPR 向 CYP3A4 的第一电子转移，将还原的 CYP3A4 作为其 Fe(II)-CO 络合物捕获，并测量特征吸收变化。在存在睾酮的情况下观察到快速电子转移，快相代表总吸收变化的 90%，其速率为 14±2 s(-1)。在蛋白脂质体中以 CPR/CYP3A4 的各种摩尔比进行第一电子转移测量；速率不受影响，与第一电子转移发生在相对稳定的 CPR-CYP3A4 复合物中的模型一致。还测量了 NADPH 氧化和 6β-羟睾酮形成的稳态速率作为蛋白脂质体中 CPR/CYP3A4 摩尔比的函数。这些速率随着 CPR/CYP3A4 比的增加而增加，呈双曲线依赖性，表明 Kd（app）约为 0.4 μM。这表明 CPR-CYP3A4 复合物可以在第一和第二电子转移之间解离和重新形成。
