Voice M W, Zhang Y, Wolf C R, Burchell B, Friedberg T
Biomedical Research Centre, Ninewells Hospital and Medical School, Dundee, DD1 9SY, United Kingdom.
Arch Biochem Biophys. 1999 Jun 1;366(1):116-24. doi: 10.1006/abbi.1999.1192.
Cytochrome P450s (P450) form a superfamily of membrane-bound proteins that play a key role in the primary metabolism of both xenobiotics and endogenous compounds such as drugs and hormones, respectively. To be enzymically active, they require the presence of a second membrane-bound protein, NADPH P450 reductase, which transfers electrons from NADPH to the P450. Because of the diversity of P450 enzymes, much of the work on individual forms has been carried out on purified proteins, in vitro, which requires the use of complex reconstitution mixtures to allow the P450 to associate correctly with the NADPH P450 reductase. There is strong evidence from such reconstitution experiments that, when cytochrome b5 is included, the turnover of some substrates with certain P450s is increased. Here we demonstrate that allowing human P450 reductase, CYP3A4, and cytochrome b5 to associate in an in vivo-like system, by coexpressing all three proteins together in Escherichia coli for the first time, the turnover of both nifedipine and testosterone by CYP3A4 is increased in the presence of cytochrome b5. The turnover of testosterone was increased by 166% in whole cells and by 167% in preparations of bacterial membranes. The coexpression of cytochrome b5 also resulted in the stabilization of the P450 during substrate turnover in whole E. coli, with 109% of spectrally active CYP3A4 remaining in cells after 30 min in the presence of cytochrome b5 compared with 43% of the original P450 remaining in cells in the absence of cytochrome b5.
细胞色素P450（P450）构成了一个膜结合蛋白超家族，分别在异生物素和内源性化合物（如药物和激素）的初级代谢中发挥关键作用。为了具有酶活性，它们需要第二种膜结合蛋白——NADPH P450还原酶的存在，该还原酶将电子从NADPH转移到P450。由于P450酶的多样性，许多关于单个形式的研究是在体外对纯化蛋白进行的，这需要使用复杂的重组混合物以使P450与NADPH P450还原酶正确结合。从这样的重组实验中有强有力的证据表明，当包含细胞色素b5时，某些P450对一些底物的周转率会增加。在这里我们证明，通过首次在大肠杆菌中共表达人P450还原酶、CYP3A4和细胞色素b5，使它们在类似体内的系统中结合，在细胞色素b5存在的情况下，CYP3A4对硝苯地平和睾酮的周转率均增加。在全细胞中，睾酮的周转率增加了166%，在细菌膜制剂中增加了167%。细胞色素b5的共表达还导致在整个大肠杆菌中底物周转过程中P450的稳定性增加，在细胞色素b5存在的情况下，30分钟后仍有109%的具有光谱活性的CYP3A4保留在细胞中，而在没有细胞色素b5的情况下，只有43%的原始P450保留在细胞中。
