Yamazaki H, Nakano M, Imai Y, Ueng Y F, Guengerich F P, Shimada T
Osaka Prefectural Institute of Public Health, Japan.
Arch Biochem Biophys. 1996 Jan 15;325(2):174-82. doi: 10.1006/abbi.1996.0022.
NADH-dependent testosterone 6 beta-hydroxylation and nifedipine oxidation activities could be reconstituted in systems containing cytochrome b5 (b5), NADH-b5 reductase, and bacterial recombinant cytochrome P450 (P450) 3A4 with a synthetic phospholipid mixture, cholate, MgCl2, and reduced glutathione. Replacement of NADH-b5 reductase with NADPH-P450 reductase produced an eightfold increase in testosterone 6 beta-hydroxylation activity. Further stimulation could be obtained when NADPH was used as an electron donor instead of NADH. Removal of b5 from the NADH- and NADPH-supported systems caused a 90% loss of testosterone 6 beta-hydroxylation activities in the presence of NADPH-P450 reductase but resulted in complete loss of the activities in the absence of NADPH-P450 reductase. These results suggested that about 10% of the activities was due to electron flow from NADPH-P450 reductase to P450 3A4 in the absence of b5. In the presence of testosterone and MgCl2, P450 3A4 was reduced by b5 and NADH-b5 reductase, although the rate of P450 3A4 reduction was much slower than that by NADPH-P450 reductase. Anti-human b5 immunoglobulin G (IgG) (purified using rabbit b5 affinity chromatography) inhibited testosterone 6 beta-hydroxylation activity catalyzed by human liver microsomes more strongly in NADH- than in NADPH-supported reactions. However, anti-rat NADPH-P450 reductase IgG inhibited microsomal activities in both NADH- and NADPH-supported systems to similar extents. Addition of NADH enhanced NADPH-supported testosterone and nifedipine oxidations in human liver microsomes. MgCl2 stimulated rates of reduction of b5 by NADPH-P450 reductase, but not by NADH-b5 reductase, in reconstituted systems. These results suggest that b5 is an essential component in P450 3A4-catalyzed testosterone hydroxylation and nifedipine oxidation in human liver microsomes. Our previous observation that rates of reduction of ferric P450 3A4 by NADPH-P450 reductase are accelerated by complexation with substrates and b5 is supported in this study.
在含有细胞色素b5（b5）、NADH - b5还原酶、细菌重组细胞色素P450（P450）3A4以及合成磷脂混合物、胆酸盐、MgCl2和还原型谷胱甘肽的体系中，可以重建依赖NADH的睾酮6β - 羟基化和硝苯地平氧化活性。用NADPH - P450还原酶替代NADH - b5还原酶后，睾酮6β - 羟基化活性增加了8倍。当使用NADPH作为电子供体而非NADH时，可获得进一步的刺激。在存在NADPH - P450还原酶的情况下，从NADH和NADPH支持的体系中去除b5会导致睾酮6β - 羟基化活性损失90%，但在不存在NADPH - P450还原酶时则导致活性完全丧失。这些结果表明，在不存在b5的情况下，约10%的活性是由于电子从NADPH - P450还原酶流向P450 3A4所致。在存在睾酮和MgCl2的情况下，P450 3A4被b5和NADH - b5还原酶还原，尽管P450 3A4的还原速率比被NADPH - P450还原酶还原的速率慢得多。抗人b5免疫球蛋白G（IgG）（使用兔b5亲和层析纯化）在NADH支持的反应中比在NADPH支持的反应中更强烈地抑制人肝微粒体催化的睾酮6β - 羟基化活性。然而，抗大鼠NADPH - P450还原酶IgG在NADH和NADPH支持的体系中对微粒体活性的抑制程度相似。添加NADH可增强NADPH支持的人肝微粒体中睾酮和硝苯地平的氧化。在重组体系中，MgCl2刺激NADPH - P450还原酶对b5的还原速率，但不刺激NADH - b5还原酶对b5的还原速率。这些结果表明，b5是人肝微粒体中P450 3A4催化的睾酮羟基化和硝苯地平氧化的必需成分。我们之前观察到，NADPH - P450还原酶对铁离子形式的P450 3A4的还原速率会因与底物和b5络合而加快，本研究支持了这一观察结果。
