Shimada Tsutomu, Mernaugh Raymond L, Guengerich F Peter
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, 638 Robinson Research Building, 23rd and Pierce Avenues, Nashville, TN 37232-0146, USA.
Arch Biochem Biophys. 2005 Mar 1;435(1):207-16. doi: 10.1016/j.abb.2004.12.008.
An immobilized system was developed to detect interactions of human cytochromes P450 (P450) with the accessory proteins NADPH-P450 reductase and cytochrome b(5) (b(5)) using an enzyme-linked affinity approach. Purified enzymes were first bound to wells of a polystyrene plate, and biotinylated partner enzymes were added and bound. A streptavidin-peroxidase complex was added, and protein-protein binding was monitored by measuring peroxidase activity of the bound biotinylated proteins. In a model study, we examined protein-protein interactions of Pseudomonas putida putidaredoxin (Pdx) and putidaredoxin reductase (PdR). A linear relationship (r(2)=0.96) was observed for binding of PdR-biotin to immobilized Pdx compared with binding of Pdx-biotin to immobilized PdR (the estimated K(d) value for the Pdx.PdR complex was 0.054muM). Human P450 2A6 interacted strongly with NADPH-P450 reductase; the K(d) values (with the reductase) ranged between 0.005 and 0.1muM for P450s 2C19, 2D6, and 3A4. Relatively weak interaction was found between holo-b(5) or apo-b(5) (devoid of heme) with NADPH-P450 reductase. Among the rat, rabbit, and human P450 1A2 enzymes, the rat enzyme showed the tightest interaction with b(5), although no increases in 7-ethoxyresorufin O-deethylation activities were observed with any of the P450 1A2 enzymes. Human P450s 2A6, 2D6, 2E1, and 3A4 interacted well with b(5), with P450 3A4 yielding the lowest K(d) values followed by P450s 2A6 and 2D6. No appreciable increases in interaction between human P450s with b(5) or NADPH-P450 reductase were observed when typical substrates for the P450s were included. We also found that NADPH-P450 reductase did not cause changes in the P450.substrate K(d) values estimated from substrate-induced UV-visible spectral changes with rabbit P450 1A2 or human P450 2A6, 2D6, or 3A4. Collectively, the results show direct and tight interactions between P450 enzymes and the accessory proteins NADPH-P450 reductase and b(5), with different affinities, and that ligand binding to mammalian P450s did not lead to increased interaction between P450s and the reductase.
开发了一种固定化系统，采用酶联亲和方法检测人细胞色素P450（P450）与辅助蛋白NADPH - P450还原酶和细胞色素b5（b5）之间的相互作用。首先将纯化的酶结合到聚苯乙烯板的孔中，然后加入生物素化的伴侣酶并使其结合。加入链霉亲和素 - 过氧化物酶复合物，通过测量结合的生物素化蛋白的过氧化物酶活性来监测蛋白质 - 蛋白质结合。在一项模型研究中，我们检测了恶臭假单胞菌的恶臭假单胞菌素还原蛋白（Pdx）和恶臭假单胞菌素还原酶（PdR）之间的蛋白质 - 蛋白质相互作用。与生物素化的Pdx与固定化的PdR的结合相比，观察到生物素化的PdR与固定化的Pdx的结合呈线性关系（r2 = 0.96）（Pdx.PdR复合物的估计Kd值为0.054μM）。人P450 2A6与NADPH - P450还原酶强烈相互作用；P450 2C19、2D6和3A4与还原酶的Kd值（与还原酶）在0.005至0.1μM之间。发现全b5或脱辅基b5（不含血红素）与NADPH - P450还原酶之间的相互作用相对较弱。在大鼠、兔和人P450 1A2酶中，大鼠酶与b5的相互作用最紧密，尽管任何一种P450 1A2酶的7 - 乙氧基试卤灵O - 脱乙基活性均未增加。人P450 2A6、2D6、2E1和3A4与b5相互作用良好，P450 3A4的Kd值最低，其次是P450 2A6和2D6。当加入P450的典型底物时，未观察到人P450与b5或NADPH - P450还原酶之间的相互作用有明显增加。我们还发现NADPH - P450还原酶不会导致根据兔P450 1A2或人P450 2A6、2D6或3A4的底物诱导的紫外 - 可见光谱变化估计出的P450 - 底物Kd值发生变化。总的来说，结果表明P450酶与辅助蛋白NADPH - P450还原酶和b5之间存在直接且紧密的相互作用，具有不同的亲和力，并且配体与哺乳动物P450的结合不会导致P450与还原酶之间的相互作用增加。
