Inano H, Tamaoki B
National Institute of Radiological Sciences, Chiba-shi, Japan.
J Enzyme Inhib. 1985;1(1):47-59. doi: 10.3109/14756368509031281.
NADPH-cytochrome P-450 reductase (EC 1.6.2.4) purified from rat hepatic microsomal fraction was inactivated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), a specific agent for modification of carboxyl groups in a protein. The inactivation exhibited pseudo-first order kinetics with a reaction order approximately one and a second-order-rate constant of 0.60 M-1 min-1 in a high ionic strength buffer and 0.08 M-1 min-1 in a low ionic strength buffer. By treatment of NADPH-cytochrome P-450 reductase with EDC, the pI value changed to 6.5 from 5.0 for the native enzyme, and the reductase activity for cytochrome c, proteinic substrate, was strongly inactivated. When an inorganic substrate, K3Fe(CN)6, was used for assay of the enzyme activity, however, no significant inactivation by EDC was observed. The rate of inactivation by EDC was markedly but not completely decreased by NADPH. Also, the inactivation was completely prevented by cytochrome c, but not by K3Fe(CN)6 or NADH. The sulfhydryl-blocked enzyme prepared by treatment with 5,5'-dithio-bis(2-nitrobenzoic acid), which had no activity, completely recovered its activity in the presence of dithiothreitol. When the sulfhydryl-blocked enzyme was modified by EDC, the enzyme in which the carboxyl group alone was modified was isolated, and its activity was 35% of the control after treatment with dithiothreitol. In addition, another carboxyl reagent, N-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward reagent K), decreased cytochrome c reductase activity of NADPH-cytochrome P-450 reductase. These results suggest that the carboxyl group of NADPH-cytochrome P-450 reductase from rat liver is located at or near active-site and plays a role in binding of cytochrome c.
从大鼠肝脏微粒体部分纯化得到的NADPH - 细胞色素P - 450还原酶（EC 1.6.2.4），被1 - 乙基 - 3 -（3 - 二甲基氨基丙基）碳二亚胺（EDC）灭活，EDC是一种用于修饰蛋白质中羧基的特异性试剂。在高离子强度缓冲液中，灭活呈现假一级动力学，反应级数约为1，二级速率常数为0.60 M⁻¹ min⁻¹；在低离子强度缓冲液中，二级速率常数为0.08 M⁻¹ min⁻¹。用EDC处理NADPH - 细胞色素P - 450还原酶后，其pI值从天然酶的5.0变为6.5，并且对蛋白质底物细胞色素c的还原酶活性被强烈灭活。然而，当使用无机底物K₃Fe(CN)₆来测定酶活性时，未观察到EDC导致的显著灭活。NADPH可使EDC的灭活速率显著但未完全降低。此外，细胞色素c可完全阻止灭活，但K₃Fe(CN)₆或NADH则不能。用5,5'-二硫代双（2 - 硝基苯甲酸）处理制备的巯基封闭酶无活性，在二硫苏糖醇存在下其活性完全恢复。当巯基封闭酶用EDC修饰后，分离出仅羧基被修饰的酶，用二硫苏糖醇处理后其活性为对照的35%。此外，另一种羧基试剂N - 乙基 - 5 - 苯基异恶唑 - 3'-磺酸盐（伍德沃德试剂K）降低了NADPH - 细胞色素P - 450还原酶的细胞色素c还原酶活性。这些结果表明，大鼠肝脏NADPH - 细胞色素P - 450还原酶的羧基位于活性位点或其附近，并且在细胞色素c的结合中起作用。
