Inano H, Kurihara S, Tamaoki B
National Institute of Radiological Sciences, Chiba-shi, Japan.
J Steroid Biochem. 1988 Feb;29(2):227-32. doi: 10.1016/0022-4731(88)90270-1.
Rat testicular NADPH-cytochrome P-450 reductase was inactivated by treatment with 2,4,6-trinitrobenzene sulfonate (TNBS) or with 2',3'-dialdehyde derivatives of 5'-ATP and NADP+. The inactivation rates were dependent on reaction time and followed pseudo-first order kinetics. The rate of inactivation of cytochrome c reducing activity by TNBS was faster than that of reducing activities for K3Fe(CN)6 and for dichlorophenol indophenol (DCPIP). Cytochrome c and DCPIP prevented NADPH-cytochrome P-450 reductase from inactivation by TNBS, but NADP(H) protected to a lesser extent. Stoichiometry indicated that two residues of amino acid modified with TNBS were essential for the enzyme activity. The 2',3'-dialdehyde derivatives of 5'-ATP and NADP+ were specific ligands for the modification of lysine residues, whereas TNBS would possibly modify residues of lysine and/or cysteine. By differential and sequential modification by 5,5'-dithio-bis(2-nitrobenzoic acid), TNBS and dithiothreitol, the residues of lysine and cysteine were identified in the active site of NADPH-cytochrome P-450 reductase. These results suggest that lysyl and cysteinyl residues are located at or near the active region of NADPH-cytochrome P-450 reductase from the rat testicular microsomal fraction.
大鼠睾丸NADPH - 细胞色素P - 450还原酶经2,4,6 - 三硝基苯磺酸（TNBS）或5'-ATP和NADP +的2',3'-二醛衍生物处理后会失活。失活速率取决于反应时间，并遵循假一级动力学。TNBS使细胞色素c还原活性的失活速率快于使铁氰化钾（K3Fe(CN)6）和二氯酚靛酚（DCPIP）还原活性的失活速率。细胞色素c和DCPIP可防止NADPH - 细胞色素P - 450还原酶被TNBS失活，但NADP(H)的保护作用较小。化学计量表明，被TNBS修饰的两个氨基酸残基对酶活性至关重要。5'-ATP和NADP +的2',3'-二醛衍生物是修饰赖氨酸残基的特异性配体，而TNBS可能修饰赖氨酸和/或半胱氨酸残基。通过5,5'-二硫代双（2 - 硝基苯甲酸）、TNBS和二硫苏糖醇的差异和顺序修饰，在NADPH - 细胞色素P - 450还原酶的活性位点鉴定出了赖氨酸和半胱氨酸残基。这些结果表明，赖氨酸残基和半胱氨酸残基位于大鼠睾丸微粒体部分NADPH - 细胞色素P - 450还原酶的活性区域或其附近。
