Hackett C S, Novoa W B, Ozols J, Strittmatter P
J Biol Chem. 1986 Jul 25;261(21):9854-7.
The soluble catalytic domain of NADH-cytochrome b5 reductase was radiolabeled with [14C]N-ethylmaleimide. Reaction for a limited time resulted in incorporation of 0.41 eq of N-ethylmaleimide and loss of 36% of the enzyme activity. Chromatography on a 5'-ADP affinity column separated the reductase which was modified with N-ethylmaleimide from the unreacted enzyme; the isolated derivative constituted 37% of the total material, was completely inactivated, and contained 1.00 eq of N-ethylmaleimide. Cyanogen bromide cleavage of the derivative demonstrated that radioactivity was limited to a single peptide which contained both Cys-283 and Cys-297. Tryptic hydrolysis of this cyanogen bromide peptide showed that the radioactivity was associated with Cys-283. Automated sequenator analysis confirmed that Cys-283 was the radiolabeled residue. These data demonstrate unambiguously that Cys-283 provides the essential thiol group of cytochrome b5 reductase.
用[14C]N-乙基马来酰亚胺对NADH-细胞色素b5还原酶的可溶性催化结构域进行放射性标记。在有限时间内进行反应，结果掺入了0.41当量的N-乙基马来酰亚胺，酶活性丧失了36%。在5'-ADP亲和柱上进行色谱分离，将用N-乙基马来酰亚胺修饰的还原酶与未反应的酶分开；分离得到的衍生物占总物质的37%，完全失活，且含有1.00当量的N-乙基马来酰亚胺。对该衍生物进行溴化氰裂解表明，放射性仅限于一个同时含有半胱氨酸-283和半胱氨酸-297的单一肽段。对该溴化氰肽段进行胰蛋白酶水解表明，放射性与半胱氨酸-283相关。自动测序仪分析证实半胱氨酸-283是被放射性标记的残基。这些数据明确表明，半胱氨酸-283提供了细胞色素b5还原酶的必需巯基。
