Lipsky R H, Hylemon P B
Biochim Biophys Acta. 1980 Apr 11;612(2):328-36. doi: 10.1016/0005-2744(80)90115-1.
flavin oxidoreductase was partially purified (seven-fold) from an intestinal Eubacterium species V.P.I. 12708 using Bio-Gel A 0.5-M and DEAE-cellulose column chromatography. Enzyme activity was measured spectrophotometrically at 340 nm under anaerobic conditions. A molecular weight of 260 000 was estimated by gel filtration chromatography. The partially purified enzyme preparation exhibited single displacement kinetics with respect to the substrates NADH and FAD. The pH optimum under these conditions was 6.8. NADH:flavin oxidoreductase showed an absolute specificity for NADH as electron donor. However, methylene blue, 2,6-dichlorophenolindophenol, K3Fe(CN)6, menadione, riboflavin, FMN and molecular oxygen served as alternate electron acceptors with varying degrees of efficiency. Acriflavin, rotenone, o-phenanthroline, p-chloromercuribenzoate, dicoumarol and 2,4-dinitrophenol inhibited enzyme activity. Surprisingly, 0.1 mM cholic acid, but not 0.1 mM deoxycholic acid, rapidly induced NADH:flavin oxidoreductase activity in growing cultures.
利用Bio-Gel A 0.5-M和DEAE-纤维素柱色谱法，从肠道真细菌V.P.I. 12708菌株中部分纯化（纯化了7倍）出一种NADH：黄素氧化还原酶。在厌氧条件下，于340 nm处通过分光光度法测定酶活性。通过凝胶过滤色谱法估计其分子量为260000。部分纯化的酶制剂对底物NADH和FAD呈现单置换动力学。在这些条件下，最适pH为6.8。NADH：黄素氧化还原酶对作为电子供体的NADH表现出绝对特异性。然而，亚甲蓝、2,6-二氯酚靛酚、铁氰化钾、甲萘醌、核黄素、FMN和分子氧作为替代电子受体，其效率各不相同。吖啶黄素、鱼藤酮、邻菲罗啉、对氯汞苯甲酸、双香豆素和2,4-二硝基苯酚抑制酶活性。令人惊讶的是，0.1 mM胆酸而非0.1 mM脱氧胆酸能在生长培养物中迅速诱导NADH：黄素氧化还原酶活性。
