Del Bello B, Maellaro E, Sugherini L, Santucci A, Comporti M, Casini A F
Instituto di Patologia Generale, Università di Siena, Italy.
Biochem J. 1994 Dec 1;304 ( Pt 2)(Pt 2):385-90. doi: 10.1042/bj3040385.
Rat liver cytosol has been found to reduce dehydroascorbic acid (DHAA) to ascorbic acid in the presence of NADPH. The enzyme responsible for such activity has been purified by ammonium sulphate fractionation, DEAE-Sepharose, Sephadex G-100 SF and Reactive Red column chromatography, with an overall recovery of 27%. SDS/PAGE of the purified enzyme showed one single protein band with an M(r) of 37,500. A similar value (36,800) was found by gel filtration on a Sephadex G-100 SF column. The results indicate that the enzyme is a homogeneous monomer. The Km for DHAA was 4.6 mM and the Vmax. was 1.55 units/mg of protein; for NADPH Km and Vmax. were 4.3 microM and 1.10 units/mg of protein respectively. The optimum pH was around 6.2. Several typical substrates and inhibitors of the aldo-keto reductase superfamily have been tested. The strong inhibition of DHAA reductase effected by steroidal and non-steroidal anti-inflammatory drugs, together with the ability to reduce 5 alpha-androstane-3,17-dione strongly, suggest the possibility that DHAA reductase corresponds to 3 alpha-hydroxysteroid dehydrogenase. Microsequence analysis performed on the electro-transferred enzyme band shows that the N-terminus is blocked. Internal primary structure data were obtained from CNBr-derived fragments and definitely proved the identity of NADPH-dependent DHAA reductase with 3 alpha-hydroxysteroid dehydrogenase.
已发现大鼠肝脏胞质溶胶在NADPH存在的情况下可将脱氢抗坏血酸（DHAA）还原为抗坏血酸。负责这种活性的酶已通过硫酸铵分级分离、DEAE-琼脂糖、葡聚糖G-100 SF和活性红柱色谱法进行了纯化，总回收率为27%。纯化酶的SDS/PAGE显示一条单一的蛋白带，其相对分子质量（M(r)）为37,500。在葡聚糖G-100 SF柱上进行凝胶过滤得到了类似的值（36,800）。结果表明该酶是一种均一的单体。DHAA的米氏常数（Km）为4.6 mM，最大反应速度（Vmax）为1.55单位/毫克蛋白；NADPH的Km和Vmax分别为4.3 microM和1.10单位/毫克蛋白。最适pH约为6.2。已经测试了醛酮还原酶超家族的几种典型底物和抑制剂。甾体和非甾体抗炎药对DHAA还原酶有强烈抑制作用，同时该酶具有强烈还原5α-雄烷-3,17-二酮的能力，这表明DHAA还原酶可能对应于3α-羟基类固醇脱氢酶。对电转移的酶带进行的微序列分析表明N端被封闭。从溴化氰衍生的片段获得了内部一级结构数据，明确证明了NADPH依赖性DHAA还原酶与3α-羟基类固醇脱氢酶的同一性。
