PURIFICATION AND PROPERTIES OF A NADH-DEPENDENT 5,10-METHYLENETETRAHYDROFOLATE REDUCTASE FROM PEPTOSTREPTOCOCCUS-PRODUCTUS
PURIFICATION AND PROPERTIES OF A NADH-DEPENDENT 5,10-METHYLENETETRAHYDROFOLATE REDUCTASE FROM PEPTOSTREPTOCOCCUS-PRODUCTUS
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DOI：
10.1111/j.1432-1033.1990.tb19242.x
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发表时间：
1990-09-11
期刊：
影响因子：
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通讯作者：
DIEKERT, G
中科院分区：
文献类型：
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作者：
WOHLFARTH, G;GEERLIGS, G;DIEKERT, G
从利用一氧化碳的同型产乙酸消化链霉菌（菌株马尔堡）中获得的亚甲基四氢叶酸还原酶已被纯化至表观均一性。纯化的酶以380 μ mol·cm-1的比活性催化以亚甲基四氢叶酸作为电子受体的NADH的氧化。min-1 mg蛋白质-1（37 ℃）。C; pH 5.5）。NADH的表观Km接近10 μ M。通过凝胶过滤测定酶的表观分子量为约250.0 kDa。该酶由8个相同的亚基组成，分子量为32 kDa。其含有4 FAD/mol八聚体，其被酶以NADH作为电子供体还原;铁不能被检测到。氧气对酶没有影响。细胞提取物的超离心显示，约40%的酶活性被回收的颗粒部分，这表明该酶与膜。该酶还催化亚甲基四氢叶酸还原亚甲基蓝作为人工电子供体。甲基四氢叶酸的氧化介导的亚甲基蓝作为电子受体，无论是NAD+或紫精染料不能取代亚甲基蓝在这个反应。NADP（H）或FAD（H2）在任一方向上都不用作反应的底物。纯化的酶的活性，这是建议参与跨细胞质膜的钠转运，不受影响的情况下或存在的添加钠。该酶的性质不同于那些铁氧还蛋白依赖亚甲基四氢叶酸还原酶的同型产乙酸梭菌甲酸乙酸和NADP+依赖还原酶的真核生物迄今为止的调查。
The methylenetetrahydrofolate reductase from the carbon-monoxide-utilizing homoacetogen Peptostreptococcus productus (strain Marburg) has been purified to apparent homogeneity. The purified enzyme catalyzed the oxidation of NADH with methylenetetrahydrofolate as the electron acceptor at a specific activity of 380 .mu.mol .cntdot. min-1 mg protein-1 (37.degree. C; pH 5.5). The apparent Km for NADH was near 10 .mu.M. The apparent molecular mass of the enzyme was determined by gel filtration to be .apprxeq. 250.0 kDa. The enzyme consists of eight identical subunits with a molecular mass of 32 kDa. It contains 4 FAD/mol octamer which were reduced by the enzyme with NADH as the electron donor; iron could not be detected. Oxygen had no effect on the enzyme. Ultracentrifugation of cell extracts revealed that about 40% of the enzyme activity was recovered in the particulate fraction, suggesting that the enzyme is associated with the membrane. The enzyme also catalyzed the methylenetetrahydrofolate reduction with methylene blue as an artificial electron donor. The oxidation of methyltetrahydrofolate was mediated with methylene blue as the electron acceptor; neither NAD+ nor viologen dyes could replace methylene blue in this reaction. NADP(H) or FAD(H2) were not used as substrates for the reaction in either direction. The activity of the purified enzyme, which was proposed to be involved in sodium translocation across the cytoplasmic membrane, was not affected by the absence or presence of added sodium. The properties of the enzyme differ from those of the ferredoxin-dependent methylenetetrahydrofolate reductase of the homoacetogen Clostridium formicoaceticum and of the NADP+-dependent reductase of eucaryotes investigated so far.