哈氏贝内克氏菌中NADH和NADPH特异性FMN氧化还原酶的纯化及性质

Purification and properties of the NADH and NADPH specific FMN oxidoreductases from Beneckea harveyi.

作者信息

Jablonski E, DeLuca M

出版信息

Biochemistry. 1977 Jun 28;16(13):2932-6. doi: 10.1021/bi00632a020.

Abstract

The NADH and NADPH specific FMN oxidoreductases from Beneckea harveyi have been purified to homogeneity as judged by single bands on sodium dodecyl sulfate gel electrophoresis. The overall purification for the NADH specific enzyme is 3000-fold and 4000-fold for the NADPH specific enzyme from a crude extract. The final step in the purification procedure is chromatography on a 5'-AMP-Sepharose 4B affinity column which results in approximately a 50-fold purification to a final specific activity of 31 mumol of NADH oxidized min-1 (mg of protein)-1 for the NADH specific FMN reductase. The NADPH specific reductase has been purified to a final specific activity of 51 mumol of NADPH oxidized min-1 (mg of protein)-1 using a NADP agarose affinity column, which results in a 70-fold purification. Molecular weights of 30 000 and 40 000 and Km's of 4.75 X 10(-5) M NADH and 4.0 X 10(-5) M NADPH have been determined for the pure NADH and NADPH specific FMN reductases, respectively. The NADPH specific FMN reductase does not utilize NADH, while the NADH specific enzyme does dehydrogenate NADPH with a maximal velocity one-tenth of that for NADH. Separate NADH and NADPH specific FMN reductases from Photobacterium fischeri could not be demonstrated.

摘要

哈维贝内克氏菌（Beneckea harveyi）中NADH和NADPH特异性FMN氧化还原酶经纯化后达到均一性，这通过十二烷基硫酸钠凝胶电泳上的单一条带得以判断。从粗提物中纯化NADH特异性酶的总纯化倍数为3000倍，NADPH特异性酶为4000倍。纯化步骤的最后一步是在5'-AMP-琼脂糖4B亲和柱上进行层析，这使得NADH特异性FMN还原酶最终比活性达到31 μmol NADH氧化·min⁻¹（mg蛋白质）⁻¹，纯化倍数约为50倍。使用NADP琼脂糖亲和柱将NADPH特异性还原酶纯化至最终比活性为51 μmol NADPH氧化·min⁻¹（mg蛋白质）⁻¹，纯化倍数为70倍。已分别测定出纯的NADH和NADPH特异性FMN还原酶的分子量为30000和40000，以及Km值分别为4.75×10⁻⁵ M NADH和4.0×10⁻⁵ M NADPH。NADPH特异性FMN还原酶不利用NADH，而NADH特异性酶虽能使NADPH脱氢，但其最大速度仅为NADH的十分之一。未能证明费氏发光杆菌（Photobacterium fischeri）中存在单独的NADH和NADPH特异性FMN还原酶。