Watanabe H, Hastings J W
Mol Cell Biochem. 1982 May 14;44(3):181-7. doi: 10.1007/BF00238506.
Three different NAD(P)H-FMN reductases were extracted from Beneckea harveyi MB-20 cells and separated by DEAE-Sephadex A50 column chromatography. Further purification was achieved by affinity chromatography. In determinations of Km values for NADH, NADPH, and FMN, these three reductases exhibited different specificities and kinetic parameters. One reductase utilizes NADH, whereas a second one utilizes NADPH as the preferred substrate. The third, a newly described reductase species, exhibits about the same reaction rates with NADH and NADPH. The reaction mechanisms of the three enzyme forms have been deduced by steady state kinetic analysis. The highly pure (based on gel electrophoresis) NADPH-FMN reductase still exhibited a low (approximately 2%) activity for NADH, which activity was increased upon storage at 4 degrees but suppressed completely by the replacement of the phosphate buffer with sodium citrate buffer. This high specificity of NADPH-FMN reductase for NADPH under these conditions is useful for the assay of NADPH, notably in systems coupled to bacterial luciferase.
从哈维贝内克氏菌MB - 20细胞中提取了三种不同的NAD(P)H - FMN还原酶，并通过DEAE - 葡聚糖A50柱色谱法进行分离。通过亲和色谱法进一步纯化。在测定NADH、NADPH和FMN的Km值时，这三种还原酶表现出不同的特异性和动力学参数。一种还原酶利用NADH，而另一种则以NADPH作为首选底物。第三种是新描述的还原酶种类，对NADH和NADPH表现出大致相同的反应速率。通过稳态动力学分析推导了这三种酶形式的反应机制。高度纯化的（基于凝胶电泳）NADPH - FMN还原酶对NADH仍表现出较低（约2%）的活性，该活性在4℃储存时增加，但用柠檬酸钠缓冲液替代磷酸盐缓冲液后完全被抑制。在这些条件下，NADPH - FMN还原酶对NADPH的这种高特异性可用于NADPH的测定，特别是在与细菌荧光素酶偶联的系统中。
