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

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Abstract
The NADH and NADPH specific FMN oxidoreductases from B. harveyi were 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 about a 50-fold purification to a final specific activity of 31 .mu.mol of NADH oxidized min-1 (mg of protein)-1 for the NADH specific FMN reductase. The NADPH specific reductase was purified to a final specific activity of 51 .mu.mol of NADPH oxidized min-1 (mg of protein)-1 using a NADP agarose affinity column, which results in a 70-fold purification. MW of 30,000 and 40,000 and Km of 4.75 .times. 10-5 M NADH and 4.0 .times. 10-5 M NADPH were 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 dehydrogenates NADPH with a maximal velocity 1/10 of that for NADH. Separate NADH and NADPH specific FMN reductases from Photobacterium fischeri could not be demonstrated.