Microbial lipoamide dehydrogenase purification and some characteristics of the enzyme derived from selected microorganisms

1. Lipoamide dehydrogenase (NADH:lipoamide oxidoreductase, EC 1.6.4.3) was partially purified from selected prokaryotic and eukaryotic organisms. The enzymes from these sources were found to differ in their sensitivity to inhibition by NADH. Lipoamide dehydrogenase from Serratia marcescens, like the enzyme from Escherichia coli, failed to catalyse a significant initial reduction of lipoamide by NADH in the absence of added NAD+. This inhibition may be overcome by the prior addition of NAD+ to the reaction medium. In contrast, the enzymes isolated from Pseudomonas fluorescens, Bacillus subtilis and Azotobacter agilis were unaffected by substrate levels of NADH and the enzymatic activity was unchanged by the addition of NAD+ to the reaction medium. Eukaryotic lipoamide dehydrogenase was inhibited by NADH, but to a lesser extent than the enzyme from E. Coli or S. Marcescens. 2. The enzymes isolated from Saccharomyces cerevisiae and Neurospora crassa displayed electrophoretic heterogeneity, having at least three diaphorase-reactive bands. In contrast, the enzymes isolated from prokaryotes formed either a single band or a predominant single band upon electrophoresis. Molecular weights of the enzyme isolated from these organisms were estimated by gel filtration and polyacrylamide gel electrophoresis. The molecular weights of the enzyme isolated from both prokaryotic and eukaryotic organisms were found to be 110 000 ± 4400.