Date of Award
This study was designed to investigate the presence of selected reduced pyridine nucleotide oxidase systems in a streptomycin bleached strain of Euglena gracilis var. bacillaris. The enzymes studied were NADH and NADPH diaphorases, NADH and NADPH cytochrome c reductases, NADH and NADPH oxidases and NADH lipoyl dehydrogenase. For experimental purposes the cells were grown axenically in a medium in which tryptone and sodium acetate were the sole source of carbon. Cell-free extracts prepared via centrifugation were used as the source of all enzyme assays. The activities of enzymes were measured in a Beckman DB-G recording spectrophotometer. In the presence of substrate and enzyme the activity of diaphorases were determined by measuring the reduction of dichlorophenol indophenol (DCPIP) at 600 nm and the cytochrome c reductase activity was measured by the reduction of cytochrome c (increase in optical density) at 550 nm. The activity of oxidases was determined by following the oxidation of reduced nicotinamide adenine dinucleotide (NADH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) spectrophotometrically at 340 nm. In the presence of enzyme and DL-lipomide the determination of lipoyl dehydrogenase activity was based on the oxidation of NADH (decrease in optical density) at 340 nm. The data obtained revealed the presence of NADH and NADPH diaphorases, NADH and NADPH cytochrome c reductases, NADH and NADPH oxidases and NADH lipoyl dehydrogenase in cell-free extracts of E. gracilis var. bacillaris. The effects of pH, protein concentration, substrate concentration, storage at -5 C, and the effects of metabolic inhibitors were determined spectrophotometrically. Utilizing polyacrylamide gel disc electrophoresis, NADH and NADPH diaphorase were shown to be molecularly heterogeneous. Results obtained show that there are numerous pathways for the oxidation of reduced pyridine nucleotides.
Mohanty, Mahendra K., "Reduced pyridine nucleotide linked oxidase systems in Euglena gracilis var. bacillaris" (1973). ETD Collection for AUC Robert W. Woodruff Library. DP14663.