A glutathione reductase from Escherichia coli.

Earlier studies had demonstrated that a flavoprotein capable of mediating a triphosphopyridine nucleotide (TPN)-linked reduction of Furacin (5-nitro-2-furaldehyde semicarbazone) was present in cell-free extracts of Furacin-susceptible strains of Escherichia coli, but was absent in extracts of resistant strains (I). Dr. Philip E. Hartman, who was then associated with our department, suggested to us that the flavoprotein involved might be a TPN-linked reductase for oxidized glutathione (GSSG). The presence of such a GSSG reductase had already been demonstrated in cell-free extracts of yeast (a), plant tissues (3-5), mammalian tissues (6), and Neurospora (7), although these reductases had not been identified specifically as flavoproteins. A partially purified preparation of our Furacin-reducing flavoprotein was indeed found to contain a very active TPN-linked GSSG reductase. However, it quickly became evident that this reductase was a flavoprotein distinctly different from the Furacin-reducing one which we have been studying. Thus the latter flavoprotein was (a) absent in cell-free extracts of resistant strains, (b) after treatment with acid could be almost completely reactivated by 3 X 1CF M riboflavin phosphate (FP) but not by an equivalent concentration of flavin adenine dinucleotide (FAD), and was (c) completely destroyed by being heated for 10 minutes at 70”. The GSSG reductase, on the other hand, was (a) present in amounts with high activity in extracts of both susceptible and resistant strains of E. coli, (b) could be reactivated after acid treatment by 3 X 1e6 M FAD, but not by as much as 50 times this concentration of FP, and (c) lost only 10 to 15 per cent of its activity when heated to 70” for 10 minutes. Since such treatment enabled us to achieve a relatively high degree of separation of the GSSG reductase, some further characterization studies of this enzyme were made and are here presented.