Purification and properties of the glutathione reductase of Chromatium vinosum.
The Chromatium vinosum glutathione reductase [NAD(P)H: glutathione disulfide oxidoreductase, EC 1.6.4.2] was purified to apparent homogeneity. The enzyme was found to require reduced nicotinamide adenine dinucleotide (NADH) as a reductant and to be specific for oxidized glutathione (GSSG). The polypeptide molecular weight in sodium dodecyl sulfate was found to be 52,000. Incubation of enzyme with NADH in the absence of GSSG resulted in a significant loss in activity. The enzyme was stimulated by phosphate and sulfate ion, but was inhibited by chloride ion, heavy metals, and sulfhydryl reagents. Adenylate nucleotides were inhibitory, and the data suggested that they were acting as competitive inhibitors of flavin adenine dinucleotide (FAD). The Km values of 7 X 10-3 for GSSG and 6 X 10-5 M for NADH were the highest reported of any previously investigated glutathione reductase. The order of addition of components markedly affected the response of the enzyme to FAD. A requirement for FAD (Km 5.2 X 10-7 M) was seen if the enzyme was incubated with NADH prior to GSSG addition, whereas no FAD was required if the order was reversed.
D000241:Adenosine / Q000494:pharmacology; D002844:Chromatium / Q000201:enzymology*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005978:Glutathione / Q000494:pharmacology; D005980:Glutathione Reductase / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D009243:NAD / Q000494:pharmacology; D009711:Nucleotides / Q000494:pharmacology; D010710:Phosphates / Q000494:pharmacology; D012492:Salts / Q000494:pharmacology; D013431:Sulfates / Q000494:pharmacology; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D008670:Metals; D009711:Nucleotides; D010710:Phosphates; D012492:Salts; D013431:Sulfates; D013439:Sulfhydryl Reagents; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D005980:Glutathione Reductase; D005978:Glutathione; D000241:Adenosine