Purification and immunological studies of glutathione reductase from rat liver. Evidence for an antigenic determinant at the nucleotide-binding domain of the enzyme.
Glutathione reductase has been purified to homogeneity by a method which is an improvement of an earlier procedure (Carlberg, I. and Mannervik, B. (1975) J. Biol. Chem. 250, 5475-5480). The new steps in the purification scheme include affinity chromatography on 2',5' ADP-Sepharose 4B. Antibodies to glutathione reductase from rat liver were raised in rabbits and used for analysis of the enzyme by quantitative 'rocket' immunoelectrophoresis. Glutathione reductase from human erythrocytes, porcine erythrocytes, and calf-liver gave precipitin lines showing partial identity with the rat liver enzyme in Ouchterlony double diffusion experiments. Enzyme from spinach, yeast (Saccharomyces cerevisiae), and the photosynthetic bacterium Rhodospirillum rubrum did not give precipitates with the antibodies to the enzyme from rat liver. Titration of glutathione reductase from the different sources with antibodies confirmed the cross-reactivity of the mammalian enzymes; the human enzyme giving the strongest heterologous reaction. No reaction was observed with the enzyme from spinach, yeast, and Rhodospirillum rubrum. NADPH, NADP+, and 2',5' ADP were found to inhibit the interaction between antibodies and glutathione reductase from rat liver and human erythrocytes. NADH, glutathione, or glutathione disulfide did not protect the enzyme from reacting with the antibodies. It is concluded that glutathione reductase has an antigenic binding site for the antibodies at the pyridine nucleotide-binding site of the enzyme molecule.
D000818:Animals; D001665:Binding Sites; D003600:Cytosol / Q000201:enzymology; D000939:Epitopes / Q000032:analysis*; D005980:Glutathione Reductase / Q000276:immunology* / Q000302:isolation & purification; D005779:Immunodiffusion; D008099:Liver / Q000201:enzymology*; D009249:NADP / Q000378:metabolism; D051381:Rats
D000939:Epitopes; D009249:NADP; D005980:Glutathione Reductase