Purification and properties of glutathione reductases from bovine ciliary body.
Two glutathione reductases, designated GR-I and GR-II, were purified from bovine ciliary body to apparent homogeneity by ion-exchange chromatography on DEAE-agarose and gel filtration on Sephacryl S-200. The two enzymes were isolated whether protease inhibitors were included or not in the medium during tissue homogenization. GR-I was found to have a molecular weight of about 140,000 by gel filtration and be composed of two identical subunits. On the other hand, GR-II was shown to exist as aggregates with molecular weights larger than 670,000. The subunit molecular weight of GR-II was estimated to be about 45,000 by SDS-polyacrylamide gel electrophoresis. The high molecular aggregate dissociated into smaller species by treatment with mercaptoethanol. The isoelectric points of GR-I and GR-II were determined to be 5.8 and 6.4, respectively. The K'm values for NADPH and oxidized glutathione (GSSG) were 22- and 84 microM for GR-I and 11- and 98 microM for GR-II. Coenzyme A-glutathione disulfide served as a substrate, though it was much less reactive than oxidized glutathione, for both enzymes. S-alkyl glutathiones were inactive as substrates. Nitrofurantoin was a non-competitive inhibitor of the enzymes. 1,3-bis(2 chloroethyl)-1-nitrosourea (BCNU) inhibited GR-I and GR-II irreversibly. The two enzymes are similar, if not identical, in their amino-acid composition. The two enzymes are also very similar on the basis of the relative position of tryptic peptides separated in two-dimensional peptide maps. From the tissue content and turnover numbers of these enzymes it is concluded that bovine ciliary body has a sufficient capability for rapid reduction of oxidized glutathione produced in the tissue during glutathione-dependent peroxide detoxification.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002847:Chromatography, Agarose; D002851:Chromatography, High Pressure Liquid; D002924:Ciliary Body / Q000201:enzymology*; D005980:Glutathione Reductase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D007700:Kinetics; D008970:Molecular Weight; D010449:Peptide Mapping
D000596:Amino Acids; D005980:Glutathione Reductase