Purification and characterization of glutathione reductase from Rhodospirillum rubrum.
Glutathione reductase (NAD(P)H:GSSG oxidoreductase EC 1.6.4.2.) was purified 1160-fold to homogeneity from the nonsulfurous purple bacteria Rhodospirillum rubrum (wild type). Specific activity of the pure preparation was 102 U/mg. The enzyme displayed a typical flavoprotein absorption spectrum with maxima at 274,365, and 459 nm and an absorbance ratio A280/A459 of 7.6. The amino acid analysis revealed an unusually high content of glycine and arginine residues. Titration of the enzyme with 5,5'-dithiobis(2-nitrobenzoic acid) showed a total of two free thiol groups per subunit, one of which is made accessible only under denaturing conditions. An isoelectric point of 5.2 was found for the native enzyme. Km values, determined at pH 7.5, were 6.1 and 90 microM for NADPH and GSSG, respectively. NADH was about 2% as active as NADPH as an electron donor. The enzyme's second choice in disulfide substrate was the mixed disulfide of coenzyme A and glutathione, for which the specific activity and Km values were 5.1 U/mg and 3.4 mM, respectively. A native molecular weight of 118,000 was found, while denaturing electrophoresis gave a value of 54,400 per subunit, thus suggesting that R. rubrum glutathione reductase exists as a dimeric protein. Other physicochemical constants of the enzyme, such as Stokes radius (4.2 nm) and sedimentation coefficient (5.71 S), were also consistent with a particle of 110,000.
Libreros-Minotta
C A
CA
Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, D.F. México.
Pardo
J P
JP
Mendoza-Hernández
G
G
Rendón
J L
JL
D000596:Amino Acids / Q000032:analysis; D002384:Catalysis; D005978:Glutathione / Q000378:metabolism; D005980:Glutathione Reductase / Q000737:chemistry / Q000302:isolation & purification*; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D012247:Rhodospirillum rubrum / Q000201:enzymology*; D013057:Spectrum Analysis; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry
D000596:Amino Acids; D013438:Sulfhydryl Compounds; D009243:NAD; D009249:NADP; D005980:Glutathione Reductase; D005978:Glutathione