Characterization and partial purification of a cytoplasmic glutathione: disulfide oxidoreductase (thioltransferase) from adenohypophysis.
A glutathione-dependent thioltransferase (thiol : disulfide oxidoreductase) has been partially purified (70-fold) from anterior pituitary cytosol, and characterized. Purification was effected by differential centrifugation, precipitation between 30 and 60% (NH4)2SO4, and sequential chromatography on Sepharose 6B, DEAE-cellulose, and CM-cellulose. Enzyme activity, monitored by the disappearance of NADPH, was associated with a protein of molecular weight 170 000 both by gel filtration and by polyacrylamide gel electrophoresis in SDS. There was apparent charge heterogeneity after the gel filtration step, and only the major DEAE-cellulose peak was further purified on CM-cellulose. When SDS-polyacrylamide gel electrophoresis was carried out in the presence of mercaptoethanol, the two predominant bands seen in its absence were converted to five major bands, all of different apparent molecular weights from the originals. Isoelectric focusing yielded two major peaks of enzyme activity, at pI 7.0 and pI 4.5-5.0. These peaks were shown to be interconvertible upon reelectrofocusing. Both low- and high-molecular weight disulfides could be reduced. The pH optimum was sharp, at pH 8.2. The Km values for glutathione and cystine (the standard assay disulfide) were 0.57 and 0.062 mM, respectively, each in the presence of saturating concentrations of the other substrate. N-Ethylmaleimide at 0.1 and 1.0 mM inhibited enzyme activity non-competitively, suggesting a non-catalytic role of enzyme thiol(s) for maintenance of optimal activity.
D000818:Animals; D003593:Cytoplasm / Q000201:enzymology; D054477:Glutaredoxins; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010088:Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D010903:Pituitary Gland, Anterior / Q000201:enzymology*; D011490:Protein Disulfide Reductase (Glutathione); D013552:Swine
D054477:Glutaredoxins; D010088:Oxidoreductases; D011490:Protein Disulfide Reductase (Glutathione)