Preparation of homogeneous pig liver thioltransferase by a thiol:disulfide mediated pI shift.
An enzyme catalyzing thiol-disulfide exchange, thioltransferase, was purified to homogeneity from pig liver. By taking advantage of the relatively large pI shift of the enzyme between its reduced and disulfide forms, the purification procedure, which included a heat step, ammonium sulfate precipitation, Sephadex G-75 and G-50 gel chromatography, and two CM-Sepharose chromatography separations, resulted in a 32% overall yield. The purified enzyme was demonstrated to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and high-performance liquid chromatography. The protein had a Mr of approximately 11,000 and, in the reduced form, a pI of 6.4. The amino acid composition of the enzyme was similar to that of rat liver thioltransferase and calf thymus glutaredoxin and the N-terminus of the protein was blocked. The optimal pH for the enzyme activity was 9.0. The plots of thioltransferase activity as a function of S-sulfocysteine, 2-hydroxyethyl disulfide, and reduced glutathione concentrations did not display Michaelis-Menten kinetics. The enzyme was very sensitive to a sulfhydryl alkylating reagent. Preincubation of the enzyme with its disulfide substrates prevented the inactivation of the enzyme by iodoacetic acid while the other substrate, GSH, did not provide such protection. The results suggest that the active center of thioltransferase is cysteine dependent.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002845:Chromatography / Q000379:methods; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D054477:Glutaredoxins; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000302:isolation & purification*; D011490:Protein Disulfide Reductase (Glutathione); D013438:Sulfhydryl Compounds; D013552:Swine
D000596:Amino Acids; D004220:Disulfides; D054477:Glutaredoxins; D013438:Sulfhydryl Compounds; D010088:Oxidoreductases; D011490:Protein Disulfide Reductase (Glutathione)