Purification and properties of thioltransferase.
A protein, previously designated thioltransferase (Askelof, P., Axelsson, K., Eriksson, S., and Mannervik, B. (1974) FEBS Lett. 38, 263-267) was purified to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and flatbed gel isoelectric focusing. The preparative procedure, a modification of that of Axelsson et al. (Axelsson, K., Eriksson, S., and Mannervik, B. (1978) Biochemistry 17, 2978-2984) and Hatakeyama et al. (Hatakeyama, M., Tanimoto, Y., and Mizoguchi, T. (1984) J. Biochem. (Tokyo) 95, 1811-1818) was faster and higher-yielding than the previous procedures. The purified enzyme has a molecular weight of 11,700 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a pI of 8.8. The amino acid composition of thioltransferase is reported, and it closely resembles that of calf thymus glutaredoxin. The optimal pH for this enzyme was 8.5 when S-sulfocysteine was used as a substrate. The plots of the activity of thioltransferase as a function of S-sulfocysteine and 2-hydroxyethyl disulfide concentrations showed sigmoidal relationships. The K0.5 for S-sulfocysteine was 0.6 mM. The enzyme was very sensitive to sulfhydryl alkylating reagents. Preincubation of the enzyme with disulfide compounds prevented the enzyme from inactivation by iodoacetamide but inhibited the thioltransferase activity in the absence of iodoacetamide. The results suggest that the active center of thioltransferase is cysteine dependent and that substrates may form mixed disulfides with the enzyme. Based on the iodoacetamide inactivation and disulfide protection of thioltransferase activity, a model for the catalytic mechanism of the thiol-disulfide oxidoreduction is proposed.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D003545:Cysteine / Q000031:analogs & derivatives / Q000378:metabolism; D003593:Cytoplasm / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D054477:Glutaredoxins; D005978:Glutathione / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007460:Iodoacetamide / Q000494:pharmacology; D007525:Isoelectric Focusing; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008970:Molecular Weight; D010088:Oxidoreductases / Q000302:isolation & purification*; D011490:Protein Disulfide Reductase (Glutathione); D051381:Rats
D000596:Amino Acids; D054477:Glutaredoxins; C011119:S-sulphocysteine; D010088:Oxidoreductases; D011490:Protein Disulfide Reductase (Glutathione); D005978:Glutathione; D003545:Cysteine; D007460:Iodoacetamide