S-thiolation of cytoplasmic cardiac creatine kinase in heart cells treated with diamide.
Two methods for quantitation of protein S-thiolation, by isoelectric focusing or by enzyme activity, were used for studying S-thiolation of cytoplasmic cardiac creatine kinase. With these methods, creatine kinase was identified as a major S-thiolated protein in both bovine and rat heart. In rat heart cell cultures, creatine kinase became 10% S-thiolated during a 10 min incubation with 0.2 mM diamide. This enzyme became S-thiolated more slowly than other heart cell proteins and it also dethiolated more slowly. Two sequential additions of diamide at a 25 min interval caused twice as much S-thiolation after the second addition as compared to the first. This increased sensitivity to the second diamide treatment may have resulted from glutathione loss during the first addition which produced a higher GSSG-to-GSH ratio after the second treatment. The GSSG-to-GSH ratio was highest prior to the maximum S-thiolation of creatine kinase, but, in general, the time course of glutathione was similar to the S-thiolation of creatine kinase. This study demonstrates that cytoplasmic creatine kinase is S-thiolated and, therefore, inhibited during a diamide-induced oxidative stress in heart cells. Implications for regulation of cardiac metabolism during oxidative stress are discussed.
D000818:Animals; D001391:Azo Compounds / Q000494:pharmacology*; D002417:Cattle; D002478:Cells, Cultured; D003402:Creatine Kinase / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D003958:Diamide / Q000494:pharmacology*; D005978:Glutathione / Q000378:metabolism; D006321:Heart / Q000187:drug effects*; D009206:Myocardium / Q000378:metabolism; D051381:Rats; D013438:Sulfhydryl Compounds / Q000378:metabolism
D001391:Azo Compounds; D013438:Sulfhydryl Compounds; D003958:Diamide; D003402:Creatine Kinase; D005978:Glutathione