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.