S-Thiolation of cytoplasmic cardiac proteins in vivo

Beating neonatal heart cell cultures were treated with diamide or t-butyl hydroperoxide (tBuOOH), and changes in glutathione oxidation, cell beating, and protein S-thiolation (protein mixed-disulfide formation) were examined. Both compounds caused extensive oxidation of glutathione. Cells treated with diamide stopped beating within 2 min, and beating returned to normal after 30-45 min. Cells stopped beating 25 min after addition of tBuOOH, and beating did not resume. tBuOOH caused S-thiolation of a variety of proteins, but only one protein, of molecular mass 23 kDa, was extensively modified. Diamide caused extensive modification of proteins with molecular masses of 97, 42, and 23 kDa as well as three proteins of about 35 kDa. Though the GSSG content of cell cultures returned to normal by 15 min after diamide treatment, S-thiolation of several proteins persisted. These studies show that S-thiolation of proteins is an important metabolic response in cells exposed to an oxidative challenge by tBuOOH or diamide, and that the specificity of the response depends on the agent used;Two methods for quantitation of protein S-thiolation, by isoelectric focusing or by enzyme activity after reaction with NEM, 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. S-Thiolation of creatine kinase with GSSG led to inhibition of enzyme activity. In rat heart cell cultures, creatine kinase became 8% 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. When two sequential additions of 0.2 mM diamide were made at a 25 min interval, the second addition produced twice as much S-thiolation as 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. This study demonstrates that cytoplasmic creatine kinase is inhibited by S-thiolation during a diamide-induced oxidative stress in heart cells. Implications of these results for cardiac function during reperfusion injury are discussed