REDUCTION (DETHIOLATION) OF PROTEIN MIXED-DISULFIDES - DISTRIBUTION AND SPECIFICITY OF DETHIOLATING ENZYMES AND N,N'-BIS(2-CHLOROETHYL)-N-NITROSOUREA INHIBITION OF AN NADPH-DEPENDENT CARDIAC DETHIOLASE

The S-thiolated proteins phosphorylase b (Phb) and carbonic anhydrase III (CAIII) were prepared with [3H]glutathione in a reaction initiated with diamide. These substrates were used to measure the rate of reduction (dethiolation) of protein mixed-disulfides by enzymes with properties similar to those of thioredoxin and glutaredoxin. This enzyme activity is termed a dethiolase since the identities of the enzymes are still unknown. The dethiolation of either S-[3H]glutathiolated Phb or S-[3 H]glutathiolated CAIII was employed in tissue assays and for study of two partially purified dethiolases from cardiac tissue. NADPH-dependent dethiolase activity was most abundant except in rat liver and muscle. Total dethiolase activity was approximately 10-fold higher in neutrophils, 3T3-L1 cells, and Escherichia coli than in other sources. Rat skeletal muscle had 3- to 4-fold higher dethiolase activity than rat heart or liver. These data indicate that protein dethiolase activity is ubiquitous and that normal expression of the two dethiolase activities varies considerably. A partially purified cardiac NADPH-dependent dethiolase acted on Phb approximately 1.5 times faster than CAIII, and a glutathione (GSH)-dependent dethiolase acted on Phb 3 times faster than CAIII. The Km for glutathione for the GSH-dependent dethiolase was 15 μm with Phb as substrate and 10 μm with CAIII. Thus, the GSH-dependent dethiolase is probably not affected by normal changes in the cardiac glutathione content (normally approximately 3 mm). Partially purified cardiac NADPH-dependent dethiolase was inactivated by BCNU (N,N′-bis(2-chloroethyl)-N-nitrosourea) and the GSH-dependent dethiolase was unaffected under similar conditions. In a soluble extract from bovine heart, 200 μm BCNU inhibited NADPH-dependent dethiolase by more than 60% but did not affect GSH-dependent activity. These results demonstrate that BCNU is a selective inhibitor of the NADPH-dependent dethiolase. © 1991.