Reduction (dethiolation) of protein mixed-disulfides; distribution and specificity of dethiolating enzymes and N,N'-bis(2-chlorethyl)-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-[3H]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 microM with Phb as substrate and 10 microM 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 microM 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.
Miller
R M
RM
Department of Biochemistry and Biophysics, Iowa State University, Ames 50011.
Park
E M
EM
Thomas
J A
JA
D000818:Animals; D002330:Carmustine / Q000494:pharmacology*; D002417:Cattle; D004220:Disulfides / Q000378:metabolism*; D054477:Glutaredoxins; D051379:Mice; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D009928:Organ Specificity; D010084:Oxidation-Reduction; D010088:Oxidoreductases; D000091402:Prohibitins; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D012995:Solubility; D013379:Substrate Specificity; D013879:Thioredoxins / Q000378:metabolism
D004220:Disulfides; C516010:Glrx2 protein, rat; D054477:Glutaredoxins; C000717332:Phb protein, rat; D000091402:Prohibitins; D011506:Proteins; D013879:Thioredoxins; D009249:NADP; D010088:Oxidoreductases; D009247:NADH, NADPH Oxidoreductases; D002330:Carmustine