Dephosphorylation of a purified bovine cardiac phosphorylated glycogen synthase can be inhibited by glycogen. Aging glycogen-free glycogen synthase in the absence of reducing agent or incubating it with oxidized glutathione (GSSG) at -20(DEGREES)C causes desensitization of the dephosphorylation by E. coli alkaline phosphatase to glycogen inhibition. The alteration in the sensitivity of glycogen synthase dephosphorylation to glycogen can be reversed by a short incubation of the enzyme with dithiothreitol (DTT), but not by dialysis or gel filtration. Treatment of glycogen-free glycogen synthase with GSSG (or other low molecular weight disulfides) also causes enzyme inactivation. This effect is used to study the reactivity of glycogen synthase sulfhydryls. The GSSG-mediated enzyme inactivation is time- and concentration-dependent, and can also be reversed by a short incubation with DTT. Reaction of GSSG with a single sulfhydryl group per subunit results in loss of 60-70% of the enzyme activity. Further loss of protein sulfhydryls has little effect on the enzyme activity. There is no evidence for formation of intra- or inter-subunit disulfides;Reaction of glycogen synthase with low molecular weight disulfides forms protein mixed-disulfides through protein sulfhydryl-disulfide exchange reaction. Uridine 5'-diphosphate glucose and glycogen prevent inactivation of glycogen-free glycogen synthase with GSSG, but glucose-6-phosphate only retards the rate of inactivation. Reduction (reactivation) of GSSG-oxidized glycogen synthase requires strong reducing agents. Physiological reductants such as cysteine and reduced glutathione are not effective at neutral pH;To examine the possible physiological significance of sulfhydryl modification of glycogen synthase, the effects of glycogen on glycogen synthase dephosphorylation in heart extracts are studied. The dephosphorylation of glycogen synthase in rat heart extracts can be studied with E. coli alkaline phosphatase in the presence of potassium fluoride. Dephosphorylation in heart extracts from normal, fasted, or fasted-refed rats responds to added glycogen differently. Dephosphorylation in extracts from fasted hearts is the most sensitive, and that from fasted-refed animals is the least sensitive to glycogen inhibition. The different responses may be caused by a stable modification, sulfhydryl modification (formation of protein mixed-disulfides).