Glucagon activation of the thiol:Protein disulfide oxidoreductase in isolated, rat, hepatic microsomes

The hepatic, microsomal, thiol:protein disulfide oxidoreductase catalyzes the glutathione (GSH) reduction of protein disulfides to sulfhydryl groups. In the presence of physiological concentrations of glucagon this activity increased from 2.3 to 6.4 fold in isolated microsomes. The stimulation had a P₅₀ for glucagon of 7.8 × 10^-10 M which was only observed at microsomal protein concentrations of less than 100 ug/ml and in the presence of a GSH reducing system. This latter observation suggests that the stimulation may be inhibited by the presence of oxidized glutathione. These data support the hypothesis that glucagon may act in part by stimulating the reduction of protein disulfides by the thiol:protein disulfide oxidoreductase. There is an active interest in elucidating the mechanism of the transduction of hormonal action. Among the mechanisms which have been most actively studied have been those schemes which involve changes in the phosphorylation status of critical enzymes in various metabolic pathways (1). Recently Ziegler (2,3) has suggested an alternative mechanism in which the regulation of the activity of these pathways is mediated by changes in the oxidation-reduction state of various enzymes in the particular metabolic pathway. Even though to date there have been no published reports presenting data directly supporting this mechanism (3), a recent study from our laboratory would suggest that changes in the ratio of protein disulfides to sulfhydryl groups may be important in hormone action (4). In this studies we observed that in isolated, hepatic microsomes, the ATP-dependent calsium pump is inactivated through direct oxidation by a NADPH-dependent system (5). This inhibition appears to be catalyzed by a cytochrome P-450 dependent pathway. Further, the pump activity can be partially restored by reduced glutathione, presumably through a microsomal, thiol:protein disulfide oxidoreductase (5, MS submited). Finally we have found that physiological concentrations of norepinephrine (10^-8 M), when added to the isolated microsomal preparation, can significantly increase this NADPH-dependent inhibition of the calcium pump (4). Together these observations are consistent with the hypothesis that the hormonal action of norepinephrine in the hepatocyte may be mediated by inactivation of the calcium pump and that this inactivation is due to the reversible oxidation of critical sulfhydryls in one or more components of the calcium pump system. Since our previous studies suggested that the modulation of oxidative processes could mediate the transduction of the action of norepinephrine, it is possible that the activities of reductive enzymes are also modulated by hormones (3). In the current study, we have therefore examined in incubations of hepatic microsomes, the effect of various concentrations of glucagon on the activity of the GSH dependent microsomal enzyme, thiol:protein disulfide oxidoreductase (EC 1.8.4.2). This enzymes catalyzes the GSH reduction of protein disulfides to sulfhydryls. We find that in this preparation the addition of physiological concentrations the hormone (10^-9) stimulated from 2.3-6.4 fold the GSH reduction of endogenous and exogenous protein disulfides.