TIME-DEPENDENT INHIBITION OF GLUCOSE 6-PHOSPHATASE BY 3-MERCAPTOPICOLINIC ACID

3-Mercaptopicolinate (3-MP) inhibits D-glucose-6-phosphate (G6P) phosp hohydrolase activity of the glucose-6-phosphatase system (Bode et al. (1993) Biochem. Cell Biol. 71, 113-121). We therefore attempted to max imize the inhibition by varying the physical state of microsomes, the concentration of 3-MP, and the time of preliminary incubation of 3-MP with the enzyme. The inhibition was irreversible and time- and inhibit or-concentration-dependent, with G6P phosphohydrolase activity of inta ct rat liver microsomes, but there was no inhibition with detergent-tr eated microsomes. The effectiveness of 3-MP as a time-dependent inhibi tor of glucose 6-phosphatase was demonstrated in situ by measuring gly cogenolysis in isolated, perfused livers from fed rats. We first expos ed the livers to 2 mM 3-MP for 40 min, and then assessed the inhibitor y effects on glycogenolysis. It was lowered by 50%. These observations establish that 3-MP at the mM level may be useful as an experimental probe in the study of the role(s) of G6P in the regulation of glycogen olysis as well as glycogenesis. Further, they validate the use of much lower (mu M) concentrations of 3-MP to block gluconeogenesis (at the phosphoenolpyruvate carboxykinase step) without interfering with gluco se 6-phosphatase. We also explored the mechanism of 3-MP inhibition. T he time-dependent inhibition of carbamoyl-phosphate:glucose phosphotra nsferase activity with microsomes incubated with 1 mM 3-MP for 60 or 9 0 min and then assayed with 1 mM carbamoyl phosphate and 180 mM glucos e was modest compared with inhibition of G6P phosphohydrolase. When G6 P production by carbamoyl-phosphate:glucose phosphotransferase was red uced by decreasing glucose concentration to 60 mM, no inhibition by 3- MP was discernible. There was no inhibition of inorganic pyrophosphata se activity. These studies support the model of time-dependent, irreve rsible reaction of 3-MP with the G6P translocase component of the gluc ose-6-phosphatase system.