Inhibition of cyclic AMP-dependent protein kinase-induced changes in the kinetic properties of hepatic pyruvate kinase by the specific cyclic AMP antagonist, the (Rp)-diastereomer of adenosine cyclic 3',5'-phosphorothioate.

Abstract

The effects of the diastereomers of adenosine cyclic 3',5'-phosphorothioate, (Sp)- and (Rp)-cAMPS, on the kinetic properties of pyruvate kinase were studied in hepatocytes isolated from fed rats. Incubation of the cells with the cAMP-dependent protein kinase agonist, (Sp)-cAMPS, produced a concentration-dependent increase in S0.5 for phosphoenolpyruvate, but had no effect on Vmax. The (Sp)-cAMPS-treated enzyme was more sensitive to inhibition by alanine and ATP and, under the same conditions, was less responsive to activation by fructose-1,6-bisphosphate when assayed at a subsaturating phosphoenolpyruvate concentration. Incubation of the hepatocytes with only the cAMP-dependent protein kinase antagonist, (Rp)-cAMPS, produced no change in any kinetic parameters, but did suppress the (Sp)-cAMPS- or glucagon-induced increase in the S0.5 for phosphoenolpyruvate with IC50 values of 10 microM and 5 microM (Rp)-cAMPS. (Rp)-cAMPS is exerting an effect on the kinetic properties of pyruvate kinase through inhibition of cAMP-dependent protein kinase.