Effects of prostaglandin E1 and adenosine on metal and metal-ATP kinetics of platelet adenylate cyclase.

The regulation of platelet adenylate cyclase by free metal and metal-ATP was studied. The effects on that regulation of adenosine, under stimulatory or inhibitory conditions, and of prostaglandin El, were evaluated. Under conditions when CAMP formation was linear with respect to time and protein concentration, double reciprocal plots with respect to either free (excess) Mg+ or MgATP were linear and intersecting to the left of the ordinate. Replots of the slopes and intercepts of these plots were also linear. The kinetic behavior of this membrane-bound adenylate cyclase conformed to that of a bireactant, random sequential mechanism in which MgATP2is substrate and free Mg+ is a requisite activator. This behavior was similar to that of the detergent-dispersed enzyme from cerebellum (Garbers, D. L., and Johnson, R. A. (1975) J. Biol. Chem. 250, 8449-8456), and suggested that the membrane environment per se does not imply anomalous kinetic behavior. Moreover, since the kinetic behavior of the platelet enzyme was similarly linear with either adenosine or prostaglandin El (PGE,), stimulation of the cyclase by these agents was not accompanied by induced apparent cooperativity with respect to either M$+ or MgATP. The kinetic constants for basal adenylate. cyclase were ZC, (MS’) 1.13 mM, ZCi (M$‘) 1.17 mM, K, (MgATP) 54 FM, and Ki (MgATP) 45 PM. Stimulation of the enzyme la-fold by 1 pM prostaglandin El was not accompanied by a significant decrease in the Michaelis constant for Mg’+, whereas stimulation 2-fold by 30 PM adenosine was accompanied by a 2to 3-fold increased affinity for free M$+. However, this effect of adenosine to alter metal ion affinity may have been a consequence of the inhibitory and not of the stimulatory effect of the nucleoside. Increasing the divalent cation concentration had the effect of diminishing stimulation by adenosine and enhancing inhibition. Neither adenosine nor PGEl had a great effect on the respective constants for MgATP. Stimulation and inhibition by adenosine were seen in the presence of a maximally stimulatory concentration of guanyl-5’-yl(P-y-imino)diphosphate (GMP-P(NH)P).