Role of Mg-ATP in norepinephrine biosynthesis in intact chromaffin granules

Dopamine beta-monooxygenase converts dopamine to norepinephrine in intact chromaffin granules using intragranular ascorbic acid as a cosubstrate. Mg-ATP with external ascorbic acid is required for maximal norepinephrine biosynthesis. Mechanisms to explain these requirements were investigated specifically using intact granules. The effect of Mg-ATP was independent of membrane potential (delta psi) because norepinephrine biosynthesis was unchanged whether delta psi was positive or collapsed. Furthermore, the effect of Mg-ATP was independent of absolute intragranular and extragranular pH as well as the pH difference across the chromaffin granule membrane (delta pH). Nevertheless, norepinephrine biosynthesis was inhibited by N-ethylmaleimide, 4-chloro-7-nitrobenzofurazane, and N,N-dicyclohexylcarbodiimide, specific inhibitors of the secretory vesicle ATPase that may directly affect proton pumping. Biosynthesis occurred normally with other ATPase inhibitors that do not inhibit the ATPase in secretory vesicles. The data indicate that the effect of Mg-ATP with ascorbic acid is mediated by the granule membrane ATPase but independent of maintaining delta psi and delta pH. An explanation of these findings is that Mg-ATP, via the granule ATPase, may change the rate at which protons or dopamine are made available to dopamine beta-monooxygenase.