Phosphorylation of the Membrane Components of Chromaffin Granules: Synthesis of Diphosphatidylinositol and Presence of Phosphatidylinositol Kinase in Granule Membranes

Abstract

Adenosine triphosphate (ATP) induces the release of catecholamines, endogenous ATP, and soluble protein from chromaffin granules isolated from the adrenal medulla. When ATP exerts this action, it is hydrolyzed by enzymes present in the granule membrane, and part of the P_i liberated from ATP is transferred to the protein and lipid of the granule membrane. The phosphorylated lipid component, which was identified by thin-layer and ion-exchange chromatography as diphosphatidylinositol, was formed from ATP and monophosphatidylinositol. This latter phospholipid was the substrate for the enzyme phosphatidylinositol kinase. Both substrate and enzyme are components of the granule membranes, because they have a similar subcellular distribution as dopamine β-hydroxylase (a granule membrane marker). The formation of diphosphatidylinositol was Mg²⁺-dependent, it was further stimulated by Mn²⁺, it was inhibited by N-ethylmaleimide and the reaction had an optimal pH of 5. The synthesis of diphosphatidylinositol was also shown to occur in chromaffin granules "in situ" during the stimulation of the adrenal medulla by acetylcholine.