Identification of a Membrane-Bound, Glycol-Stimulated Phospholipase A₂ Located in the Secretory Granules of the Adrenal Medulla

Chromaffin granule membranes prepared from bovine adrenal medullae showed Ca²⁺-stimulated phospholipase A₂ (PLA₂) activity when assayed at pH 9.0 with phosphatidylcholine containing an [¹⁴C]-arachidonyl group in the 2-position. However, the activity occurred in both soluble and particulate subcellular fractions, and did not codistribute with markers for the secretory granule. PLA₂ activity in the granule membrane preparation was stimulated dramatically by addition of glycerol, ethylene glycol, or poly(ethylene glycol). This glycol-stimulated PLA₂ activity codistributed with membrane-bound dopamine β-hydroxylase, a marker for the granule membranes, through the sequence of differential centrifugation steps employed to prepare the granule membrane fraction, as well as on a sucrose density gradient which resolved the granules from mitochondria, lysosomes, and plasma membrane. The glycol-stimulated PLA₂ of the chromaffin granule was membrane-bound, exhibited a pH optimum of 7.8, retained activity in the presence of EDTA, and was inactivated by p-bromophenacyl bromide. When different ¹⁴C-labeled phospholipids were incorporated into diarachidonylphosphatidylcholine liposomes, 1-palmitoyl-2-arachidonylphosphatidylcholine was a better substrate for this enzyme than 1-palmitoyl-2-oleylphosphatidylcholine or 1-acyl-2-arachidonyl-phosphatidylethanolamine, and distearoylphosphatidylcholine was not hydrolyzed.