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 beta-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.
Hildebrandt
E
E
Pharmacology Department, University of Texas Southwestern Medical Research Center, Dallas 75235.
Albanesi
J P
JP
D000098:Acetophenones / Q000494:pharmacology; D000313:Adrenal Medulla / Q000201:enzymology*; D000818:Animals; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D002451:Cell Compartmentation; D002837:Chromaffin Granules / Q000201:enzymology*; D004299:Dopamine beta-Hydroxylase / Q000378:metabolism; D006018:Glycols / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000378:metabolism; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054467:Phospholipases A2; D013379:Substrate Specificity
D000098:Acetophenones; D001095:Arachidonic Acids; D006018:Glycols; D016718:Arachidonic Acid; D004299:Dopamine beta-Hydroxylase; D010741:Phospholipases A; D054467:Phospholipases A2; C006821:4-bromophenacyl bromide; D002118:Calcium