ATP-mediated activation of Ca²⁺-independent phospholipase A₂ in secretory granular membranes from rat parotid gland

We characterized the Ca²⁺-independent, membrane-associated phospholipase A₂ (PLA₂) from rat parotid secretory granules. Among four phosphatidylcholine species with different fatty acyl (palmitoyl, oleoyl, linoleoyl, and arachidonoyl) groups at the sn-2 position, 2-arachidonoyl-phosphatidylcholine was the preferred substrate. Such specificity was also apparent even when 2-arachidonoyl-phosphatidylcholine coexisted with another species. The various well-documented inhibitors of PLA₂s, bromoenol lactone, arachidonyl trifluoromethyl ketone, methyl arachidonyl fluorophosphate, and diisopropyl fluorophosphate, did not inhibit granular PLA₂ activity. The granular PLA₂ was activated markedly by ATP, and to a lesser extent by GTP and ATPγS. GTP also partially suppressed the ATP-mediated activation. UTP, CTP, GTPγS, and the hydrolyzed products of ATP and GTP showed little activation of the enzyme. Neither addition of K-252a nor depletion of Mg²⁺ affected ATP-mediated activation. Although this enzyme was located in the granular membranes, the granular soluble contents or BSA were required for the full activity and full ATP-mediated activation. These results suggested that the PLA₂ located in granular membranes may participate in the liberation of arachidonic acid in parotid cells and be regulated through a mechanism mediated by ATP.