An apical-enriched plasma membrane fraction (A-PM) was prepared from rat parotid gland by Mn2+ precipitation. In this fraction, phosphatidylcholine (PC) labelled at the sn-2 position was mainly decomposed into two labelled compounds (free fatty acid and 1,2-diacylglycerol) under Ca2+-free conditions. Studies using double-labelled PC and 2,3-diphosphoglycerate (as a phospholipase D inhibitor) showed that they were produced through different pathways: free fatty acid was released by phospholipase A2 (PLA2) while 1,2-diacylglycerol may be produced by sequential action of phospholipase D and phosphatidate phosphatase. The PLA2 in A-PM did not require Ca2+ for its activity and was highly activated by Triton X-100 and ATP. The inhibitor of the well-documented Ca2+-independent PLA2, bromoenol lactone, did not inhibit the PLA2 activity in A-PM. Although PLA2 activity was detected in other subcellular fractions, the highest specific activity was in A-PM. Its distribution among various fractions was roughly similar to that of the marker enzyme of apical plasma membranes. These findings suggested that Ca2+-independent PLA2 activity is present in apical plasma membranes from rat parotid gland. In addition, to clarify the involvement of the PLA2 in exocytosis, the fusion of exogenous PLA2-treated membranes with secretory granules was examined by fluorescence dequenching assay. This study clearly demonstrated the facilitation of fusion by PLA2 treatment, which suggests some involvement of apical PLA2 in saliva secretion.
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