Ca2+-independent phospholipase A(2) activity in apical plasma membranes from the rat parotid gland

An apical-enriched plasma membrane fraction (A-PM) was prepared from rat pa rotid gland by Mn2+ precipitation. In this fraction. phosphatidylcholine (P C) labelled at the sn-2 position was mainly decomposed into two labelled co mpounds (free fatty acid and 1,2-diacylglycerol) under Ca2+-free conditions . Studies using double-labelled PC and 2,3-diphosphoglycerate (as a phospho lipase D inhibitor) showed that they were produced through different pathwa ys: free fatty acid was released by phospholipase A(2) (PLA(2)) while 1,2-d iacylglycerol may be produced by sequential action of phospholipase D and p hosphatidate phosphatase. The PLA(2) in A-PM did not require Ca2+ for its a ctivity and was highly activated by Triton X-100 and ATP. The inhibitor of the well-documented Ca2+-independent PLA(2), bromoenol lactone, did not inh ibit the PLA(2) activity in A-PM. Although PLA(2) 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 ma rker enzyme of apical plasma membranes. These findings suggested that Ca2+- independent PLA(2) activity is present in apical plasma membranes from rat parotid gland. In addition, to clarify the involvement of the PLA(2) in exo cytosis, the fusion of exogenous PLA(2)-treated membranes with secretory gr anules was examined by fluorescence dequenching assay. This study clearly d emonstrated the facilitation of fusion by PLA(2) treatment, which suggests some involvement of apical PLA(2) in saliva secretion. (C) 2001 Elsevier Sc ience Ltd. All rights reserved.