Complete discrimination of docosahexaenoate from arachidonate by 85 kDa cytosolic phospholipase A2 during the hydrolysis of diacyl- and alkenylacylglycerophosphoethanolamine

Abstract In our previous report (Shikano, M., Masuzawa, Y. and Yazawa, K. (1993) J. Immunol. 150, 3525–3533), we described that the enrichment of docosahexaenoic acid (DHA, 22:6( n − 3) reduces both arachidonic acid (AA, 20:4( n − 6)) release and platelet-activating factor (PAF) synthesis in human eosinophilic leukemia cells, Eol-1. Since no DHA release was observed in response to Ca-ionophore stimulation, we presumed that the phospholipase A 2 (PLA 2 ) responsible for AA release and PAF synthesis can not hydrolyze the DHA moiety of phospholipids. In the present paper, we examined whether DHA-containing diacyl- and alkenylacylglycerophosphoethanolamine (DHA-diacylGPE and DHA-alkenylacyGPE) are susceptible to the action of AA-preferential 85 kDa cytosolic phospholipase A 2 (cPLA 2 ) from rabbit platelets in comparison with AA and eicosapentaenoic acid (EPA, 20:5( n − 3)) derivatives. When diacylGPE was used as a substrate, DHA release was almost negligible under the assay condition that allowed AA and EPA to be liberated at the rates of 4.3 μmol/min per mg protein and 2.5 μmol/min per mg protein, respectively. On the other hand, 14 kDa type II PLA 2 hydrolyzed DHA-diacylGPE as well as AA-diacylGPE and EPA-diacylGPE. When DHA-diacylGPE and AA-diacylGPE were mixed at equimolar concentrations, DHA release by cPLA 2 was not observed and AA release was reduced to 32% in the case without DHA-diacylGPE. This indicated that DHA-diacylGPE is a poor substrate but possesses the inhibitory activity for cPLA 2 . cPLA 2 does not clearly discriminate between AA-alkenylacylGPE and AA-diacylGPE. As in the case using diacylGPE as a substrate, DHA-alkenylacylGPE was completely discriminated from AA-alkenylacylGPE by cPLA 2 . The roles of DHA and cPLA 2 in the synthesis of lipid mediators will be discussed in relation to the new aspects of the substrate specificity of cPLA 2 provided here.