One of the major aims of this renewal application is to characterize a novel closely-integrated CoA-independent transacylase and phospholipase A2 (PLA2) activities that are involved in the production of platelet activating factor (PAF) in the remodeling pathway and in the movement of arachidonate among phospholipids. These experiments are focused on the substrate specificities of the enzymes for the donor and acceptor phospholipid molecules that participate in transacylation reactions involving arachidonate (and other polyenoates) and the accompanying production of PAF. The mechanism of the transacylation process and the kinetics of the 20:4 movement in intact cells and isolated microsomes will be high priority projects in this proposal. The complex series of reaction that account for the formation of lyso-PAF and PAF during transacylation include a) a PLA2 (I) to form a lyso-phospholipid acceptor from ethanolamine plasmalogens or other choline- and ethanolamine-containing glycerophosphatides, b) a transacylase/PLA2 (II) that forms lyso-PAF and transfers arachidonate or other polyenoates from the donor phospholipid (alkylarachidonoylglycerophosphocholine) to the lyso-phospholipid acceptor generated by PLA2 (I) and c) the lyso-PAF acetyltransferase that forms PAF. The enzyme activities [PLA2 (I), PLA2 (II), and transacylase] could represent one, two, or three individual catalytic proteins. Therefore, to address this issue we will attempt to purify the enzymes involved in the CoA-independent transacylation reactions. A second significant aim is to investigate factors responsible for the regulatory control of the metabolism and levels of PAF. These studies include and investigation of the role of hormones, cellular differentiation, and membrane vs. cytosolic forms of acetylhydrolase on PAF metabolism. Finally, we plan to examine the compartmentalization (subcellular location and topographical mapping) of the PAF cycle enzymes, products (PAF and the corresponding acyl and plasmalogen analogs), and PAF precursors (lyso and diradyl forms of phospholipids). The membrane topography studies are very closely integrated with the experiments on PAF regulation. Experimental strategies to accomplish these aims utilize intact and permeabilized cells, isolated membrane fractions or organelles, and purified/partially purified enzymes. Cultured HL-60 cells are the primary cell model; however, rabbit platelets and rat alveolar macrophages and peritoneal neutrophils will be used for comparative purposes. Results of this research are expected to provide a major contribution to our understanding of the complex factors that regulate PAF metabolism and of the very close interrelationships between ether-linked phospholipids and eicosanoid mediators.
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