The studies in this proposal are designed to investigate the mechanism by which the catabolic and anabolic processes contribute to the composition and turnover of phospholipid species. Our approach is to measure the specificities of the acylation and deacylation reactions in vitro and correllated these results with studies to determine the turnover rates of individual species in intact cells. We will use cultures of a kidney cell line (MDEK cells) as a model system for both enzymatic and cellular studies. Experiments at the cellular level will measure the rates at which fatty alcohols, fatty acids and glycerol are incorporated into individual species of phospholipids. The method we have developed for the quantitative analysis of phospholipid species makes it possible to follow the specific radioactivities in each molecular species during radiolabelling of cells. We can thereby assess the turnover of different moieties of particular molecules of phospholipid. These studies will provide information on 1) the contribution of the de novo pathway to the synthesis of ether and diacyl glycerolipid species, 2) the influence of the sn-1 linkage, sn-1 aliphatic group and polar headgroup on the turnover of the acyl moiety at position 2 of phospholipids, and 3) the species involved in the mobilization of arachidonic acid between phospholipid classes. Molecular species analysis will be used to examine the specificity of acylation and deacylation reactions in vitro. The ability to follow the catalysis of individual species in a complex mixture of natural substrates provides a means of determining the specificity of enzymes for each moiety of the phospholipid molecule. We will examine the acyl-CoA acyltranferase an CoA-independent transacylase reactions in MDCK cell membranes as well as other tissues. The specificity of deacylation of phospholipid species in MDCK cell membranes will also be examined. A comparison of the specificities of these enzymes with the turnover of acyl groups in different species of phospholipids in intact cells will indicate their contributions to the synthesis and turnover of phospholipids.
