The major objective of the proposed research is to study the properties and physiological function of the enzymes catalyzing the formation of phosphatidic acid, the key precursor of glycerides and phosphoglycerides. Detailed studies will be made on the enzymes of the acyl dihydroxyacetone phosphate (acyl DHAP) pathway, namely DHAP acyltransferase, alkyl DHAP synthase and acyl/alkyl DHAP reductase. The acyl DHAP pathway has been shown to play a role in the biosynthesis of glycerolipids, especially the containing ether linkages (glycerol-ether lipids and plasmalogens). In recent years, a number of discoveries such as the presence of acyl DHAP pathway enzymes in microbodies (peroxisomes and microperoxisomes), the demonstration that in patients with Zellweger cerebrohepatorenal syndrome both the microbodies and glycerol-ether lipids are absent, and the discovery that an ether lipid (platelet activating factor), has a potent biomessenger role, all point towards the importance of the acyl DHAP pathway in lipid metabolism. In the proposed studies, our aim is to purify the membrane-bound acyl DHAP pathway enzymes to homogeneity and study in detail their properties and reaction mechanisms. Attempts will be made to raise specific antibodies against these enzymes and then to use these antibodies to estimate and to localize the enzymes in different tissues. The activity of these enzymes under different physiological conditions and the contributions of the acyl DHAP pathway to the biosynthesis of non-ether glycerolipids will be studied. Different analogs of acyl/alkyl DHAP will be chemically synthesized to test their possible use as inhibitors or substrates for different enzymes. The role of ether lipids will be investigated by studying their distribution, turnover, and transport in different systems. The incorporation of exogenously added ether lipid precursors to membrane lipids of skin fibroblasts from Zellweger syndrome patients and in whole animals will also be studied. The deficiency of different acyl DHAP pathway enzymes in the cultured fibroblasts from patients suffering from Zellweger syndrome, neonatal adrenoleukodystrophy and related diseases will be studied in detail.
