The long-term objectives of this research are to elucidate the metabolic relationships between lipids and to study the enzymatic control of membrane lipid biogenesis. The specific objective of this proposal is to characterize the enzymes of the acyl dihydroxyacetone phosphate (acyl DHAP) pathway which catalyze the biosynthesis of the key membrane lipid precursors, phosphatidic acid and its ether analog. Several recent discoveries, such as the presence of acyl DHAP pathway enzymes in microbodies (peroxisomes and microperoxisomes), the identification of a number of congenital peroxisomal disorders which result in a deficiency of tissue ether lipids, and the recognition of biomessenger and second messenger roles for ether lipids, have emphasized the importance of the acyl DHAP pathway enzymes in membrane lipid biogenesis. Two of these enzymes, DHAP acyltransferase and alkyl DHAP synthase, are deficient in a number of genetic diseases involving peroxisomal disorders, such as Zellweger cerebrohepatorenal syndrome (ZS), Rhizomelic chondrodysplasia punctata (RCDP), neonatal adrenoleukodystrophy, infantile Refsum disease, etc. Individuals with these diseases suffer from progressive neurological disorders and mental retardation, and are diagnosed by the deficiency of their cellular DHAP acyltransferase. In this application we intend to make thorough studies of the molecular properties and cellular regulation of two of these acyl DHAP pathway enzymes. The following specific aims are proposed: 1) To purify the key membrane-bound enzyme, DHAP acyltransferase (DHAPAT), to homogeneity. Following purification, the properties, catalytic reaction mechanisms, and regulation of its activity by membrane lipids will be investigated. 2) To generate specific antibodies against DHAPAT and acyl/alkyl DHAP reductase (which has already been purified to homogeneity), which will be used to study the tissue distribution and subcellular localization of the enzymes, the developmental profile of the enzymes in brain, and to detect the presence of mutant enzymes in peroxisomal genetic diseases, such as ZS and RCDP. 3) To study the molecular properties, regulation and biogenesis of acyl/alkyl DHAP reductase by determining its primary structure by cloning and sequencing the corresponding cDNA. The results obtained from these studies will not only enhance our understanding of membrane lipid biogenesis and the function of ether lipids, but will also provide knowledge regarding the molecular defects in aberrant peroxisomal lipid synthesis that may lead to treatments for diseases involving these disorders.
