The long-term objective of this proposal is to elucidate the molecular mechanisms which function to regulate the amounts and types of membrane phospholipids synthesized. The experimental approach is one of biochemistry and genetics. Each of the specific aims focuses on either the cytoplasmic membrane-bound sn-glycerol 3-phosphate acyltransferase of Escherichia coli or its structural gene, plsB. During the last grant period, the plsB gene was cloned and the sn-glycerol 3-phosphate acyltransferase was solubilized, reconstituted, and purified.
The specific aims are to sequence the plsB gene; to elucidate the primary structure of the enzyme; to determine the efficiency and specificity of reconstitution with phospholipids; to characterize the reconstituted enzyme kinetically with emphasis on acyl-CoA and acyl-ACP utilization; to elucidate the structure of the enzyme by focusing on its topography within the transverse plane of the membrane; to generate defined mutants to probe the structure, function, and regulation of activity. this enzyme catalyzes the committed step of pathways producing 10-90% the dry weight of cells. Ours are the first pure preparations. Knowledge of its structure, function, and regulation are relevant to fundamental biological problems (membrane biogenesis, assembly of pospholipid bilayers, and protein-lipid interactions) and to significant problems of human health (cardivasular and digestive diseases; obesity, hypertention, and stroke; liporpotein and bile metabolic disorders; alcholoism; respiratory distress syndrome; and cancer).
