The long term objectives are to elucidate the molecular mechanisms by which cells regulate the synthesis, integration, translocation, and turnover of membrane phospholipids. The proposal focuses on the membrane-bound diacylglycerol kinase of Escherichia coli and its putative structural gene, dgkA. The specific aims are: to elucidate its primary structure; to investigate the phospholipid dependent reconstitution of activity in mixed micellar and vesicular systems; to kinetically characterize the reconstituted enzyme with emphasis on diacylglycerol utilization; to determine its secondary, and quarternary structure; to elucidate the topography of specific domains within the transverse plane of the membrane; to locate ATP, diacylglycerol and phospholipid binding sites; to determine the mechanism of regulation by a transacting regulatory mutation, dgkR; and to generate a set of molecularly defined mutant diacylglycerol kinases to further knowledge of structure and function. These aims are to be achieved by biochemical-genetic techniques that will exploit our previous studies on the cloning and DNA sequence analysis of the dgkA locus, construction of plasmids which overproduce diacylglycerol kinase activity over a hundred fold, and the development of novel extraction and purification (HPLC) techniques. In depth knowledge of the enzymes of glycerolipid metabolism is fundamental to understanding the molecular biology of membrane biogenesis and maintenance, to the structure and function of membrane bound enzymes and proteins, to cellular regulation, and to elucidation of the molecular basis of disease. Disturbance of glycerolipid metabolism is known to occur in cardiovascular disease and digestive diseases; in obesity, hypertension and stroke; in lipoprotein and bile disorders, and in respiratory distress syndrome.