In an effort to understand how penicillin interacts with cell surfaces and kills bacteria, the proposed research will focus on the problem of purifying and characterizing the penicillin sensitive membrane receptor target enzymes in Escherichia coli and Staphylococcus aureus. Particular emphasis will be placed upon the isolation and identification of the transpeptidase since this enzyme is thought to be the killing site of the penicillins and related cephalosporin antibiotics. The techniques used for isolation and characterization of the target proteins and lipoproteins will be those of the protein chemist supplemented by methods such as detergent treatment and lipid activation, frequently used for the purification of membrane proteins. Subsequent to physical and chemical studies, attempts would be made either to reconstitute the soluble proteins to depleted membrane structures by combining the soluble protein with lipid and divalent cations. With this knowledge in hand and with the establishment of an analytical method of identifying the proteins, quantitative studies on penicillin uptake and binding, using radioactive penicillin, will be performed with purified enzymes, whole cells, protoplasts, disrupted membrane fragments, and reconstituted materials. The knowledge obtained will be correlated with penicillin inhibition of cell wall synthesis and attempts will be made to locate these receptor proteins on the membranes of the bacteria. Bibliographic references: Pollock, J. J., M. Nguyen-Disteche, J. M. Ghuysen, R. Linder, and M. R. J. Salton. The DD-Carboxypeptidase- Transpeptidase System in Escherichia coli K12 Mutant Strain 44. Annals N.Y. Acad. Sci., 235 (1974) 225; Pollock, J. J., M. Nguyen-Disteche, J. M. Ghuysen, J. Coyette, R. Linder, M. R. J. Salton, K. S. Kim, H. R. Perkins and P. Reynolds. Fractionation of the DD-Carboxypeptidase- Transpeptidase Activities Solubilized from Membranes of Escherichia coli K12, Strain 44, Eur, J. Biochem., 41 (1974) 439.