Membrane-Bound Transpeptidase and Penicillin Binding Sites in <em>Streptomyces</em> Strain R61.

High-affinity penicillin binding sites from which the antibiotic could not be removed by washings at 4 °C in 0.017 M K₂HPO₄ or 0.05 M Tris-HCl pH 7.5, were shown to occur in the isolated membranes of Streptomyces R61. These sites caused the attachment of 25 picomoles of [¹⁴C]benzylpenicillin per milligram membrane protein. Penicillins and cephalosporins competed for the same binding sites. The antibiotic concentrations which excluded [¹⁴C]benzylpenicillin from 50% of the binding sites were those which inhibited by 50% the membrane-bound transpeptidase. The same rate constant (about I × 10^-4s^-1) for the dissociation of the benzylpenicillin membrane complex at 37 °C and in 0.017 M K₂HPO₄, was calculated either from the release of the radioactivity (using [¹⁴C]benzylpenicillin) or from the recovery of the transpeptidase activity. These observations supported the conclusion that the high-affinity binding sites in the isolated membranes were the transpeptidase molecules. All the complexes formed between the membranes and the various penicillins and cephalosporins examined were reversible at 37 °C and in 0.017 M K₂HPO₄ at least with regard to the transpeptidase. Depending upon the antibiotics, the rate constants for the dissociation of these complexes varied from 3.3 × 10^-3 to 0.73 ×10^-4 s^-1. The radioactivity released through the dissociation of [¹⁴C]benzylpenicillin membrane complex occurred mainly in the form of a compound which behaved as [¹⁴C]-benzylpenicilloic acid both by paper electrophoresis and thin-layer chromatography. It was impossible to choose between several possible mechanisms for the release of the antibiotic molecule. [ABSTRACT FROM AUTHOR]