The Separation and Properties of Two Penicillin-Binding Proteins from Salmonella typhimurium

Abstract

The cell envelope of Salmonella typhimurium SW 1061 contains five proteins which covalently bind benzyl-[14C]penicillin, numbered 1–5 in order of decreasing molecular weight. Proteins 1. 4 and 5 were extracted from membranes of the organism by treatment with Genapol X-100 in the presence of 1 M LiCl. This extract catalysed a dd-carboxypeptidase reaction, the synthesis of a cross-linked dimer from l-Ala-d-Glu-(ms-[1,7−14C]A2pm-d-Ala) in a natural model transpeptidation reaction, and the concomitant hydrolysis of this dimer, i.e. an endopeptidase reaction. Proteins 1. 4 and 5 have been separated by ion-exchange chromatography of the LiCl/Genapol X-100 extract on DEAE-Sepharose CL-6B, and protein 4 was found to possess the majority of the recovered dd-carboxypeptidase activity, and to be an efficient transpeptidase. However, this procedure yielded unstable preparations of protein 5 (the major penicillin-binding protein in the cell), which had very low enzymic activity but retained the ability to bind benzyl-[14C]penicillin. Stable, pure preparations of protein 5 were obtained by covalent affinity chromatography on ampicillin-affinose of material solubilized from membranes using Genapol X-100 in the absence of LiCl. Such preparations possessed dd-carboxypeptidase activity, and also catalysed the natural model transpeptidation reaction but with a lower efficiency than protein 4. Proteins 4 and 5 released benzylpenicillin with a concomitant recovery of catalytic activity, the half-times for this recovery being approximately 90 min and 5 min respectively at 37 °C. Protein 4 was found to have the highest affinity of the five membrane-bound penicillin-binding proteins for benzyl-[14C]penicillin. The complete separation of protein 1 from proteins 4 and 5 has not been achieved; however, our preliminary findings suggest that this protein also catalyses both the dd-carboxypeptidase and natural model transpeptidase activities.