Abstract
Penicillin-binding proteins (PBPs) 5 and 6 in the cytoplasmic membranes of Escherichia coli K12, which had previously been co-purified as a penicillin-sensitive D-alanine carboxypeptidase IA (Tamura, T., Imae, Y., and Strominger, J. L. (1976) J. Biol. Chem. 251, 414-423), were each purified to protein homogeneity. Purification involved selective solubilization of PBPs 1a, 5, and 6 from membranes by Triton X-100 at low ionic strength, covalent penicillin affinity chromatography, and CM-cellulose column chromatography. Purified PBP 5 and PBP 6 each catalyzed a D-alanine carboxypeptidase I activity using various natural and synthetic substrates including linear uncross-linked peptidoglycan. PBP 5 showed 3- to 4-fold higher specific activities toward these substrates than PBP 6. Both PBPs also catalyzed a model transpeptidase activity using glycine as a transpeptidation acceptor, and showed similar pH profiles and MgCl2 sensitivities for their D-alanine carboxypeptidase I activities. Both PBPs bound a stoichiometric amount of [14C]penicillin G at saturation. Peptide mapping by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after partial proteolysis by proteases and cyanogen bromide demonstrated that these PBPs are distinct polypeptides.
Highlights
G was incubated in 100 pl of 80 mM KP,. 1 R Triton X-100 with 30 pg/ml of [‘4C]penicillin G a t 25°C for 20 min
Covalent [“C]penicillin G.PBP complex was precipitated by addition of trichloroacetic acid, collected by centrifugation a t 7000 X g for 15 min, and washed once with 200 pl of acetone
Assays were carried out under the standard assay conditionsusing ["Clalanine-labeled linear, uncrosslinked peptidoglycan and 0.35 pgof P B P 5 (Mor)0.28 pg of P B P 6 (MA) fter.incubation for the time indicated, the reaction mixture was boiled for 3 min and treated with lysozyme, and the digests were subjected to paper electrophoresiast pH 4.0
Summary
Covalent [“C]penicillin G.PBP complex was precipitated by addition of trichloroacetic acid (final 58), collected by centrifugation a t 7000 X g for 15 min, and washed once with 200 pl of acetone. Assays were carried out under the standard assay conditionsusing ["Clalanine-labeled linear, uncrosslinked peptidoglycan (containing 19 p~ of disaccharide-pentapeptide unit, 4.4 X 10' cpm) and 0.35 pgof P B P 5 (Mor)0.28 pg of P B P 6 (MA) fter.incubation for the time indicated, the reaction mixture was boiled for 3 min and treated with lysozyme, and the digests were subjected to paper electrophoresiast pH 4.0.
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