PMID:7002918
| Citation |
Amanuma, H and Strominger, JL (1980) Purification and properties of penicillin-binding proteins 5 and 6 from Escherichia coli membranes. J. Biol. Chem. 255:11173-80 |
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| 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. |
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| Keywords |
Alanine/metabolism; Amino Acids/analysis; Bacterial Proteins; Carboxypeptidases/metabolism; Carrier Proteins/isolation & purification; Carrier Proteins/metabolism; Cell Membrane/metabolism; Escherichia coli/metabolism; Glycine/pharmacology; Hexosyltransferases; Kinetics; Molecular Weight; Muramoylpentapeptide Carboxypeptidase; Penicillin-Binding Proteins; Penicillins/isolation & purification; Penicillins/metabolism; Peptide Fragments/analysis; Peptidyl Transferases; Substrate Specificity |
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