Characterization of penicillin-binding protein 2 of Staphylococcus aureus: deacylation reaction and identification of two penicillin-binding peptides.
Penicillin-binding protein (PBP) 2 is the major PBP of five that have been identified in susceptible strains of Staphylococcus aureus. Beta-lactam antibiotic binding to PBP 2 is important for the antibacterial effect. Antibiotic binding to PBP 2 in strain 209P was examined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis in competition assays using [3H]penicillin as the radiolabel. Clavulanic acid, which is specifically bound by PBP 2, and cefaclor, which is specific for PBP 3, were studied. Cefaclor, which alone appeared not to bind PBP 2, in combination inhibited PBP 2 binding of clavulanic acid. By varying the temperature during radiolabeling with [3H]penicillin in cefaclor competition assays and in direct radiolabeling assays with [3H]cefaclor, it was shown that cefaclor was bound by PBP 2 with high affinity (50% inhibitory concentration, less than or equal to 0.1 microgram/ml) and that the apparent low-affinity binding (50% inhibitory concentration, greater than 10 micrograms/ml) in competition assays performed at 37 degrees C was due to rapid deacylation. Two penicillin-binding peptides of PBP 2 also were identified in fluorographs of PBPs separated by nonequilibrium pH gradient gel and two-dimensional electrophoresis. Rapid deacylation for some antibiotics and the presence of two penicillin-binding peptides are two properties of PBP 2 that should be considered when correlating results of binding assays with effects of beta-lactam antibiotics on S. aureus.
Chambers
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Medical Service, San Francisco General Hospital, California 94110.
Miick
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D000900:Anti-Bacterial Agents / Q000378:metabolism* / Q000494:pharmacology; D001426:Bacterial Proteins; D001667:Binding, Competitive; D002352:Carrier Proteins; D002433:Cefaclor / Q000378:metabolism* / Q000494:pharmacology; D002440:Cefoxitin / Q000378:metabolism / Q000494:pharmacology; D019818:Clavulanic Acid; D002969:Clavulanic Acids / Q000378:metabolism* / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D006602:Hexosyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D008712:Methicillin / Q000378:metabolism / Q000494:pharmacology; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism*; D002267:Muramoylpentapeptide Carboxypeptidase; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases / Q000302:isolation & purification / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000187:drug effects* / Q000378:metabolism
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; D002969:Clavulanic Acids; D009097:Multienzyme Complexes; D046915:Penicillin-Binding Proteins; D019818:Clavulanic Acid; D002433:Cefaclor; D002440:Cefoxitin; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase; D008712:Methicillin