Resistance of Escherichia coli to penicillins. VI. Purification and characterization of the chromosomally mediated penicillinase present in ampA-containing strains.
The chromosomally mediated penicillinase present in three strains of Escherichia coli K-12 has been purified and characterized. Two of the strains carried the ampA gene and the third the wild-type allele. The purification involves release of the enzyme by spheroplast formation, dialysis, chromatography on sulfoethyl cellulose, and chromatography on hydroxylapatite. Enzyme from the two mutants appeared homogeneous in polyacrylamide gel electrophoresis. Enzyme from the wild-type strain gave two bands. Immunologically, the enzymes from all three strains were identical. Ultracentrifugation gave a homogeneous peak with a sedimentation coefficient of 3.4S. Gel filtration gave an estimated molecular weight of 29,000. The N-terminal amino acid residue was found to be alanine. Complete amino acid analysis showed a lack of cysteine. Ultraviolet spectra were recorded at three different pH values. The extinction coefficient at 280 nm is 21.0 for a 1% solution at pH 6.8. The optimal pH is 7.3. With enzyme from one of the resistant mutants, the following K(m) and turnover number values were obtained: for penicillin G, 12 mum and 2,080; for d-ampicillin, 6 mum and 83; for cephalosporin C, 217 mum and 18,400. The effect of different salts on the enzyme activity was tested. Under many conditions the enzyme was found to be unstable.
D000483:Alleles / Q000187:drug effects; D000596:Amino Acids / Q000032:analysis; D000667:Ampicillin / Q000494:pharmacology; D002511:Cephalosporins / Q000494:pharmacology; D002712:Chlorides / Q000494:pharmacology; D002845:Chromatography; D002876:Chromosomes, Bacterial / Q000032:analysis* / Q000201:enzymology; D003956:Dialysis; D004586:Electrophoresis; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D009154:Mutation; D010400:Penicillin G / Q000494:pharmacology; D010403:Penicillin Resistance; D010405:Penicillinase / Q000302:isolation & purification*; D011523:Protoplasts / Q000378:metabolism; D014461:Ultracentrifugation
D000596:Amino Acids; D002511:Cephalosporins; D002712:Chlorides; D000667:Ampicillin; D010405:Penicillinase; D008274:Magnesium; D010400:Penicillin G