A spectrophotometric assay of β-lactamase action on penicillins

Waley, S. G.

doi:10.1042/bj1390789

Cited by 223 publications

(131 citation statements)

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“…Hydrolysis of P-lactams by crude and purified Plactamase preparations was examined by UV spectrophotometric assay (O'Callaghan et al, 1969;Waley, 1974). The antibiotic solutions were prepared in 0.1 M phosphate buffer, pH 7.0, and hydrolysis rates were measured at 37°C.…”

Section: Hydrolysis and Inhibition Assaysmentioning

confidence: 99%

Chromosomal -lactamase expression and antibiotic resistance in Enterobacter cloacae

Yang

Livermore

Williams

1988

Journal of Medical Microbiology

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Summary. The activities of P-lactam antibiotics were compared against Enterobacter cloacae clinical isolates and mutants which had inducible, stably-derepressed, and basal expression of a PI 8.4 subtype of the Ia chromosomal P-lactamase. These activities were correlated with the results of studies of the P-lactamase-lability and /?-lactamase-inducer-power of the antibiotics. Cefoxitin and ampicillin were labile, and induced p-lactamase production strongly at concentrations below their MIC values. Consequently, /?-lactamase-inducible and p-lactamase-stably-derepressed organisms were highly resistant (MIC > 256 mg/L) to these antibiotics, whereas enzyme-basal strains and mutants were much more susceptible (MIC 1-16 mg/L). Imipenem also induced p-lactamase production strongly at concentrations below its MIC, but was more stable than ampicillin and cefoxitin. It was active against enzyme-inducible and stably-derepressed organisms at 0-25-0.5 mg/L and against P-lactamase-basal organisms at 0.06-0-25 mg/L. Thus the P-lactamase afforded only very low-level protection against imipenem ; this appeared to be by a non-hydrolytic mechanism, with the enzyme binding to the antibiotic in a relatively stable complex. This complex, which probably was an intermediate in a hydrolytic pathway, was isolated by gelfiltration chromatography and shown to have a breakdown half-life of 47 f 2 min. Cefotaxime, ceftriaxone and mezlocillin were labile to the PI 8.4 P-lactamase but induced p-lactamase production weakly at concentrations below their MIC values. Consequently, P-lactamase-inducible and P-lactamase-basal organisms remained equally susceptible (MIC 0.06-4 mg/L), but stably-derepressed organisms were considerably more resistant (MIC > 64 mg/L) to these antibiotics.

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Section: Hydrolysis and Inhibition Assaysmentioning

confidence: 99%

Chromosomal -lactamase expression and antibiotic resistance in Enterobacter cloacae

Yang

Livermore

Williams

1988

Journal of Medical Microbiology

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“…The fractions were assayed for phosphotransferase (Haas & Dowding, 1975), glucose-6 phosphate dehydrogenase (Deutsch, 1983) and fl-lactamase activity (Waley, 1974). Subcellular site for phosphorylation of neomycin in E. coli JM103IpMS5J…”

Section: N-terminal Sequence Of the Phosphotransferasementioning

confidence: 99%

Cloning of aminoglycoside phosphotransferase (APH) gene from antibiotic-producing strain of Bacillus circulans into a high-expression vector, pKK223-3. Purification, properties and location of the enzyme

1991

Biochemical Journal

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The aminoglycoside phosphotransferase gene from a butirosin-producing strain of Bacillus circulans was cloned in a highexpression vector (pKK223-3) to give the recombinant plasmid pMS5. Escherichia coli harbouring the plasmid, E. coli JM103[pMS5], was characterized, and several features of the expression of the phosphotransferase were studied. The phosphotransferase activity was best expressed in a medium lacking glucose, and the highest levels of the enzyme were found between 12 and 24 h of growth. The induction of the phosphotransferase expression with isopropyl /3-D-thiogalactopyranoside (inducer) was found to be undesirable as the overproduction of the enzyme led to the killing of the bacteria. The subcellular location of the phosphotransferase, and also the site in vivo of the phosphorylation of neomycin, was found to be in the cytoplasm. The phosphotransferase was purified to homogeneity in good yield (17 mg of purified -protein/3 litres of culture) and was shown to be a monomer of Mr 30000-32000. The N-terminal amino acid sequence was in agreement with that predicted from the gene sequence and confirmed the absence of any signal sequence. The regiospecificity of the phosphotransferase reaction was studied by m.s. and by 'H-, 13C-and 31P-n.m.r. using ribostamycin as the substrate, and it was found that the antibiotic was phosphorylated at the 3'-hydroxy group.

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“…Beta-lactamase I activity was determined by the spectrophotometric procedure with benzylpenicillin as substrate (36). In some experiments, activity measurements were also performed using N-(2-furyl) acryloyl penicillin as substrate (10).…”

Section: Measurement Of Activitymentioning

confidence: 99%

Bacillus cereus 569/H β-lactamase I: Structure-function relationship

Durkin

Viswanatha

1980

Carlsberg Res. Commun.

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Various functional groups of I~-lactamase I were modified using appropriate group specific reagents. Modification of five of the ten arginine residues and approximately fifteen of twenty-one lysine residues present in the protein could readily be achieved by reaction with diacetyl trimer and trinitrobenzene sulfonic acid respectively, without any adverse effect on catalytic activity. Reaction with N-bromosuccinimide revealed that two of the three tryptophan residues could be oxidized without impairment of enzymatic activity. Treatment with tetranitromethane resulted in the modification of two of the seven tyrosine residues of the protein with approximately 30 % diminution in activity. Reaction of the enzyme with a water soluble carbodiimide resulted in a rapid inactivation, complete loss of activity being accompanied by the modification of five of the carboxylic functions. Conversion of all the four histidine residues of the enzyme to the corresponding N-carbethoxy derivatives had no effect on the catalytic function. All these observations suggest that chemical modification of the enzyme in the presence of substrate or its analog is essential for the identification and/or localization of functional groups participating in the catalytic mechanism.Abbreviations: DCC = l-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide; DEP = diethylpyrocarbonate; DT = trimer of 2,3-butane dione, NBS = N-bromosuccinimide, TNBS = trinitrobenzene sulfonic acid; TNM = tetranitromethane.

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A spectrophotometric assay of β-lactamase action on penicillins

Abstract: A new method for measuring the enzymic hydrolysis of the beta-lactam ring in penicillins is described. The change in extinction in the u.v. region is determined. The method is sensitive (50mum-benzylpenicillin can be used) and convenient.

Cited by 223 publications

References 9 publications

Chromosomal -lactamase expression and antibiotic resistance in Enterobacter cloacae

Chromosomal -lactamase expression and antibiotic resistance in Enterobacter cloacae

Cloning of aminoglycoside phosphotransferase (APH) gene from antibiotic-producing strain of Bacillus circulans into a high-expression vector, pKK223-3. Purification, properties and location of the enzyme

Bacillus cereus 569/H β-lactamase I: Structure-function relationship

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