A Sensitive Procedure for Screening Microorganisms for the Presence of Penicillin Amidase

Baker, William L.

doi:10.1071/bi9840257

Cited by 10 publications

(7 citation statements)

References 11 publications

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“…The enzyme activities of the conjugates were determined using penicillin-V as a substrate [2]. Competitive antigen-binding activity was measured against fluorescein-isothiocyanate (FITC)-labeled L6 on H2981 cells and against FITC-labeled 1F5 on Daudi cells using a Coulter Epics-C flourescence analyzer.…”

Section: Methodsmentioning

confidence: 99%

Antibody-penicillin-V-amidase conjugates kill antigen-positive tumor cells when combined with doxorubicin phenoxyacetamide

Kerr

Senter

Burnett

et al. 1990

Cancer Immunol Immunother

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The two monoclonal antibodies (mAb), L6 (anti-carcinoma), and 1F5 [anti-(B-cell-lymphoma)], were chemically linked to the enzyme penicillin-V amidase (PVA), which hydrolyzes phenoxyacetamides, to explore the potential of using mAb-enzyme conjugates for the localization of chemotherapeutic drugs at tumor cells. The phenoxyacetamide derivatives of doxorubicin and melphalan were prepared, yielding the less toxic amides, doxorubicin-N-p-hydroxyphenoxyacetamide (DPO) and melphalan-N-p-hydroxyphenoxyacetamide (MelPO). These were hydrolyzed by PVA to doxorubicin and melphalan respectively. In vitro studies with the L6-positive lung carcinoma cell line, H2981, and the 1F5-positive B-cell lymphoma line, Daudi, showed that DPO was 80-fold less toxic to H2981 cells and 20-fold less toxic to Daudi cells than doxorubicin, and its toxicity was substantially increased when the H2981 cells were pretreated with L6-PVA or the Daudi cells were pretreated with 1F5-PVA. The cytotoxic effect was antigen-specific, since only the binding mAb-enzyme conjugate increased the cytotoxicity of the prodrug. MelPO was more than 1000-fold less toxic than melphalan to H2981 cells and more than 100-fold less toxic than melphalan to Daudi cells. Pretreatment with the mAb-PVA conjugates did not enhance the toxicity of MelPO in either cell line, because PVA hydrolyzes the phenoxyacetamide bond of MelPO too slowly to generate a toxic level of melphalan.

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Section: Methodsmentioning

confidence: 99%

Antibody-penicillin-V-amidase conjugates kill antigen-positive tumor cells when combined with doxorubicin phenoxyacetamide

Kerr

Senter

Burnett

et al. 1990

Cancer Immunol Immunother

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“…ampicillin) can be measured by an increase in fluorescence due to the 6-APA formed (Baker 1984). Conversion of 6-APA to 6-APOA by a broad spectrum 8-lactamase, commonly found in strains of Klebsiella aerogenes, would be accompanied by a fall in fluorescence which can also be measured quantitatively.…”

Section: Discussionmentioning

confidence: 99%

A sensitive enzymic assay for benzylpenicillin

Baker

1985

Journal of Applied Bacteriology

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A method has been developed for the determination of sodium benzylpenicillin concentrations in the range 3.3-33 micrograms/ml. 6-Aminopenicillanic acid is released from the benzylpenicillin by the action of the enzyme penicillin acylase and is estimated from its reaction with fluorescamine at pH 4. 7-Aminocephalosporanic acid shows a similar trend to 6-aminopenicillanic acid in its reaction at pH 4. The open beta-lactam ring form of each compound shows little fluorescence with fluorescamine at pH 4 but shows strong fluorescence in the pH range 7-9. 6-Aminopenicillanic acid and its open beta-lactam ring form give different fluorescent responses to increasing volumes of a solution of the fluorigenic agent at pH 7.8. This effect can be used to estimate concentrations in a mixture of the two components providing other amino material is absent.

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“…Tubes were shaken at 37°C and samples (0.1 ml) taken at specific times, vigorously mixed with 0.1 ml of 1 : 1000 cetyltrimethyl ammonium bromide (CTAB) solution, left for 5 min and diluted with 0.5 mol/l acetate buffer pH 4. One ml samples of the diluted solution were treated with fluorescamine (Baker 1983a(Baker , 1984 and the fluorescence of 6-APA formed estimated against a 6-APA standard curve after removal of cell debris by centrifugation. The 6-APA substrate was used to detect p-lactamases which act on 6-APA.…”

Section: Detection and Estimation Of Penicillin Acyiase Activitymentioning

confidence: 99%

Co‐existence of β‐lactamase and penicillin acylase in bacteria; detection and quantitative determination of enzyme activities

Baker

1992

Journal of Applied Bacteriology

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Twenty-six bacteria were examined for the presence of penicillin acylase and beta-lactamase. A copper reducing assay, which was sensitive in the analytical range 2-20 micrograms/ml, was used for determination of penicilloates and a fluorescamine assay was used to determine 6-aminopenicillanic acid concentrations when both substances were produced by the action of the enzymes on a single substrate. Seventeen bacteria contained beta-lactamases, six contained penicillin acylases and four contained both enzymes. Two bacteria contained a Type 1 penicillin acylase and four bacteria contained a Type II enzyme. No ampicillin acylases were detected. All beta-lactamases were constitutive enzymes in those organisms where both enzymes co-existed. Bacillus subtilis and B. cereus produced inducible and extracellular beta-lactamases. Acinetobacter calcoaceticus ATCC 21288 produced a constitutive beta-lactamase which was detected extracellularly.

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A Sensitive Procedure for Screening Microorganisms for the Presence of Penicillin Amidase

Cited by 10 publications

References 11 publications

Antibody-penicillin-V-amidase conjugates kill antigen-positive tumor cells when combined with doxorubicin phenoxyacetamide

Antibody-penicillin-V-amidase conjugates kill antigen-positive tumor cells when combined with doxorubicin phenoxyacetamide

A sensitive enzymic assay for benzylpenicillin

Co‐existence of β‐lactamase and penicillin acylase in bacteria; detection and quantitative determination of enzyme activities

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