Nuclear magnetic resonance spectrometric assay of beta-lactamase

Kono, Megumi; Ohara, Koji; Shiomi, Yoko

doi:10.1128/aac.17.1.16

Cited by 7 publications

(5 citation statements)

References 13 publications

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“…The intensity was compared with that of the proton (H)-signal of non-degraded side chains such as the phenyl group in the case of ABPC or PCG. The decrease of intensity of the signal related to the /3-1actam ring agreed with the rate of degradation of the fl-lactam ring in the same way as described in a previous paper [7].…”

Section: Analytical Methods With a Suspension Of Bacterial Cells Usinsupporting

confidence: 89%

“…The NMR measurement method already reported by our laboratory [7,10] was modified in accordance with the following conditions: 10 mg of antibiotic, bacterial cells from colonies on a nutrient agar (Eiken Chemical Co. Ltd., Tokyo) plate, and 0.4 ml of 0.1 M phosphate buffer (pH 7.0 for Gram-negative bacteria) with D20 were mixed in an NMR tube (5 mm diam.). The deuterated buffer was made as follows: 0.4 ml of phosphate buffer (0.1 M, pH 7.0) was lyophilized and the powdered buffer dissolved with 0.4 ml of D20.…”

Section: Analytical Methods With a Suspension Of Bacterial Cells Usinmentioning

confidence: 99%

“…The following strains were used: Escherichia coli K-12 W3110 rif (RP1) was a rifampicin-resistant mutant (rif) harboring a drug-resistant plasmid RP1 [7]. The MIC [12,15] of ABPC to the strain was > 800/~g/ml.…”

Section: Organisms and Minimum Inhibitory Concentrations (Mics)mentioning

confidence: 99%

“…A series of NMR studies has shown that the change in 1H-NMR signals of fl-lactam antibiotics degraded by a hydrolyzing enzyme (fl-lactamase or acylesterase) obtained from bacteria gives valuable information for the measurement of enzyme activity [4][5][6][7][8][9][10][11][12]. These methods were applied to the measurement of the activity of a fl-lactamase inhibitor [13].…”

Section: Introductionmentioning

confidence: 99%

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NMR spectrometric assay for determining enzymatic hydrolysis of Î²-lactam antibiotics with bacteria in aqueous solution

Ohara¹,

Shiomi²,

Kono³

1984

FEMS Microbiology Letters

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An application of a nuclear magnetic resonance (NMR) spectrometer for the measurement of β‐lactamase activity in clinical material containing bacteria is presented. By means of proton (1H)‐NMR, it was easy to measure quantitatively β‐lactamase activity in human bacteriuria, without performing any such pretreatment as isolation of bacteria or extraction of crude enzymes and without preparing special reagents for the detection. This is the first report on the application of 1H‐NMR analysis of structural changes for determining hydrolysis of β‐lactam antibiotics with β‐lactamase‐producing bacteria in aqueous solution.

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Section: Analytical Methods With a Suspension Of Bacterial Cells Usinsupporting

confidence: 89%

Section: Analytical Methods With a Suspension Of Bacterial Cells Usinmentioning

confidence: 99%

Section: Organisms and Minimum Inhibitory Concentrations (Mics)mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

NMR spectrometric assay for determining enzymatic hydrolysis of Î²-lactam antibiotics with bacteria in aqueous solution

Ohara¹,

Shiomi²,

Kono³

1984

FEMS Microbiology Letters

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“…Bacillus subtilis ATCC 6633 (20) and Staphylococcus aureus 209P (13) were used as test strains in microbioassays for determination of the potencies of macrolides and lincosamide antibiotics, respectively. E. coli K-12 W3110 Rif' (14), which is susceptible to macrolides, was used as a reference strain.Media. Nutrient broth (Eiken Chemical Co. Ltd., Tokyo, Japan) was used as a liquid medium, and nutrient agar was used as a solid medium.…”

mentioning

confidence: 99%

Purification and characterization of macrolide 2'-phosphotransferase from a strain of Escherichia coli that is highly resistant to erythromycin

Ohara¹,

Kanda²,

Ohmiya³

et al. 1989

Antimicrob Agents Chemother

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Macrolide 2'-phosphotransferase [MPH(2')] was purified 90-fold from an erythromycin-resistant strain of Escherichia coli, and its enzymatic properties were investigated. MPH(2') is an inducible intracellular enzyme which showed high levels of activity with 14-member-ring macrolides and extremely low levels with 16-member-ring macrolides. The optimum pH for inactivation of oleandomycin was 8.2, and the optimum temperature of the reaction was 40°C. Enzyme activity was lost by heat treatment at 50°C for 1 min. The isoelectric point and molecular weight of the enzyme were 5.3 and 34,000, respectively. Purine nucleotides, such as GTP, ITP, and ATP, were effective as cofactors in the inactivation of macrolides. Iodine, EDTA, or divalent cations inhibited MPH(2') activity.In general, erythromycin is much better known for its activity against gram-positive bacteria and Mycoplasma pneumoniae than for its activity against gram-negative bacteria. However, in Europe, macrolides, in particular erythromycin, have been recommended for prophylaxis of septicemia in immunocompromised patients (1, 2) and for prevention of traveler's diarrhea (4). Recently, members of the family Enterobacteriaceae which were highly resistant to erythromycin were isolated from patients in a hematologyoncology unit (3). Colonization of the intestinal tract by members of the family Enterobacteriaceae that are highly resistant to erythromycin is usually associated with previous long-term oral therapy with the drug (2-4). It has been reported that high-level resistance to erythromycin in members of the family Enterobacteriaceae results from production of erythromycin esterases and rRNA methylases (1,5,(6)(7)(8)24).We reported previously that the structure of inactivated oleandomycin generated by erythromycin-resistant (MIC, 1,600 ,ug/ml) Escherichia coli Tf481A, which was isolated from a patient in Japan in 1983, was oleandomycin 2'-phosphate (22). This paper describes the purification and characterization of a new macrolide-inactivating enzyme, macrolide 2'-phosphotransferase [MPH(2')]. MATERIALS AND METHODSBacterial strains. A clinical isolate of E. coli Tf481A was used for determination of drug resistance and for preparation of crude extracts. Bacillus subtilis ATCC 6633 (20) and Staphylococcus aureus 209P (13) were used as test strains in microbioassays for determination of the potencies of macrolides and lincosamide antibiotics, respectively. E. coli K-12 W3110 Rif' (14), which is susceptible to macrolides, was used as a reference strain.Media. Nutrient broth (Eiken Chemical Co. Ltd., Tokyo, Japan) was used as a liquid medium, and nutrient agar was used as a solid medium.Antibiotics and reagents. Erythromycin, oleandomycin, and spiramycin were purchased from Sigma Chemical Co., * Corresponding author.St. Louis, Mo.; and leucomycin was purchased from Wako Pure Chemical Industries Ltd., Osaka, Japan. Erythromycin A was a gift from Shionogi & Co. Ltd., Osaka, Japan; josamycin was a gift from Yamanouchi Pharmaceutical Co.Ltd., Tokyo, Japan; midecamycin...

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Purification and characterization of macrolide 2′-phosphotransferase type II from a strain of Escherichia coli highly resistant to macrolide antibiotics

Kono¹

1992

FEMS Microbiology Letters

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The resistance mechanism of Escherichia coli BM2506 to macrolides was found to be due to inactivation. Inactivated oleandomycin was identified as oleandomycin 2'-phosphate by thin-layer chromatography. A new type of macrolide-phosphorylating enzyme, macrolide 2'-phosphotransferase type II (MPH(2')II), was detected, purified 95-fold and its enzymological properties investigated. MPH(2')II was a constitutive intracellular enzyme which showed high levels of activity with both 14-member-ring and 16-member-ring macrolides. The optimum pH for the inactivation of oleandomycin was 8.2 and the optimum temperature of the reaction was 40 degrees C. Enzyme activity was lost by heat treatment at 60 degrees C for 1 min. The isoelectric point and M(r) of the enzyme were 5.3 and 48,000, respectively. Purine nucleotides, such as ITP, GTP and ATP, were effective as cofactors in the inactivation of macrolides. An inhibitory effect of iodine, EDTA, or divalent cations on MPH(2')II activity was observed.

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Nuclear magnetic resonance spectrometric assay of beta-lactamase

Cited by 7 publications

References 13 publications

NMR spectrometric assay for determining enzymatic hydrolysis of Î²-lactam antibiotics with bacteria in aqueous solution

NMR spectrometric assay for determining enzymatic hydrolysis of Î²-lactam antibiotics with bacteria in aqueous solution

Purification and characterization of macrolide 2'-phosphotransferase from a strain of Escherichia coli that is highly resistant to erythromycin

Purification and characterization of macrolide 2′-phosphotransferase type II from a strain of Escherichia coli highly resistant to macrolide antibiotics

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