Biological characterization of a new radioactive labeling reagent for bacterial penicillin-binding proteins

Journal Article · Tue May 01 04:00:00 EDT 1990 · Antimicrobial Agents and Chemotherapy; (USA)

DOI:https://doi.org/10.1128/AAC.34.5.718· OSTI ID:6642412

Preston, D A; Wu, C Y; Blaszczak, L C; Seitz, D E; Halligan, N G ^[1]

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1. Eli Lilly and Co., Indianapolis, IN (USA)

Radiolabeled penicillin G is widely used as the imaging agent in penicillin-binding protein (PBP) assays. The disadvantages of most forms of labeled penicillin G are instability on storage and the long exposure times usually required for autoradiography or fluorography of electrophoretic gels. We investigated the utility of radioiodinated penicillin V as an alternative reagent. Radioiodination of p-(trimethylstannyl)penicillin V with ({sup 125}I)Na, using a modification of the chloramine-T method, is simple, high yielding, and site specific. We demonstrated the general equivalence of commercially obtained ({sup 3}H)penicillin G and locally synthesized ({sup 125}I)penicillin V (IPV) in their recognition of bacterial PBPs. Profiles of PBPs in membranes from Bacteroides fragilis, Escherichia coli, Providencia rettgeri, Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis, and Enterococcus faecium labeled with IPV or (3H)penicillin G were virtually identical. Use of IPV as the imaging agent in competition experiments for determination of the affinities of various beta-lactam antibiotics for the PBPs of E. coli yielded results similar to those obtained in experiments with ({sup 3}H)penicillin G. Dried electrophoretic gels from typical PBP experiments, using IPV at 37.3 Ci/mmol and 30 micrograms/ml, exposed X-ray film in 8 to 24 h. The stability of IPV on storage at 4{degrees}C was inversely proportional to specific activity. At 37.3 Ci/mmol and 60 micrograms/ml, IPV retained useful activity for at least 60 days at 4{degrees}C. IPV represents a practical and stable reagent for rapid PBP assays.

• OSTI ID: 6642412
• Journal Information: Antimicrobial Agents and Chemotherapy; (USA), Journal Name: Antimicrobial Agents and Chemotherapy; (USA) Vol. 34:5; ISSN AMACC; ISSN 0066-4804
• Country of Publication: United States
• Language: English

Related Subjects

550601* -- Medicine-- Unsealed Radionuclides in Diagnostics
62 RADIOLOGY AND NUCLEAR MEDICINE
ANTI-INFECTIVE AGENTS
ANTIBIOTICS
AUTORADIOGRAPHY
BACTERIA
BETA DECAY RADIOISOTOPES
DAYS LIVING RADIOISOTOPES
DRUGS
ELECTRON CAPTURE RADIOISOTOPES
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPES
LABELLING
MICROORGANISMS
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
PENICILLIN
PROTEINS
RADIOISOTOPES
REAGENTS