Biological characterization of a new radioactive labeling reagent for bacterial penicillin-binding proteins.
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 [125I]Na, using a modification of the chloramine-T method, is simple, high yielding, and site specific. We demonstrated the general equivalence of commercially obtained [3H]penicillin G and locally synthesized [125I]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 [3H]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.
Preston
D A
DA
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285.
Wu
C Y
CY
Blaszczak
L C
LC
Seitz
D E
DE
Halligan
N G
NG
D001345:Autoradiography; D001426:Bacterial Proteins / Q000032:analysis*; D002352:Carrier Proteins / Q000032:analysis*; D055598:Chemical Phenomena; D002621:Chemistry; D006602:Hexosyltransferases; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D002267:Muramoylpentapeptide Carboxypeptidase / Q000032:analysis*; D010404:Penicillin V / Q000032:analysis; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases
D001426:Bacterial Proteins; D002352:Carrier Proteins; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase; D010404:Penicillin V