Monitoring beta-lactamase activity in vivo by 13C nuclear magnetic resonance spectroscopy.
A 13C-labeled cephalothin, 7 beta-(2-thienylacetamido)-3-[acetoxy-13C1]methyl-3-cephem-4- carboxylate (compound 1), has been prepared and used to monitor beta-lactamase activities by 13C nuclear magnetic resonance spectroscopy. Time-elapsed spectral analysis of the reaction of the labeled cephalothin with the TEM-2 beta-lactamase purified from Escherichia coli revealed the progressive loss of the cephalothin acetyl resonance at 176.8 ppm and accumulation of an acetate signal at 184.3 ppm. Spectral results identical to those observed in the in vitro experiment were obtained when compound 1 was incubated with cell suspensions of E. coli JSR-O (pBR322), which contains the plasmid-encoded TEM-2 beta-lactamase, and Enterobacter cloacae strains that contain a class I chromosomal beta-lactamase. Pseudo-first-order rate constants for the lactamase-catalyzed formation of acetate from cephalothin in vivo were obtained by integration of the 13C-acetyl resonances of compound 1 during timed incubations with cell preparations. These results constitute the first demonstration of the ability to monitor beta-lactamase activity in viable cells by nuclear magnetic resonance spectroscopy.
Mobashery
S
S
Department of Chemistry, Searle Chemistry Laboratory, University of Chicago, Illinois 60637.
Lerner
S A
SA
Johnston
M
M
D002512:Cephalothin / Q000138:chemical synthesis / Q000378:metabolism; D055598:Chemical Phenomena; D002621:Chemistry; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D001618:beta-Lactamases / Q000378:metabolism*
D001618:beta-Lactamases; D002512:Cephalothin