Coupled spectrofluorometric assay for aminoglycoside phosphotransferases.
In order to make accurate kinetic measurements for the substrates of aminoglycoside (AG) phosphotransferases (APHs), we have developed an assay which overcomes many of the limitations of currently used assays. We have adapted the coupled spectrophotometric assay (P. R. Goldman and D. B. Northrop (1976) Biochem. Biophys. Res. Commun. 68, 230-236) for use in a spectrofluorometer. At an excitation wavelength of 340 nm, NADH will emit an intensity peak at 450 nm; NAD does not emit under these conditions. Our assay can accurately measure differences of 0.25 microM. For the APH(3')-II encoded on Tn5, we have redetermined the Km's for the AGs, amikacin (AK), kanamycin (KM), and ribostamycin (Rib), and for ATP. Our values for AK (76 microM) were lower than those derived from the spectrophotometric assay; for KM and Rib we obtained Km values of 5.1 and 3.6 microM, respectively. These values were well below the limit of accuracy (10 microM) for the spectrophotometric assay. In addition, we have begun characterization of an APH from a clinical isolate with a low Km for AK. Thus far, we have determined that this enzyme has Km's of approximately 1 microM for both AK and KM. These results show that the assay is well suited for accurate determinations of kinetic constants for low Km substrates of APH enzymes.
Perlin
M H
MH
Department of Biology, University of Louisville, Kentucky 40292.
McCarty
S C
SC
Greer
J P
JP
D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000201:enzymology; D019868:Kanamycin Kinase; D010770:Phosphotransferases / Q000032:analysis* / Q000235:genetics; D010957:Plasmids; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D013050:Spectrometry, Fluorescence / Q000379:methods
D010770:Phosphotransferases; D019868:Kanamycin Kinase