Abstract
An improved enzymatic assay has been developed for the rapid quantitative measurement of gentamicin or tobramycin in serum and other biological fluids. The assay uses a crude enzyme from Escherichia coli W677/HJR66 that adenylates gentamicin or tobramycin in the presence of adenosine-5'-triphosphate (ATP). Enzymatic adenylation of gentamicin is optimal at 8 mM Mg2+, and the Michaelis constant (K,,) of the enzyme for ATP is 0.043 mM. The modifications introduced here increase the sensitivity and decrease the background radioactivity of the previously described assay. The amount of 14C-ATP required per assay is also reduced, thereby decreasing the cost of the assay. The accuracy of the present method has been demonstrated both in control experiments and with 96 specimens of serum from 59 patients treated with gentamicin. Ten other commonly used antibiotics structurally unrelated to aminoglycosides were tested and do not interfere with the assay. The present method is not suitable for kanamycin assays because the crude enzyme used contains activities that catalyze two different ATP-dependent reactions leading to phosphorylation or adenylation of kanamycin.
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