Gentamicin assay by enzymatic adenylylation and the application of a double osmotic shock procedure to prepare gentamicin adenine mono-nucleotide transferase.

The release of gentamicin adenine mono-nucleotide transferase (GAdT) during single cold osmotic shock treatment of E. coli K12 W677/HJR66 is not always maximal. The yield of GAdT could not be improved by using E. coli harvested at different stages of growth, by prolonging the exposure to the different steps of the shock procedure, by changing the sucrose concentration, or the magnesium chloride volume. The quantity of GAdT in osmotic extracts could be increased when a double shock procedure was performed. Using an aliquot (30 microliter) of the extract, an accurate and quick assay for gentamicin, sisomicin and tobramycin in microvolumes of serum (30 microliter) can be accomplished. To avoid high background activity in the assay, the extracts should be prepared from E. coli grown in gentamicin-free medium.