| Assay Method Information | |
| | Inhibition of Dog Renal Na+/K+ ATPase Activity |
| Description: | Na+/K+ ATPase activity was assayed in vitro by measuring the release of 32P from 32P-ATP, as described (Ferrandi M. et al., Hypertension 1996, 28:1018-25). Increasing concentrations of the standard ouabain, or tested compound, were incubated with 0.3 μg of purified dog kidney enzyme for 10 min at 37° C. in 120 μl final volume of a medium containing 140 mM NaCl, 3 mM MgCl2, 50 mM Hepes-Tris, 3 mM ATP, pH 7.5. Then, 10 μl of a solution containing 10 mM KCl and 20 nCi of 32P-ATP (3-10 Ci/mmol, Perkin Elmer) were added. The reaction was allowed to continue for 15 min at 37° C. and then stopped by acidification with 20% v/v ice-cold perchloric acid. 32P was separated by centrifugation with activated Charcoal (Norit A, Serva) and the radioactivity was counted. The inhibitory activity was expressed as percent of the control samples carried out in the absence of ouabain, or tested compound. The concentration of compound causing 50% inhibition of the Na+/K+ ATPase activity (IC50) was calculated by using a multiple parameter non-linear regression best fitting program (Kaleidagraph™, Sinergy Software). |
| Affinity data for this assay | |
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