| Assay Method Information | |
| | Selective Inhibition Assays of Isolated Na,K-ATPase |
| Description: | To screen for isoform selectivity of the digoxin derivatives we compared inhibition of Na,K-ATPase activity of purified detergent-soluble human isoform complexes α1β1FXYD1, α2β1FXYD1, α2β2FXTD1 and α2β3FXYD1. Although all the preparations and assays were conducted with FXYD1 in order to stabilize the complexes, the FXYD1 suffix is omitted in naming of isoform complexes for simplicity.Na,K-ATPase activity of α/βPFXYD1 complexes was measured over one hour at 37° C. in a medium containing 130 mM NaCl, 5 mM KCl, 3 mM MgCl2, 1 mM EGTA, 25 mM Histidine, pH 7.4 and 1 mM ATP using the PiColor Lock gold malachite green assay (Inova Biosciences).The Na,K-ATPase activities were α1β1, 21.5±5.3 μmoles/min/mg; α2β1, 18.7±1.8 μmoles/min/mg, and α2β3, 10.7±1.9 μmoles/min/mg protein. As discussed below, an important kinetic property in relation to inhibition by cardiac glycosides is K0.5 for activation by K: α1β1-1.25±0.05 mM, α2β1-2.7±0.14 mM and α2β3 6.4±0.50 mM, respectively.Selectivity of the compounds for various isolated isoforms of human Na,K-ATPase was determined essentially as described before [Katz, A. et al., J Biol Chem., 2010, 285(25), pp. 19582-19592].ATPase activity assays as well as titrations with NaCl, KCl and vanadate were performed as described in Lifshitz-2007 and Loayza-1998 using PiColorLock malachite green assay (Inova Bioscience). Inhibitor assays were performed as described in Katz-2010. [3H]ouabain binding and K+-[3H]digoxin displacement assays were performed as described in Katz-2010.The percent inhibition VCG/V0 was calculated and Ki values were obtained by fitting the data to the function VCG/V0=Ki/([CG]+Ki)+c (CG stands for cardiac glycoside). Inhibition was estimated in 3-5 separate experiments and average Ki values±standard error of the mean (SEM) were calculated. The ratios Ki α1β1/α2β1, α1β1/α2β2 and α1β1/α2β3 was calculated for each compound. |
| Affinity data for this assay | |
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