| Assay Method Information | |
| | In Vitro Inhibition Activity |
| 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, α2β2FXYD1 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/βFXYD1 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 moles/min/mg; α2β1, 18.7±1.8 moles/min/mg, and α2β3, 10.7±1.9 moles/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. |
| Affinity data for this assay | |
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