Complete quantification of the total concentration of rat skeletal-muscle Na⁺ + K⁺-dependent ATPase by measurements of [³H]ouabain binding

In the standard [³H]ouabain-binding assay for quantification of the Na,K-ATPase (Na⁺ + K⁺-dependent ATPase) concentration in rat skeletal muscles, samples are incubated for 2 x 60 min in 1 μM-[³H]ouabain at 37°C followed by a wash-out for 4 x 30 min at 0° C. To obtain accurate determinations, values determined by this standard assay should be corrected for (1) non-specific uptake and retention of [³H]ouabain (11% overestimation), (2) loss of specifically bound [³H]ouabain during wash-out (21% underestimation), (3) evaporation from muscle samples during weighing (4% overestimation), (4) impurity of [³H]ouabain (5% underestimation) and (5) incomplete saturation of [³H]ouabain binding sites (6% underestimation). Thus corrected the standard [³H]ouabain-binding assay determines the total Na,K-ATPase concentration. Hence, in the soleus muscle of 12-week-old rats the total [³H]ouabain-binding-site concentration is 278 ± 20 pmol/g wet wt. This is at variance with the evaluation of the Na,K-ATPase concentration from Na,K-ATPase activity measurements in muscle membrane fractions, where the recovery of Na,K-ATPase is only 2-18%. Quantification of the total Na,K-ATPase concentration is of particular importance since it is a prerequisite for the discussion of quantitative aspects of the Na,K-ATPase.