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  • Title: Complete quantification of the total concentration of rat skeletal-muscle Na+ + K+-dependent ATPase by measurements of [3H]ouabain binding.
    Author: Kjeldsen K.
    Journal: Biochem J; 1986 Dec 15; 240(3):725-30. PubMed ID: 3030284.
    Abstract:
    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 microM-[3H]ouabain at 37 degrees C followed by a wash-out for 4 X 30 min at 0 degree C. To obtain accurate determinations, values determined by this standard assay should be corrected for non-specific uptake and retention of [3H]ouabain (11% overestimation), loss of specifically bound [3H]ouabain during wash-out (21% underestimation), evaporation from muscle samples during weighing (4% overestimation), impurity of [3H]ouabain (5% underestimation) and 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.
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