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 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.
D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D005260:Female; D009132:Muscles / Q000201:enzymology*; D010042:Ouabain / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D011919:Rats, Inbred Strains; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis* / Q000378:metabolism; D014316:Tritium
D014316:Tritium; D010042:Ouabain; C045349:3-O-methyl-fluorescein phosphatase; D000254:Sodium-Potassium-Exchanging ATPase