Homogeneity of [3H]ouabain-binding sites in rat soleus muscle.

Homogeneity or heterogeneity of rat soleus-muscle Na,K-ATPase (Na+ + K+-dependent ATPase) with respect to affinity for 3H]ouabain was evaluated. Since the standard method for measuring specific 3H]ouabain binding to rat skeletal-muscle samples includes subtraction of a value for non-specific 3H]ouabain uptake and retention, and a wash-out in the cold to remove 3H]ouabain from the extracellular phase, it was possible that these procedures could hide a class of 3H]ouabain-binding sites either with low affinity or with a rapid dissociation of 3H]ouabain. However, measurements of 3H]ouabain uptake and retention over the range 0.1-5 mM, as well as the omission of wash-out, gave no evidence for heterogeneity of 3H]ouabain-binding sites in rat soleus muscle. Furthermore, the observation of agreement between the uptake and retention of non-specific 3H]ouabain and of 14C]sucrose gave no evidence for the existence of a major pool of 3H]ouabain-binding sites with low affinity for 3H]ouabain. Assuming homogeneity, the total concentration of 3H]ouabain binding sites in soleus-muscle samples from 12-week-old rats is 278-359 pmol/g wet wt.