Ouabain binding and Na +K + transport in rat muscle cells and adipocytes

Abstract

1. 1. The accumulation and the release of [ 3H]ouabain has been characterized in isolated intact soleus muscles and free fat cells of the rat. The number of ouabain-binding sites was determined and compared with the rates of active Na +K + transport. 2. 2. In soleus muscle [ 3H]ouabain is bound to the surface of the plasma membrane by a reversible and saturable process, which is inhibited by K +, Li +, 2,4-dinitrophenol, the omission of Na + or the addition of digoxin. 3. 3. The release of [ 3H]ouabain from preloaded muscles is accelerated by K +, 2,4-dinitrophenol and unlabelled ouabain or digoxin, presumably becuase of diminished binding of the glycoside. 4. 4. Under steady-state conditions for ouabain binding a kinetic analysis indicates that soleus muscles contain 7.2 · 10 −1 moles binding sites per g wet wt or 3350 per μm 2 of sarcolemma surface area. An apparent dissociation constant of 2.1 · 10 −7 M was found. 5. 5. Measurement of 22Na efflux and 42K influx under basal conditions indicate that the number of Na + and K + transported per ouabain-binding site correspond to respectively 500 and 325 per min. 6. 6. In isolated fat cells ouabain binding showed qualitatively the same characteristics as in soleus muscle, although the rate was considerably faster and the affinity higher (apparent dissociation constant: 1.7 · 10 −8 M. 7. 7. The fat cells were estimated to contain 2.0 · 10 −11 moles ouabain-binding sites per ml of cells or 66 per μm 2 of plasma-membrane surface. The ouabain-sensitive component of 42K influx corresponds to 3450 ions per site per min. 8. 8. It is concluded that in intact muscle cells and adipocytes, which constitutes the major portion of total body weight in mammals, the binding of ouabain is qualitatively closely similar to that described in microsomal Na + + K +- activated ATPase.