Density and apparent location of the sodium pump in frog sartorius muscle

The binding of the cardiosteroid 3H-ouabain to frog skeletal muscle was determined by studying the kinetics of its uptake and release. The amount of ouabain bound as a function of drug concentration in the external medium follows a hyperbolic relationship with a maximum binding (Bmax) of the order of 2500 molecules per square micrometer of surface membrane and an affinity constant (K) of 2.2 X 10(-7)M. The data do not suggest a drug-receptor (Na pump site) relation other than one-to-one. Ouabain molecules are released from whole muscle into ouabain-free media very slowly. The release is a single exponential function of time (tau approximately equal to 25 hr). When re-binding is prevented by the presence of unlabeled ouabain in the external medium, the loss of labeled ouabain is increased (tau approximately equal to 15 hr). Increasing [K+]O from 2.5 to 10 mM slows the time course of binding without any significant change in binding capacity of the muscle fibers. Experiments on detubulated muscles indicate that the density of pump sites is considerably higher in the surface than in the T-tubular membrane. These findings agree with the report by Narahara et al. [Narahara, H.T., Vogrin, V.G., Green, J.D., Kent, R.A., Gould, M.K. (1979) Biochim. Biophys. Acta 552:247] on the distribution of (Na+ + K+)- ATPase among different cell membrane fractions from frog skeletal muscle.