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

Abstract

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.

Quantification of the maximum capacity for active sodium‐potassium transport in rat skeletal muscle.

“…In order to assess the significance of tissue dimensions, strips weighing 5-15 mg were prepared from the lateral parts of soleus muscles obtained from 4-week-old rats. Following Na+ loading, the ouabain-suppressible 86Rb+ uptake in these strips was compared with that of whole intact muscles weighing around 30 mg. From (Clausen & Hansen, 1974;Erlij & Grimstein, 1976;Manery, Dryden, Still & Madapallimattam, 1977;Venosa & Horowicz, 1981;Clausen, Hansen, Kjeldsen & N0rgaard, 1982; for review, see Clausen, 1986 (12) 5113+828 (5) > 060 129-6…”

Mechanism of alteration of sodium potassium pump of erythrocytes from patients with chronic renal failure.

“…In the present study, the number of Na-K pump sites was quantitated by the technique of [3H]ouabain binding (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). The accurate quantitation of the number of pump sites in patients with normal and high cell Na permitted an examination of the role of alteration in the number of ion pump units in raising intracellular Na.…”

Peeled mammalian skeletal muscle fibers. Possible stimulation of Ca2+ release via a transverse tubule-sarcoplasmic reticulum mechanism.

“…Since the binding site for ouabain is on the outside of the sarcolemma (and thus the inside of the TTs) (Venosa and Horowicz, 1981) and ouabain is not very lipid-soluble (Erdmann et al, 1977;Schwartz et al, 1975), it was necessary to allow diffusion of ouabain into the TTs before peeling . Single unpeeled fibers were cut in half transversely in the relaxing solution .…”

“…Ouabain would be expected to block the polarization of any sealed TTs within the peeled fiber, and if the TT membrane is not polarized it should not respond to ionic depolarization (Hodgkin and Horowicz, 1960;Nakajima and Endo, 1973). The finding that ouabain application after peeling did not affect the Cl--induced Ca" release is consistent with ouabain's lipid insolubility (Erdmann et al ., 1977 ;Schwartz et al, 1975) and with the location of the ouabain binding sites on the interior (extracellular side) of any sealed TTs (Venosa and Horowicz, 1981). The failure to block the Cl -induced tension transient by ouabain presoak in some fibers may have been due to damage that impaired ouabain entry into TTs.…”