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The kinetics of association and dissociation for the ouabainNa+,K+-dependent ATPase complex have been studied in intact turkey erythrocytes as a function of external Na § concentration, K § concentration, and temperature. At free ligand concentrations substantially exceeding the concentration of available binding sites, the association reaction exhibits pseudo-firstorder kinetics with an association rate constant (kl) that is conveniently determined over a wide range of temperatures (5-37~The dissociation reaction exhibits strict first-order kinetics with a dissociation rate constant (k-a) that has the unusual property, in the turkey cell, of being sufficiently great to permit its direct determination even at temperatures as low as 5~Values for the equilibrium binding constant for the ouabain-ATPase complex (KA) predicted from the ratio of the association and dissociation rate constants agree closely with independently measured values of KA determined directly under conditions of equilibrium binding. KA is a sensitive function of the composition of the external ionic environment, rising with increasing Na § concentration and falling with increasing K § concentration. These changes in KA are shown to be quantitatively attributable to changes in the rate constant kl, k-1 in contrast being unaffected at any given temperature by even very large changes in Na + or K + concentration. Arrhenius plots of k~ and k-i both yield straight lines over the entire temperature range corresponding to activation energies for association and dissociation of 29.5 and 24.2 kcal/mol, respectively. These observations have made it possible to calculate the following standard values for the ouabain binding reaction in the presence of 150 mM Na+: AG ~ ------9.8 kcal/mol; ~ff_/o = +5.3 kcal/mol; AS ~ = +48.7 cal/degree/mol. The large positive value of AS ~ presumably reflects a highly ordered configuration of the ouabain-free ATPase molecule that is lost upon ouabain binding and that "drives" the reaction despite the positive value of ~//o.

A B S T R A C T We have previously reported that in hypothyroid turkeys the number of beta-adrenergic receptors in intact erythrocytes is reduced by -50% without any changes in the affinity of the receptor for the agonist, isoproterenol. In view of the physiological action of the catecholamines to stimulate bidirectional ion fluxes in these cells, we have now examined the possibility that the decrease in beta receptor number might be associated with concomitant changes in catecholamine-dependent potassium ion transport. Hypothyroid turkey erythrocytes display decreased sensitivity to isoproterenol-stimulated potassium influx.Half-maximal stimulation of potassium influx occurs at 9.2±+1.7 nM in hypothyroid cells as opposed to only 3.8±+0.4 nM in normal cells (P < 0.005). A maximal stimulatory concentration of isoproterenol (100 nM) leads to the same increment in ion flux in erythrocytes from hypothyroid and normal turkeys. Analysis of the quantitative relationship between isoproterenol concentration, receptor occupancy, and associated effects upon potassium influx shows that at low levels of isoproterenol, where occupancy is linear with agonist concentration, occupation of a given number of beta receptors leads to a stimulation of potassium transport that is identical in erythrocytes from normal and hypothyroid turkeys. Thus, decreased sensitivity to catecholamine-stimulated potassium transport in hypo-