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Department of General Physiology and Biophysics, Panum Institute, University of Copenhagen, Denmark.
1. Na(+)-HCO3- co-transport across the retinal membrane of the frog retinal pigment epithelium was studied by means of double-barrelled pH-selective microelectrodes. Transient changes in the intracellular pH were monitored in response to abrupt changes in the Na+ concentration on the retinal side of the epithelium. 2. The experiments were performed as follows. The Na(+)-HCO3- co-transport was inhibited by perfusing the retinal side of the epithelium with a Na(+)-free solution. The co-transport was then stimulated by changing the perfusate from the Na(+)-free solution to a solution which contained from 5 to 110 mM-Na+. The resulting inward Na(+)-HCO3- co-transport produced an intracellular alkalinization, the initial rate of which was used to calculate the initial rate of Na(+)-HCO3- co-transport, JHCO3-. 3. The Na+ dependence of the Na(+)-HCO3- co-transport was studied at two different values of extracellular pH (7.40 and 7.10), at constant extracellular HCO3- concentration (27.5 mM) and at two different extracellular HCO3- concentrations (27.5 mM and 55 mM) at constant extracellular pH (7.40). In these experiments, the calculated values of JHCO3- followed single Michaelis-Menten kinetics with respect to the extracellular Na+ concentration. 4. The data are consistent with a model in which the co-transporter has a single binding site for the Na+ ion with an apparent affinity constant (apparent Km) of 37 mM. The apparent affinity constant for Na+ was independent of the extracellular concentration of CO3(2-) in the range of 16-65 microM, and of the extracellular HCO3- concentration in the range 27.5-55 mM. 5. The NaCO3- ion-pair hypothesis, in which sodium binds to the co-transporter and is translocated across the cell membrane as the NaCO3- ion pair, was analysed. For stoichiometries 1:2 and 1:3 of the Na(+)-HCO3- co-transport, the NaCO3- ion-pair hypothesis was found incompatible with the data. 6. The intracellular buffer capacity as measured by the CO2 method was 15 mM.
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