Phosphate transport in human red blood cells: concentration dependence and pH dependence of the unidirectional phosphate flux at equilibrium conditions.
The concentration dependence and the pH dependence of the phosphate transport across the red cell membrane were investigated. The unidirectional phosphate fluxes were determined by measuring the 32P-phosphate self-exchange in amphotericin B (5 mumol/liter) treated erythrocytes at 25 degrees C. The flux/concentration curves display an S-shaped increase at low phosphate concentrations, a concentration optimum in the range of 150 to 200 mM phosphate and a self-inhibition at high phosphate concentrations. The apparent half-saturation concentrations, P(0.5), range from 50 to 70 mM and are little affected by pH. The self-inhibition constants, as far as they can be estimated, range from 400 to 600 mM. The observed maximal phosphate fluxes exhibit a strong pH dependence. At pH 7.2, the actual maximal flux is 2.1 X 10(-6) moles . min-1 . g cells-1. The ascending branches of the flux/concentration curves were fitted to the Hill equation. The apparent Hill coefficients were always in the range of 1.5-2.0. The descending branches of the flux/concentration curves appear to follow the same pattern of concentration response. The flux/pH curves were bell-shaped and symmetric with regard to their pH dependence. The pH optimum is at approximately pH 6.5-6.7. The apparent pK of the activator site is in the range of 7.0 to 7.2, while the apparent pK for the inactivating site is in the range of 6.2 to 6.5. The pK-values were not appreciably affected by the phosphate concentration. According to our studies, the transport system possesses two transport sites and probably two modifier sites as indicated by the apparent Hill coefficients. In addition, the transport system has two proton binding sites, one with a higher pK that activates and one with a lower pK that inactivates the transport system. Since our experiments were executed under self-exchange conditions, they do not provide any information concerning the location of these sites at the membrane surfaces.
D000666:Amphotericin B / Q000494:pharmacology; D000838:Anions; D001692:Biological Transport / Q000187:drug effects; D002712:Chlorides / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010710:Phosphates / Q000097:blood*; D014634:Valinomycin / Q000494:pharmacology
D000838:Anions; D002712:Chlorides; D010710:Phosphates; D014634:Valinomycin; D000666:Amphotericin B