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The phosphate self-exchange flux in resealed erythrocyte ghosts and in amphotericin B (5.5 microM) permeabilized erythrocytes has been studied. The phosphate self-exchange flux exhibits an S-shaped concentration dependence and a self-inhibition in permeabilized red cells while in erythrocyte ghosts no self-inhibition of the phosphate flux has been observed. The apparent half-saturation constants and the apparent Hill coefficients were assessed by the double reciprocal Hill plots of 1/JP versus 1/[P]n. The phosphate half-saturation constants amount to approx. 125 mM in ghosts and to about 75 mM in permeabilized cells while the apparent Hill coefficients amount to 1.15 and to 1.65 (pH 7.2, 25 degrees C), respectively. Both chloride and sulfate elicit a mixed-type inhibition of the phosphate self-exchange flux. In permeabilized cells, chloride and sulfate shift the flux optimum towards higher phosphate concentrations and reduce the apparent Hill coefficients. In erythrocyte ghosts, the apparent Hill coefficients are insensitive to these anions. The double reciprocal Hill plots indicate a mixed-type inhibition of the phosphate self-exchange flux by DNDS, salicylate and dipyridamole and a noncompetitive inhibition of the phosphate self-exchange flux by phlorhizin. By contrast, the Hill-Dixon plots for chloride and sulfate indicate a competitive inhibition of the phosphate self-exchange flux in erythrocyte ghosts and a mixed-type inhibition in permeabilized cells and provide Hill coefficients of greater than unity for chloride and sulfate. The Dixon plots for DNDS, salicylate, phlorhizin and dipyridamole show a noncompetitive inhibition of the phosphate flux and provide apparent Hill coefficients of 0.95-1.0 for inhibitor binding. Using the Debye-Hückel theory, the effects of ionic strength upon phosphate transport and inhibitor binding can be eliminated. The results of our studies provide strong evidence for the assumption that electrostatic forces are involved in phosphate transport and in inhibitor binding.

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.

The concentration dependence of the unidirectional chloride flux and the inhibition of the unidirectional chloride flux by sulfate were studied in human red cell ghosts. The concentration dependence of the unidirectional chloride flux and its inhibition by sulfate were asymmetric. The unidirectional chloride flux can be saturated from the inner and from the outer membrane surface. For the inner membrane surface, lower chloride half-saturation constants were obtained than for the outer membrane surface. The inhibition of the unidirectional chloride flux by sulfate is competitive. In contrast to the chloride half-saturation constants, the inhibition constants of sulfate for the inner membrane surface were higher than the inhibition constants of sulfate for the outher membrane surface. Either there are fixed anion binding sites at the inner and at the outer membrane surface which control the access of anions to a pore, or there is a mobile carrier which is in contact with both membrane surfaces. The asymmetry of the concentration response and of the inhibition of the unidirectional chloride flux suggest that the anion binding sites at the inner and at the outer membrane surface differ with respect to their affinities for chloride and for sulfate. Alternatively, the asymmetry of the chloride transport system could indicate an asymmetric distribution of a mobile anion carrier across the erythrocyte membrane.

The disulfonatostilbene spin label, NDS-TEMPO, was synthesized (purity over 96%) and the binding of the spin label to human red-cell ghosts was studied. NDS-TEMPO is readily absorbed to the membrane surface. Both pretreatment of the ghosts with FDNB and DIDS and the presence of DNDS completely prevent the binding of NDS-TEMPO to red-cell ghosts. Chloride and sulfate competitively inhibit the binding of NDS-TEMPO. Conversely, NDS-TEMPO is a strong, competitive inhibitor of chloride and of sulfate transport. The dissociation constants of NDS-TEMPO from the ESR studies were in the range 1.0-2.0 microM (pH 7.6, 20 degrees C). The inhibition constants of NDS-TEMPO as obtained from the flux experiments were in the range 0.5-2.5 microM (pH 7.3, 25 degrees C). The close accordance of the NDS-TEMPO dissociation constants from the ESR studies with the NDS-TEMPO inhibition constants from the flux measurements indicate a specific labeling of the inorganic-anion-transport system.

The DMO (5,5-dimethyloxazolidine-2,4-dione) transport was studied in human red blood cells by measuring the 14C-DMO back exchange under self exchange conditions from 14C-DMO labeled cells at 0 degrees C. the unidirectional DMO-flux was linearly related to the DMO concentration up to approx. 100 mM. The unidirectional DMO-flux increases as pH is reduced. The DMO transport is not inhibited by anion transport inhibitors like DIDS, SITS, dipyridamole, phlorhizin, salicylate or butanol. A close correlation between the unidirectional DMO-flux and DMOH, the unionized form of DMO, has been observed suggesting that DMO is transported predominantly by nonionic diffusion. the permeability of DMOH is 9.5 X 10(-6) cm/s (0 degrees C) while the permeability of DMO- cannot be assessed from our data.

The DMO (5,5-dimethyloxazolidine-2,4-dione) transport was studied in human red blood cells by measuring theC-DMO back exchange under self exchange conditions fromC-DMO labeled cells at 0°C. The unidirectional DMO-flux was linearly related to the DMO concentration up to approx. 100 mM. The unidirectional DMO-flux increases as pH is reduced. The DMO transport is not inhibited by anion transport inhibitors like DIDS, SITS, dipyridamole, phlorhizin, salicylate or butanol. A close correlation between the unidirectional DMO-flux and DMOH, the unionized form of DMO, has been observed suggesting that DMO is transported predominantly by nonionic diffusion. The permeability of DMOH is 9.5·10 cm/s (0°C) while the permeability of DMO cannot be assessed from our data. [ABSTRACT FROM AUTHOR]