Concentration dependence of the unidirectional sulfate and phosphate flux in human red cell ghosts under selfexchange and under homoexchange conditions

Abstract

The concentration dependence of the sulfate and the phosphate selfexchange and homoexchange fluxes was studied in resealed red cell ghosts (25 degrees C, pH 7.3). The selfexchange fluxes were calculated from the rate constant of the tracer back-exchange and from the intracellular substrate anion content. The homoexchange fluxes were determined from the initial cis-to-trans tracer fluxes and the initial specific substrate anion activities at the cis-membrane side. Sulfate and phosphate concentrations ranging from approx. 2-100 mM were employed. The selfexchange fluxes of sulfate and of phosphate exhibit sigmoidal flux/concentration curves. The apparent Hill coefficients were in the range of 1.2-1.4 indicating a type of positive cooperativity. Under homoexchange conditions the positive cooperativity of the flux/concentration curves disappears. The outward homoexchange fluxes of sulfate and phosphate display a saturation kinetics with Hill coefficients close to 1.0. The inward homoexchange fluxes exhibit a negative type of cooperativity with Hill coefficients smaller than 1.0. The sulfate and the phosphate half-saturation concentrations for the outer and the inner membrane surface are equal in size and amount to approx. 35 mM for sulfate and to approx. 110 mM for phosphate, respectively. The positive cooperativity of the unidirectional sulfate and phosphate fluxes under selfexchange conditions and the disappearance of the positive cooperativity under homoexchange conditions indicate a cooperativity of the translocation process. The saturation of the outward homoexchange flux and the negative cooperativity of the inward homoexchange flux suggest a substrate anion binding according to the law of mass action at the inner and a negative cooperativity of substrate anion binding at the outer membrane surface.

Characterization of the Band 3 substrate site in human red cell ghosts by NDS-TEMPO, a disulfonatostilbene spin probe: The function of protons in NDS-TEMPO and substrate-anion binding in relation to anion transport

“…Although K-citrate does not penetrate the erythrocyte membrane, it is not an ideal substitute. Below pH 7.0, K-citrate has a weak inhibitory effect on chloride and on sulfate transport which increases as pH is reduced (Milanick & Gunn, 1982;Schnell & Besl, 1984;Hautmann & Schnell, 1985;Dobler & Schnell, unpublished results). Similarly, the NDS-TEMPO/substrate-site dissociation constants and the chloride/substrate-site dissociation constants increase at decreasing pH (Table 3).…”

Deficient HCO3- Transport in an AE1 Mutant with Normal Cl- Transport Can be Rescued by Carbonic Anhydrase II Presented on an Adjacent AE1 Protomer

“…In contrast, the kinetics of erythroid dithionite/sulfate hetero-exchange (64) and of sulfate and phosphate homo-exchange (65) 3 Ϫ Transport by CA2rescued activity is mediated by the LDAAA protomer of the heterodimer. Furthermore, the LDAAA protomer of the heterodimer suffices to constitute a transbilayer anion translocation pathway.…”

Inhibition of the phosphate self-exchange flux in human erythrocytes and erythrocyte ghosts

“…As shown in Table 4 e has been observed which indicates that phosphate behaves like a divalent anion. The volume of erythrocyte ghosts from double isotonic solutions amounts to 0.49 + 0.03 ml/g cells wet weight and is smaller than the volume of erythrocyte ghosts from isotonic phosphate solutions which amounts to 0.65 -0.03 ml/g cells wet weight (Schnell & Besl, 1984), but no statistical significant differences of the phosphate self-exchange fluxes between erythrocyte ghosts from isotonic and erythrocyte ghosts from double isotonic solutions have been observed. The volume of permeabilized red cells amounts to 0.67 + 0.03 ml/g cells wet weight and exhibits a slight decrease at phosphate concentrations of greater than 200 mM.…”

“…With regard to the dielectric pore concept, self-inhibition of anion transport results from the blocking of the pore as the trans-substrate site of the dielectric pore gets occupied by substrate anions (Schnell, 1977;Tanford, 1985). For reviews see: Knauf, 1979;Macara & Cantley, 1983;Jennings, 1985Jennings, , 1989Passow, 1986. Previous studies on the concentration dependence of the phosphate transport in amphothericin B or valinomycin-permeabilized erythrocytes and in resealed erythrocyte ghosts have shown a complex pattern of concentration responsiveness of the phosphate transport (Schnell, Besl &von der Mosel, 1981;Schnell & Besl, 1984). The phosphate selfexchange flux in permeabilized red cells and in erythrocyte ghosts exhibited an S-shaped concentration response and provided apparent Hill coefficients of 1.60-1.80 in permeabilized erythrocytes and of about 1.25 in erythrocyte ghosts.…”