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

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Abstract
The concentration dependence of the sulfate and the phosphate selfexchange and homoexchange fluxes was studied in resealed red cell ghosts (25° 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.

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