Unidirectional [C-14]HCO3- and Cl-36 efflux from human red cells and ghosts was studied under self-exchange conditions at pH 7.8 and 0-degrees-C by means of the Millipore-Swinnex filtering technique. Control bicarbonate experiments showed that (CO2)-C-14 loss from the cells to the efflux medium was insignificant. The anion flux was determined under (a) symmetric variations of the anion concentration (C(i) = C(o) = 5-700 mM), and (b) asymmetric conditions with C(An) constant on one side and varied on the other side of the membrane. Simple Michaelis-Menten-like kinetics (MM fit: J(max)(eff).C/(K1/2 + C)) was used to describe anion flux dependence on C for (a) C(i) = C(o) = 5-100 mM, (b) C(i) = 6-100 mM, C(o) = constant, and (c) C(i) = constant, C(o) = 1-25 mM. At higher cellular concentrations noncompetitive self-inhibition by anion binding (inhibition constant K(i) mM) to an intracellular site was included in the model (MS fit): J(eff) = J(max)eff.C(i)/[(K1/2 + C(i)).(1 + C(i)/K(i))]. The MM fits show that the external half-saturation constant, K1/2o (= C(An)(o) for J(eff,o) = 1.2.J(max)eff,o) at C(o) = 1-25 mM is 1.5-2.4 mM (HCO3-) and 1.8-2.6 mM (Cl-). At C(o) = 1-260 mM K1/2o is 1.2-1.5 mM (HCO3-) and 1.4-1.8 mM (Cl-). The respective maximum flux, J(max)eff,o (nmol/[cm2.s]), for C(o) = 1-25 mM is 0.41-0.51 (HCO3-) and 0.28-0.38 (Cl-), and for C(o) = 1-260 mM 0.39-0.44 (HCO3-) and 0.27-0.31 (Cl-). The internal half-saturation constant, K1/2i mM is: MM fit (C(i) = 6-100 mM, C(o) = 50 mM), 18.0 mM (HCO3-) and 23.8 mM (Cl-); MS fit (C(i) = 6-920 mM, C(o) = 50 mM), 32.0 mM (HCO3-) and 45.1 mM (Cl-). The maximum flux, J(max)eff,i (nmol/[cm2.s]) is: MM fit; 0.50 (HCO3-) and 0.34 (Cl-); MS fit, 0.70 (HCO3-) and 0.50 (Cl-). The half-inhibition constants of the MS fit, K(i), are 393 mM (HCO3-) and 544 mM (Cl-). The MM fit shows that the symmetric half-saturation constant, K1/2s, is 20.2 (HCO3-) and 23.9 (Cl-) mM, and J(max)eff,s is 0.51 (HCO3-) and 0.32 (Cl-) nmol/(cm2.s). The MS fit shows that for C = 5-700 mM K1/2s is 30.4 mM (HCO3-) and 50.1 mM (Cl-), and K(i) is 541 mM (HCO3-) and 392 mM (Cl-). In both fits the K(i) values for HCO3- and Cl- are not significantly different. The functional asymmetry of the transport system was demonstrated for both anions, independent of any model, in two reversed experiments with either internal or external 50 mM anion and a varied concentration on the opposite side. The apparent affinity for both anions is approximately 10 times higher on the outside than on the inside (MM fit). According to the "ping-pong" model, K1/2o and K1/2s can be used to calculate an asymmetry factor (A). In our study A is 0.12 +/- 0.02 for both anions if the results obtained in the low concentration ranges are used; i.e., eight times more unloaded transport sites face inward than outward when C(i) = C(o). If the results of the whole concentration range are used, A(Cl) is 0.05 +/- 0.01 and A(bic) is 0.06 +/- 0.01. Hence, A does not depend on which anion is transported, but the magnitude of A depends on which concentration range and fitting procedure are used to determine the factors that can be used to calculate A.
