474 Biochimica el Biophysica Acta, 550 (1979) 474--484 © Elsevier/North-Holland Biomedical Press BBA 78260 EVIDENCE OF MULTIPLE OPERATIONAL AFFINITIES FOR D-GLUCOSE INSIDE THE HUMAN ERYTHROCYTE MEMBRANE G.F. BAKER and R.J. NAFTALIN Department of Physiology, University of London King's College, Strand, London WC2R 2LS (U.K.) (Received June 15th, 1978) Key words: D-Glucose transport; Asymmetric carrier; Pore kinetics; (Erythrocyte) Summary 1. The Michaelis-Menten parameters of labelled D-glucose exit from human erythrocytes at 2°C into external solution containing 50 mM D-galactose were obtained. The Km is 3.4 + 0.4 mM, V 17.3 + 1.4 mmol • 1-1 cell water • min -1 for this infinite-trans exit procedure. 2. The kinetic parameters of equilibrium exchange of D-glucose at 2°C are Km= 25 -+ 3.4 mM, V 30 + 4.1 mmol • 1-1 cell water • min -1. 3. The Km for net exit of D-glucose into solutions containing zero sugar is 15.8 + 1.7 mM, V 9.3 + 3.3 mmol • 1 -~ cell water • min -1. 4. This experimental evidence corroborates the previous finding of Hankin, B.L., Lieb, W.R. and Stein, W.D. [(1972) Biochim. Biophys. Acta 255, 126-- 132] that there are sites with both high and low operational affinities for D- glucose at the inner surface of the human erythrocyte membrane. This result is inconsistent with current asymmetric carrier models of sugar transport. Introduction Several paper~ recently have suggested that an asymmetric form of the mobile carrier model for sugar transport is consistent with the observed asymmetric transport of D-glucose across the human erythrocyte membrane [1--6]. The transport site at the inner surface is commonly considered to have a 10-fold lower affinity, but a 10-fold higher rate of transference to the other side of the membrane than the sites at the outer surface. This straightforward adaptation of the mobile~arrier model explains the apparent asymmetries in net entry and exit fluxes as well as the differences in operational affinity between the net entry, net exit and exchange transport systems for .sugars whose transport is facilitated across the erythrocyte membrane. The observation of Hankin et al. [7] and Ginsburg and Stein [8] that there is