Galactose transport in human erythrocytes. The transport mechanism is resolved into two simple asymmetric antiparallel carriers

Abstract

The kinetic properties of the mediated transport of galactose in human erythrocytes are investigated at 20 degrees C. Different methodological procedures are used to acquire a complete kinetic description of the system. Under zero-trans conditions the uptake of galactose is mediated by two distinctly different carriers (defined as alpha and beta) having significantly different Mic;aelis parameters: alpha K = 12.7 mM and beta K = 81.5 mM, but similar maximal velocities, approx. 40 nM.min-1. The zero-trans efflux procedure reveals apparently one single carrier with K = 74.4 mM and V = 241 mM.min-1. Under equilibrium-exchange conditions the galactose transport is mediated apparently by a single site with K = 146 mM and V = 521 mM.min-1. The data for the alpha-carrier are analyzed in terms of the simple carrier model as formulated by Lieb and Stein (Biochim. Biophys. Acta (1974) 373, 178). Application of several rejection criteria for the simple carrier failed to indicate lack of fitness of the alpha-carrier to a simple asymmetric carrier. From the analysis of the kinetic data it is inferred that the transport of galactose across the human erythrocyte membrane is mediated by two simple asymmetric carriers operating in antiparallel fashion. Using this model and the data of zero-trans and equilibrium-exchange, it is shown that the predicted half-saturation constants for both uptake and efflux in infinite-cis conditions fully agree with the experimentally derived values. Further analysis of the kinetic data indicate that the translocation of the unloaded alpha-carrier is the rate-limiting step in galactose uptake. Under equilibrium-exchange conditions the unloaded carrier is asymmetrically distributed across the membrane so that its concentration is 8 times higher on the inner side of the membrane. Using the value of 3.3.10(5) hexose carriers per cell, the turnover number of galactose exchange is 6.5.10(4) molecules/carriers per min.