A kinetic analysis of hexose transport in cultured human lymphocytes (IM-9)

Abstract

3-O-Methyl-D-glucose transport across the plasma membrane of cultured human lymphocytes of the IM-9 line was followed for net entry into sugar-free cells (zero trans entry), net exit into sugar-free medium (zero trans exit) and for equilibration of labelled sugar in cells with the same sugar concentration in the intracellular water as in the medium (equilibrium exchange). The measurements were performed at 37 degrees C (pH 7.4). Equilibrium exchange of 1 mM 3-O-methylglucose (t 1/2 about 7 S) was exponential, suggesting a homogeneous cell suspension. Initial rates of transport showed a Michaelis-Menten dependency on the sugar concentration. The transport system was found to be asymmetric with the following kinetic parameters. Zero trans entry: Km = 2.8 mM, Vmax = 10.7 mM X min-1. Zero trans exit: Km = 9.5 mM, Vmax = 37.9 mM X min-1. Equilibrium exchange: Km = 9.9 mM, Vmax = 44.0 mM X min-1. Finally, the affinity constant for the internal site was measured as approx. 1.2 mM using the infinite cis protocol.