146 Biochimica et Biophysica Acta 857 (1986) 146 154 Elsevier BBA 73096 The kinetics of glucose transport in human red blood cells Allan G. Lowe and Adrian R. Walmsley Department o/Biochemistry, Medical School, University of Manchester, Manchester, MI3 9PT (U.K.) (Received December 24th, 1985) Key words: Glucose transport; Kinetics; (Erythrocyte membrane) A quenched-flow apparatus and a newly developed automated syringe system have been used to measure initial rates of D-[ 14C]glucose transport into human red blood cells at temperatures ranging from 0 ° to 53°C. The Haldane relationship is found to be obeyed satisfactorily at both 0 and 20°C, but Arrhenius plots of maximum D-[ t4C]glucose transport rates are non-linear under conditions of both equilibrium exchange and zero trans influx. Fitting of the data by non-linear regression to the conventional model for glucose transport gives values at 0°C of 0.726 =l= 0.0498 s- t and 12.1 + 0.98 s- l for the rate constants governing outward and inward movements of the unloaded carrier molecule and 90.3 =1= 3.47 s- t and 1113 + 494 s-t for outward and inward movements of the carrier-glucose complex. Activation energies for these four rate constants are respectively 173 + 3.10, 127 + 4.78, 88.0 + 6.17 and 31.7 + 5.11 kJ • mol -i. These parameters indicate that at low temperatures the marked asymmetry of the transport mechanism arises mainly from the high proportion of inward-facing carriers and carrier-glucose complexes, and that there is a relatively small difference between the affinities of the carrier for glucose in the inward and outward-facing conformations. At high (physiological) temperatures the carrier is fairly evenly distributed between outward- and inward- facing conformations and the affinity for glucose is about 2.5-times greater outside than inside. Introduction The glucose carrier of the human red blood cell is one of the most intensively studied transport systems, with isolation of the carrier molecule [1], specific labelling with cytochalasin B [2] and fluo- rescence studies of single half turnovers [3] among recent interesting advances. The amino-acid se- quence and trans-membrane disposition of the glucose carrier in human hepatoma cells has also been determined [4]. Despite this progress, the essential kinetics of the transport mechanism re- main only partially resolved. Most kinetic studies Abbreviations: maximum rates of transport under conditions of equilibrium exchange, zero trans influx and zero trans efflux are respectively V ee, V, zt, and Vi~ t. Michaelis constants (Kin) under conditions of equilibrium exchange, zero trans influx and zero trans efflux are respectively K ee, KoCh, and Ki 2t. have involved measurements of isotope-labelled glucose exchange under equilibrium exchange con- ditions, or net movement of glucose under 'in- finite-cis' or 'infinite-trans' conditions, but studies of glucose entry into glucose-free cells have been few and restricted to relatively low temperatures, while only Brahm [5] and Miller [6] have measured initial rates of glucose efflux from cells in to glucose-free medium. The rapidity of glucose transport has also restricted most studies to well below the physiological temperature range. In this work, we describe the application of a recently developed rapid reaction technique [7] and a newly developed semi-rapid reaction tech- nique to measurements of glucose transport in human red cells under conditions of equilibrium exchange entry and zero trans entry and exit, at temperatures between 0 and 53°C. This work has enabled us to characterise the transport mecha- 0005-2736/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)