Analysis of protein-mediated 3-O-methylglucose transport in rat erythrocytes: rejection of the alternating conformation carrier model for sugar transport

Abstract

3-O-Methylglucose (3OMG) transport in rat erythrocytes (RBCs) is mediated by a low-capacity, facilitated diffusion-type process. This study examines whether the characteristics of sugar transport in rat RBCs are consistent with the predictions of two diametric, theoretical mechanisms for sugar transport. The one-site carrier describes a transport mechanism in which sugar influx and efflux substrate binding sites are mutually exclusive. The two-site carrier describes a transport mechanism in which sugar influx and efflux substrate binding sites can exist simultaneously but may interact in a cooperative fashion when occupied by substrate. Michaelis and velocity parameters for saturable 3OMG transport in rat erythrocytes at 24 degrees C were obtained from initial rate measurements of 3OMG transport. The results are incompatible with the predictions of the one-site carrier but are consistent with the predictions of a symmetric two-site carrier, displaying negligible cooperativity between substrate binding sites. This allows reduction of the two-site carrier transport equations to a form containing fewer constants than the one-site carrier equations without limiting their predictive success. While the available evidence does not prove that rat erythrocyte sugar transport is mediated by a two-site mechanism, we conclude that adoption of the formally more complex one-site model for sugar transport in rat erythrocytes is unnecessary and unwarranted. Counterflow experiments have also been performed in which the time course of radiolabeled 3OMG uptake is measured in cells containing saturating levels of 3OMG. The results of these experiments are consistent with the hypothesis [Naftalin et al. (1985) Biochim. Biophys. Acta 820, 235-249] that exchange of sugar between intracellular compartments (cell water and hemoglobin) can be rate limiting for transport under certain conditions.

ATP-dependent sugar transport complexity in human erythrocytes

“…The glucose transport protein GLUT1 catalyzes sugar transport in cells of the reticuloendothelial system (7,61) and presents an interesting experimental puzzle. The steady-state kinetics of GLUT1-mediated sugar transport in rabbit (70), rat (38,62), and avian (7,8) erythrocytes and in basal (insulin-starved) rat adipocytes (76) are consistent with classical models for carrier-mediated solute transport (5,47). GLUT1-mediated sugar transport in human red cells, however, displays a kinetic complexity that has proven difficult to reconcile with models for carrier-mediated transport (4,21,32,49,56,79).…”

“…2) If net cellular sugar import were composed of two steps (transport followed by intracellular diffusion/distribution), the diffusional step could become rate limiting if the transport step were sufficiently rapid. Evidence for nonuniform intracellular distribution of sugars has been obtained in both human and rat erythrocytes (4,36,38,62,64).According to the diffusional barrier hypothesis, human red cell net sugar import is composed of rapid transport (owing to high cellular GLUT1 content) and slow intracellular diffusion and/or distribution. The overall result is one where net sugar import is rate limited by intracellular diffusion/distribution and not by transport.…”

“…The sugar transport capacity of human red cells is 220-to 10,000-fold greater than that of rat basal adipocytes (76), rat red blood cells (38), and avian erythrocytes (24). Naftalin and Holman (63) discussed several ways by which this could give rise to transport complexity.…”

“…2) If net cellular sugar import were composed of two steps (transport followed by intracellular diffusion/distribution), the diffusional step could become rate limiting if the transport step were sufficiently rapid. Evidence for nonuniform intracellular distribution of sugars has been obtained in both human and rat erythrocytes (4,36,38,62,64).…”

Human Erythrocyte Membranes Contain a Cytochrome b561 That May Be Involved in Extracellular Ascorbate Recycling

“…Because FeCN reduction is dependent on intracellular Asc, mouse and human erythrocytes were loaded to sim- ilar high intracellular Asc concentrations by allowing them to take up and reduce DHA to Asc. A caveat in this experiment is that DHA is taken up on glucose transporters, which are expressed about 200-fold higher in human than in rat erythrocytes (and presumably in erythrocytes from the mouse) (39). Therefore, mouse cells were loaded with 4-fold higher DHA concentrations to generate similar intracellular Asc concentrations.…”