Activation Energy of the Slowest Step in the Glucose Carrier Cycle: Break at 23 °C and Correlation with Membrane Lipid Fluidity

Glucose transport in the rat erythrocyte is subject to feedback regulation by sugar metabolism at high but not at low temperatures [Abumrad et al. (1988) Biochim. Biophys. Acta 938, 222-230]. This indicates that temperature, which is known to alter membrane fluidity, also alters sensitivity of transport to regulation. In the present work, we have investigated a possible correlation between the effects of temperature on rate-limiting steps of glucose transport and on membrane fluidity. The dependences of methylglucose efflux and influx on cis and trans methylglucose concentrations were studied at temperatures between 17 and 37 °C. Membrane fluidity was monitored over the same temperature range by using electron paramagnetic resonance spectroscopy. External sugar did not affect efflux, and the K_m and V_max of sugar exit were respectively the same as the K_m and V_max of equilibrium exchange. These K_m's were relatively temperature independent, but the V_max's increased sharply with temperature. The K_m and V_max of methylglucose entry were respectively much lower than the K_m and V_max of exit and exchange. Consistent with the above, intracellular sugar greatly enhanced sugar influx, and did so by increasing the influx V_max without affecting the influx K_m. Both lines of evidence indicated that the conformational change of the empty sugar-binding site from in-facing to out-facing orientation is the rate-limiting step of sugar entry into the rat erythrocyte. This was the case at all temperatures; however, the discrepancies of coefficients declined significantly with increasing temperature. The temperature dependence of the slowest step (change from in- to out-facing empty carrier) was evaluated. An Arrhenius plot showed that this step had a much greater activation energy below 23 °C than above: 48.5 ± 6.0 kcal/mol compared to 24.1 ± 1.8 kcal/mol. The temperature dependence of membrane fluidity, monitored by electron paramagnetic resonance spectroscopy of the probe 5-nitroxylstearate, also exhibited a transition near 23 °C. Our data indicate that membrane fluidity may be a factor influencing ease of carrier conformation change.