Conventional transport assays underestimate sugar transport rates in human red cells

The time course of protein-mediated 3-O-methylglucose uptake by human red cells and by red cell ghosts containing or lacking 4 mM MgATP was measured at ice temperature and sub-saturating sugar levels by conventional sampling procedures and at 20°C by use of a quench-flow apparatus. The temporal resolution of the quench-flow apparatus (as fast as 5-ms sample times) was confirmed by analysis of alkaline hydrolysis of dinitrophenolacetate. Red cell sugar uptake at 4°C is consistent with two processes [fast (τ = 120 s) and slow (τ = 1100 s)] that occur in series. Intracellular ATP increases the size and the rate of equilibration of the fast compartment and slows the rate of filling of the slow compartment. Red cell ghost volume and protein content are unaffected by lysis/resealing in the presence of ATP. Uptake at 20°C is also consistent with two processes [fast (τ = 10 ms) and slow (τ = 15 s)] that occur in series. ATP increases the size of both compartments and the rate of filling of the small compartment at 20°C. Preliminary estimates indicate that the sugar uptake capacity of human red cells at 20°C is underestimated by as much as 8-fold by measuring sugar uptake over 2 s vs. 26 ms. We discuss the implications of multiphasic sugar uptake in the context of models for protein-mediated sugar transport.