Sugar Transport in Chick Embryo Fibroblasts

Abstract

Abstract Uptake of 2-deoxy-d-glucose in cultured chick embryo fibroblasts consists of two reactions, transport and phosphorylation (Kletzien, R. F., and Perdue, J. F. (1973) J. Biol. Chem. 248, 711–719). Further studies were initiated to examine in greater detail the mechanism of sugar uptake and to determine which of the two reactions was responsible for the differing rates of 2-deoxy-d-glucose uptake between rapidly and slowly growing cells. The uptake of 2-deoxy-d-glucose was studied as a function of time and substrate concentration to establish the conditions under which the phosphorylation of the sugar parallels uptake. The initial rates of phosphorylation of 2-deoxy-d-glucose in intact cells were dependent on the rate of cell growth. However, the initial rates of phosphorylation in cellular homogenates prepared from rapidly and slowly growing cells were identical and were 4 to 6 times greater than the rates observed in intact cells. Experiments with cytochalasin B, a sugar transport inhibitor, demonstrated that an inhibition of transport was always paralleled by an equal inhibition of sugar phosphorylation. These data indicate that a reaction prior to phosphorylation is responsible for the altered rates of sugar uptake in rapidly and slowly growing cells and that this same reaction limits the rate at which 2-deoxyglucose is phosphorylated in intact cells at early time points. The kinetic constants for the uptake and phosphorylation of 2-deoxy-d-glucose and the inhibition of this uptake and phosphorylation by competing sugars were calculated from Lineweaver-Burk plots. The Km for sugar uptake was constant at 2 mm; the Vmax was 16.7 or 33.2 nmoles per mg of protein per min, depending on the rate of cell growth. The Km for sugar phosphorylation in cellular homogenates was 0.8 mm; the Vmax was 75 nmoles per mg of protein per min and was not dependent on the rate of cell growth. The Ki values for the competitive inhibition of 2-deoxy-d-glucose uptake in intact cells and phosphorylation in cellular homogenates by d-glucose were 2 and 0.18 mm, respectively. 3-O-Methyl-d-glucose competitively inhibited 2-deoxy-d-glucose uptake with a Ki of 17 to 20 mm. The kinetic constants for 3-O-methyl-d-glucose uptake and inhibition of uptake by d-glucose were also calculated from Lineweaver-Burk plots. The Km for 3-O-methyl-glucose uptake was 3.5 to 5.0 mm, and the Vmax was 19 or 37.5 nmoles per mg of protein per min, depending on the rate of cell growth. The Ki for d-glucose inhibition of uptake was 1.8 mm. These results demonstrate that 2-deoxy-d-glucose, 3-O-methyl-d-glucose, and d-glucose are all transported by the same site, that sugar transport is the rate-limiting step in sugar uptake, and that sugar transport is a reaction distinct from phosphorylation. The latter conclusion was further substantiated by the finding that hexokinase was not associated with isolated plasma membrane. The difference in the rate of uptake of 2-deoxy-d-glucose and 3-O-methyl-d-glucose between rapidly growing and slowly growing cells was strictly attributed to differences in the Vmax for sugar transport; the Km for transport did not change with the rate of cell growth. Activation energies for the sugar transport reaction were calculated from Arrhenius plots and were found to be identical for rapidly and slowly growing cells, a result which is consistent with the interpretation that the increase in Vmax in rapidly growing cells is the result of a greater number of sugar transport sites.