Sugar Transport in Chick Embryo Fibroblasts: II. ALTERATIONS IN TRANSPORT FOLLOWING TRANSFORMATION BY A TEMPERATURE-SENSITIVE MUTANT OF THE ROUS SARCOMA VIRUS

Abstract

Abstract Chick embryo fibroblasts infected with the temperaturesensitive mutant of the Rous sarcoma virus, Ts-68, are morphologically transformed when cultured at 37° but retain their fusiform morphology when cultured at 41°. Accompanying the temperature-dependent cell shape changes are alterations in the rate of 2-deoxy-d-glucose uptake. Ts-68-infected fibroblasts cultured at 37° have a 2.5- to 4-fold higher rate of sugar uptake than fibroblasts cultured at 41°. When Ts-68-infected fibroblasts cultured at 41° are shifted to 37° or vice versa, the greatest change in the rate of sugar uptake occurs in the first 6 hours following the temperature shift. The initial rates of 2-deoxy-d-glucose uptake and intracellular phosphorylation in Ts-68-infected fibroblasts depended upon the culture temperature. However, no differences were found in the initial rates of phosphorylation in homogenates prepared from infected cells cultured at 41 and 37°. Experiments with cytochalasin B, a sugar transport inhibitor, showed that inhibition of 2-deoxy-d-glucose uptake was always paralleled by an equal inhibition of 2-deoxy-d-glucose phosphorylation. These data indicated that transport was rate-limiting in 2-deoxy-d-glucose uptake at early time points, and alterations in transport were responsible for the alterations in sugar uptake in Ts-68-infected chick embryo fibroblasts. Lineweaver-Burk plots were used to calculate the kinetic constants for 2-deoxy-d-glucose and 3-O-methyl-d-glucose transport in Ts-68-infected cells cultured at 41 and 37°. The Km remained constant at approximately 2 or 5 mm at both temperatures for 2-deoxy-d-glucose and 3-O-methyl-d-glucose, respectively, but the Vmax was more than 2-fold greater for cells cultured at 37° (17 and 40 nmoles per mg of protein per min for Ts-68-infected cells cultured at 41 and 37°, respectively). Determination of the kinetic constants in Ts-68-infected cells following a 6-hour shift in culture temperature from 41 to 37° or 37° to 41° revealed that only the Vmax for 2-deoxy-d-glucose transport was changed. The similar values calculated for activation energy, 17.9 and 17.5 Cal per mole, for the 2-deoxy-d-glucose transport reaction in Ts-68-infected cells at 41 and 37°, respectively, is consistent with the interpretation that the increased Vmax in cells cultured at 37° was the result of an increase in the number of transport sites.