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The incorporation of 2-deoxy-d-[14C]glucose by cultured Novikoff rat hepatoma cells was investigated as a function of the deoxyglucose concentration in the medium. The intracellular radioactivity was mainly associated with deoxyglucose 6-phosphate, to a small extent with 6-phosphodeoxygluconate, and the remainder with free deoxyglucose. Below a concentration of 1 to 2 mm in the medium, the initial rate of deoxyglucose incorporation followed simple Michaelis-Menten kinetics at 22, 27, 32, and 37° with an apparent Km of about 2 mm. The Vmax values increased with a Q10 of about 2.5 with an increase in temperature. Between 2 and 10 mm, on the other hand, the initial rate of incorporation increased linearly with an increase in deoxyglucose concentration and the relative increase in incorporation rate with increase in deoxyglucose concentration was about the same at the temperatures tested. Treatment of the cells with 0.5 mm p-chloromercuribenzoate for 15 min or heat shock (48.5°, 5 min) abolished the saturable uptake component without affecting the nonsaturable component. When combined, the results indicate that at low concentrations, deoxyglucose is taken up mainly by carrier-mediated transport (probably facilitated diffusion), whereas at concentrations above 2 mm, simple diffusion becomes the principal mode of entry of deoxyglucose into the cells. Results from studies on d-glucose and 3-O-methyl-d-glucose incorporation support this conclusion and suggest that all three substrates are transported by a single system. Persantine competitively inhibited deoxyglucose uptake by whole cells and decreased the metabolism of glucose without affecting the phosphorylation of the substrates. Prior treatment with p-chloromercuribenzoate or heat shock also had no effect on the hexokinase activity of the cells. Little if any hexokinase activity was associated with the plasma membrane and the kinetic properties of the in vitro hexokinase reaction differed from that for glucose or deoxyglucose uptake by whole cells. These results indicate that transport of glucose into the cell is a reaction distinct from phosphorylation and is the rate-limiting step in the metabolism of glucose by the cells. Since the cells possess an excess of hexokinase, glucose is phosphorylated as rapidly as it enters the cells and thereby trapped. At low concentrations of glucose in the medium (5 to 50 µm), the glucose 6-phosphate formed was exclusively used by the cells for macromolecular synthesis and oxidative processes without net production of lactate. The rate of CO2 production and incorporation of glucose in macromolecules approached a maximum at about 1 mm glucose in the medium and the excess glucose 6-phosphate formed, particularly at higher concentrations of glucose in the medium, was converted to lactate. |
