Deoxyglucose and 3‐O‐methylglucose transport in untreated and ATP‐depleted novikoff rat hepatoma cells. Analysis by a rapid kinetic technique, relationship to phosphorylation and effects of inhibitors

Abstract

Detailed time courses of uptake of labeled 3‐O‐methyl‐D‐glucose and 2‐deoxy‐D‐glucose by untreated and ATP‐depleted Novikoff rat hepatoma cells were determined as function of concentration (0.2–10 mM) by a rapid mixing/sampling technique which allows uptake measurements in time intervals as short as 1.5 seconds. Intracellular accumulation of 3‐O‐methylglucose in untreated and ATP‐depleted cells and of deoxyglucose in ATP‐depleted cells to equilibrium followed pseudo‐first order kinetics and initial velocities were computed from the overall time courses of substrate accumulation. Initial velocity was a Michaelis‐Menten function of exogenous substrate concentration. The estimated kinetic constants for zero‐trans transport of 3‐O‐methylglucose were about the same for untreated and ATP‐depleted cells (K = 1.73 ± 0.24 mM; V = 28.8 ± 3.6 pmoles/μl cell H₂O. sec) and were similar to those for deoxyglucose transport in ATP‐depleted cells (K = 0.65 ± 0.1 mM; V = 19.6 ± 1.6 pmoles/μl cell H₂O. sec). Similar kinetic parameters were obtained for the transport of D‐glucose and D‐galactose in ATP‐depleted cells. The transport of 3‐O‐methylglucose and deoxyglucose were inhibited by each other in a simple competitive manner with apparent K_i's similar to their transport K_m's. In untreated cells, in which deoxyglucose was phosphorylated, intracellular steady‐state levels of free deoxyglucose accumulated within 10 to 20 seconds of incubation regardless of its concentration in the medium. Thereafter, the rate of deoxyglucose incorporation into total cell material reflected the rate of phosphorylation rather than the transport rate. The rate of deoxyglucose transport exceeded the initial rate of its phosphorylation by 20–40%. The intracellular steady‐state levels observed during the first 2 minutes of incubation decreased from about 40% of equilibrium level at 0.2 mM deoxyglucose to about 8% at 10 mM. Computer fits of a kinetic equation describing transport and phosphorylation as independent processes operating in tandem to these data are consistent with the observed kinetic constants for hexose transport and hexokinase activity with deoxyglucose as substrate. Upon longer incubation (2–10 minutes) the rate of deoxyglucose uptake by the phosphorylating cells decreased progressively, concomitant with a decrease in intracellular ATP and an increase in intracellular deoxyglucose to equilibrium levels. It is demonstrated that the rate of deoxyglucose uptake, measured at two or more minutes, seriously underestimates the hexose transport rate and yields misleading conclusions regarding the extent and type of inhibition by transport inhibitors, such as Persantin or cytochalasin B. Persantin inhibited hexose transport in a simple non‐competitive manner (K_i = 20 μM) indicating that the drug affects the function of the hexose carrier.