Techniques are described by which the transport of nutrients into mammalian cells in suspension can be measured at intervals of 1.5 seconds. By application of these techniques, the existence of a saturable (Km = 85 muM), non-concentrative, transport system for thymidine was demonstrated in Novikoff rat hepatoma cells depleted of ATP. At concentrations of thymidine less than the Km, this system operated at velocities sufficient to nearly completely equilibrate intra- and extra-cellular thymidine pools within 8 seconds. In phosphorylating cells, the transport system operated with similar rapidity, so that intracellular phosphorylation was rate-limiting for the incorporation of thymidine into nucleotides. Uptake of 3-O-methylglucose occurred at comparable velocities, attaining 90% of equilibrium between internal and external pools within 25 seconds. Uptake of cytosine by simple diffusion was 100 times slower.
Detailed time courses of uptake of labeled 3-O-methyl-D-glucose and 2-deoxy-D-glycose 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 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 (Kztm = 1.73 +/- 0.24 mM; Vztmax = 28.8 +/- 3.6 pmoles/microliter cell H2O. sec) and were similar to those for deoxyglucose transport in ATP-depleted cells (Kztm = 0.65 +/- 0.1 mM; Vztmax = 19.6 +/- 1.6 pmoles/microliter 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 Ki's similar to their transport Km'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 (Ki = 20 muM) indicating that the drug affects the function of the hexose carrier.
Sugar uptake in cells in culture is inhibited by the mold metabolite cytochalasin B. Kletzien et al. (1972) demonstrated that 2-deoxyglucose and glucosamine uptake was inhibited by cytochalasin B in chick embryo fibroblasts, in a Buffalo rat liver cell line, and in a Buffalo rat liver line transformed by the Harvey strain of murine sarcoma virus. Using Novikoff hepatoma cells, Estensen and Plageman (1972) showed that cytochalasin B inhibited the uptake of glucose, 2-deoxyglucose, and glucosamine. Mizel and Wilson (1972) also reported that the uptake of glucose, 2-deoxyglucose, and glucosamine was inhibited by cytochalasin B in HeLa, 3T3, HTC rat hepatoma, and chick embryo heart cells. Zigmond and Hirsch (1972) reported cytochalasin B inhibition of the uptake of 2-deoxyglucose in L cells and 2-deoxyglucose, glucosamine and 3-0-methylglucose in horse leukocytes. The finding that 3-0-methylglucose uptake was inhibited in horse leukocytes prompted us to study the effect of cytochalasin B on the uptake of 3-0-methylglucose in sarcoma-transformed cells. Previously, Hatanaka et al. (1969) reported that, of all the sugars assayed for uptake in sarcoma-virus-infected cells, only 3-0-methylglucose exhibited a Km of comparable value to that of uninfected cells. Therefore, the possibility existed that there would be a difference between the inhibition by cytochalasin B of the uptake of 3-0-methylglucose by transformed cells and that of other hexoses. In addition, the inhibition of
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