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Transport and Metabolism of Glucosamine by Cultured Novikoff Rat Hepatoma Cells and Effects on Nucleotide Pools

Department of Microbiology, Medical School, University of Minnesota, Minneapolis, Minnesota 55455

As estimated from initial uptake rates, the uptake of glucosamine-1-¹⁴C by cultured Novikoff rat hepatoma cells in glucose-free basal medium follows normal Michaelis-Menten kinetics, with an apparent K_m of about 20 mM. Since glucosamine is not accumulated by the cells against a concentration gradient, the main mode of uptake is by facilitated diffusion, but other evidence indicates that glucosamine also enters the cells by simple diffusion. Glucosamine is probably transported by the same system as glucose. Glucosamine transport is competitively inhibited by glucose and, to about the same extent as is glucose transport, by Persantine and Cytochalasin B. Since neither of the two inhibitors affects the phosphorylation of glucose or glucosamine by cell-free preparations, we suggest that transport is a reaction distinct from phosphorylation. At low concentrations of glucosamine in the medium (0.1 mM and below), the inhibition of glucosamine transport into the cell by glucose, Persantine, or Cytochalasin B results in a proportional decrease in glucosamine incorporation into macromolecules. When cells are incubated with low concentrations of glucosamine-¹⁴C, most of the acid-soluble intracellular radioactivity is associated with uridine diphospho-N-acetylglucosamine, and, after a 30- to 60-min lag period, glucosamine enters glycoproteins and glycolipids at a rapid and constant rate. With an increase in glucosamine concentration in the medium, an increasingly greater proportion of the intracellular radioactivity accumulates in N-acetylglucosamine 6-phosphate, glucosamine 6-phosphate, and free glucosamine (in that order), and a progressively smaller proportion is incorporated into macromolecules. This inhibition of glucosamine incorporation into macromolecules at high glucosamine concentrations is correlated with a lack of uridine diphospho-N-acetylglucosamine formation and an inhibition of macromolecular synthesis. Both effects seem to result from a rapid loss of adenosine triphosphate and uridine triphosphate during incubation of the cells with high concentrations of glucosamine, which loss is caused by the rapid phosphorylation of the glucosamine taken up by the cells. The nucleotides become degraded to nucleosides and bases and are released into the culture fluid. The inhibition of protein synthesis by treatment of the cells with puromycin or cycloheximide also results in an inhibition of glucosamine incorporation into macromolecules without affecting the uptake of glucosamine or its conversion to uridine diphospho-N-acetylglucosamine.

Received 6/20/72. Accepted 11/22/72.

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