The uptake of amethopterin-3H by L1210 leukemia cells appears to be mediated, probably, via an active transport mechanism. Uptake occurred against a concentration gradient, was temperature dependent, required a readily available source of energy, exhibited a pH optimum of 7.6 and conformed to Michaelis-Menten kinetics. The maximum intracellular concentration of unbound amethopterin-3H accumulated at equilibrium was 8-fold the external concentration. Both folate and H2-folate compete with amethopterin-3H for the same system, but to a limited degree. The rate of transport was reduced 4-fold in cells of a subline (XVI4) resistant to amethopterin, 6-mercaptopurine and 5-fluorouracil. Kinetic analysis revealed an increase in the Michaelis constant (Km) for the system in these resistant cells, suggesting an alteration in the binding properties of a carrier component as the basis for the impairment. A second subline (XVI4a) was derived directly from the original subline XVI4 and was characterized by the absence of the subtelocentric chromosome and an elevated dihydrofolate reductase. The rate of transport in cells of this chromosomal variant was normal, although the Km for the system was increased as in cells of subline XVI4. Maximum intracellular accumulation of unbound amethopterin-3H by cells of subline XVI4a was greater than 11-fold the external concentration.

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This investigation was supported in part by USPHS Grant CA 08748 from the National Cancer Institute, and by Grant T-107 from the American Cancer Society.

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