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Evidence for Active Transport of Melphalan by Two Amino Acid Carriers in L5178Y Lymphoblasts in Vitro¹

Department of Medicine, University of Manitoba, and the Manitoba Institute of Cell Biology, Winnipeg, Manitoba, R3E OV9, Canada

Investigation of the mechanism of uptake of melphalan by L5178Y lymphoblasts has been extended with particular emphasis on the chemical specificity of the transport mechanism. Evidence was obtained that uptake of melphalan was an active carrier-mediated process. Uptake appeared to proceed "uphill" against a concentration gradient of approximately eight-fold and was temperature sensitive, partly sodium dependent, and inhibited by ouabain and p-hydroxymercuribenzoate. Drug uptake followed biphasic Michaelis-Menten kinetics suggesting the involvement of at least two carrier systems and was markedly inhibited by a variety of amino acids.

Melphalan uptake was strongly inhibited by ß-2-aminobicyclo[2,2,1]heptane-2-carboxylic acid, a specific inhibitor of the L amino acid transport (leucine-preferring) system but not by 2-aminoisobutyric acid or 2-(methylamino)-isobutyric acid, specific inhibitors of the A amino acid (alanine-preferring) system. Under conditions in which the L and A systems were saturated by ß-2-aminobicyclo-[2,2,1]heptane-2-carboxylic acid and 2-aminoisobutyric acid, drug uptake was inhibited by serine, an amino acid known to be transported by the ASC (alanine, serine, and cysteine) system. In reciprocal experiments with leucine as substrate, melphalan inhibited uptake of the amino acid by both the L system and a second system resembling ASC. All of the carrier-mediated uptake of melphalan could be accounted for by transport on these two systems.

In the drug concentration range of 3.33 to 20 µM, carriermediated uptake of melphalan appeared to be almost equally divided between the L system and the system resembling ASC, while in the 20 to 100 µM concentration range, uptake by the L system became increasingly dominant. Uptake of melphalan by the L carrier system had a K_m of 8.0 ± 3.0 x 10^-5 M and a V_max of 1.1 ± 0.4 x 10^-16 mol/cell/min, while uptake by the second system had a K_m of 1.0 ± 0.7 x 10^-5 M and a V_max of 2.2 ± 2.4 x 10^-17 mol/cell/min.

¹ This work was supported by a grant from the National Cancer Institute of Canada.

Received 8/ 8/78. Accepted 10/30/78.