Evidence for active transport of melphalan by two amino acid carriers in L5178Y lymphoblasts in vitro.

Investigation of the mechanism of uptake of melphalan by L5178Y lymphoblasts has been extended with particular emphasis on the chemical specificity of the transport mech anism. 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 involvementof at least two carrier systems and was markedly inhibited by a variety of amino acids. Melphalan uptake was strongly inhibited by fJ-2-aminobi cyclo[2,2,ljheptane-2-carboxylic acid, a specific inhibitor of the L amino acid transport (leucine-preferring) system but not by 2-aminoisobutyric acid or 2-(methylammno)isobutyric acid, specific inhibitors of the A amino acid (alanine-preferring) system. Under conditions in which the L and A systems were saturated by fJ-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 ac counted for by transport on these two systems. In the drug concentration range of 3.33 to 20 @M, carrier mediated uptake of melphalan appeared to be almost equally divided between the L system and the system resembling ASC, while in the 20 to 100 @Mconcentration range, uptake by the L system became increasingly domi nant. Uptake of melphalan by the L carrier system had a Km of 8.0 ±3.0 x i0@ M and a Vmaxof 1.1 ±0.4 x 10' mol/ cell/mm, while uptake by the second systemhad a K,,,of 1.0 ± 0.7 x i0-@ M and a Vmax of 2.2 ± 2.4 x 1017 mol/cell/