Transport of folate compounds, pterins and adenine in L1210 mouse leukemia cells

L1210 mouse leukemia cells provide a convenient model for examining the mechanisms and components involved in the active transport of various metabolites and drugs. One of these transport systems exhibits a broad specificity for folate compounds, including 4-amino antagonists such as methotrexate. The primary substrate for this system is 5-methyltetra-hydrofolate (Kt = 1 microM), the principal circulating form of the vitamin in mammals. 5-Formyltetrahydrofolate (Kt = 5 microM) and Methotrexate (Kt = 5 microM) are also taken up efficiently, but folate (Kt = 100 microM) is a relatively poor substrate. Vmax for this system is ca. 15 pmoles/min/mg protein. Energy for substrate internalization is provided by an anion-exchange mechanism, and regulation appears to be mediated by cyclic AMP. The system can be inhibited irreversibly by treatment of the cells with photo-activated azido AMP or carbodiimide-activated folate compounds. The latter method allows the membrane-associated binding protein to be labeled in situ, thereby providing a means for identifying it during subsequent solubilization and purification. Guidance for this latter project is provided by previous experience in the purification to homogeneity of a similar folate-binding protein from Lactobacillus casei. L1210 cells also contain an efficient system for the transport of adenine (Kt = 20 microM; Vmax = 200 pmoles/min/mg protein). Uptake of adenine is linked with its conversion to AMP via PRPP-dependent adenine phosphori-bosyltransferase. Pterins, which have a close structural similarity to adenine (as well as to a portion of the folate molecule), are also transported into L1210 cells. Transport of [3H] 6-hydroxymethylpterin (Kt = 20 microM) was inhibited by 6-formylpterin, 6-methylpterin and 6-carboxypterin with Ki values of 42, 100 and 350 microM, respectively. Adenine (Ki = 20 microM) and various other purines were also good inhibitors of pterin transport. Present evidence indicates that adenine and pterins use separate transport systems, but isolation of the components of these systems may further delineate their interrelationships.