Irreversible inactivation of the methotrexate transport system of L1210 cells by carbodiimide-activated substrates.

Abstract

Methotrexate and 1-ethyl-3(3-dimethylaminopropyl)carbodiimide react to form a relatively unstable product (activated methotrexate) that irreversibly inhibits methotrexate transport in L1210 mouse leukemia cells. The rate of methotrexate transport was reduced 50% by pretreatment of cells with activated methotrexate at a concentration of 1 microM and by 98% upon exposure to an excess (40 microM) of this agent. Specificity was demonstrated by the fact that activated methotrexate had no effect on other transport systems (i.e. leucine and phosphate) and that complete protection against inactivation was afforded by either the unlabeled substrate (methotrexate) or a competitive inhibitor (phosphate). The carbodiimide-generated derivatives of aminopterin, folate, and p-aminobenzoylglutamate were also irreversible inhibitors of methotrexate transport, and their relative effectiveness paralleled the ability of the parent compounds (aminopterin greater than methotrexate greater than folate greater than p-aminobenzoylglutamate) to act as reversible transport inhibitors. Measurements at 4 degrees C showed that activated methotrexate (Ki = 0.8 microM) was a competitive inhibitor for binding of methotrexate (KD = 0.4 microM) to the transport protein. Treatment of the cells with sufficient activated methotrexate to inhibit transport increased the KD for methotrexate to 0.9 microM but had no effect on total methotrexate-binding activity. Methotrexate efflux was also reduced but was not blocked by an excess of this reagent.