Polymorphism of dihydrofolate reductase from a methotrexate-resistant subline of L1210 cells.
Dihydrofolate reductase, purified to homogeneity (as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis), from a subline of L1210 murine leukemia cells resistant to 10(-6) M methotrexate, was resolved into two principal forms (1 and 2) by polyacrylamide gel electrophoresis at pH 8.3 or isoelectric focusing. In the latter procedure, these forms had pI values of 7.4 and 8.2, respectively; both stained for protein and catalytic activity. Form 1 appears to be a single component, comprising ca. 10% of the total protein and at least 20% of the total catalytic activity. It is also more sensitive to inhibition by MTX, more heat-stable, and less susceptible to activation than form 2. Multiple components of 2 were observed by narrowing the pH range in isoelectric focusing, and further resolution was achieved by urea denaturation. Substrate and inhibitor complexes of 1 and 2, differentiated by polyacrylamide gel electrophoresis or isoelectric focusing, provided information about the ability of the enzyme to undergo conformational changes. Interconversion of 1 with one of the components of 2 may also involve conformational isomerism. These conclusions are consistent with the well-known ability of eukaryotic dihydrofolate reductases to exhibit increased catalytic activity (attributed to transformations to more open conformations) when treated with salts, chaotropes, or cysteine-modifying agents. Treatment of the L1210/R6 enzyme preparation with one of these activating agents, 5,5'-dithiobis(2-nitrobenzoic acid), derivatized both 1 and 2 (changing their pI values to 7.3 and 6.9, respectively) and altered the enzyme such that stoichiometric inhibition for MTX was observed.
D000818:Animals; D002460:Cell Line; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004351:Drug Resistance; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000378:metabolism*; D007939:Leukemia L1210 / Q000201:enzymology* / Q000473:pathology; D046911:Macromolecular Substances; D008727:Methotrexate / Q000494:pharmacology*; D051379:Mice; D011487:Protein Conformation; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*
D007527:Isoenzymes; D046911:Macromolecular Substances; D004228:Dithionitrobenzoic Acid; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate