Properties of an altered dihydrofolate reductase encoded by amplified genes in cultured mouse fibroblasts

We have studied a line of 3T6 mouse embryo fibroblasts grown in progressively increasing concentrations of methotrexate. Resistance of cells to low concentrations of the inhibitor (<5 X 10^-5M) is attributed to selective multiplication of the genes coding for dihydrofolate reductase and the resulting elevation of enzyme content. Cells isolated at a higher methotrexate concentration (4 X 10^-4M) contain high levels of a dihydrofolate reductase with a reduced affinity for methotrexate. The altered dihydrofolate reductase exhibits a 270-fold reduction in binding affinity for methotrexate as measured by equilibrium dialysis (K(d)=5.4 X 10^-8M versus 2 X 10^-10M for the wild type enzyme). While binding to NADPH is unchanged, the K(m) for dihydrofolate is increased 3-fold over wild type enzyme and the turnover number for the reduction of dihydrofolate to tetrahydrofolate is decreased 20-fold. The altered dihydrofolate reductase shows a broader and more predominant acidic peak in its pH profile for this reaction. The molecular weights of the altered and wild type enzymes are identical as determined by sodium dodecyl sulfate-gel electrophoresis, but 2-dimensional electrophoresis reveals a significant basic shift in the migration of the altered enzyme. Studies with various folic acid analogs suggest that modifictions involving the para-aminobenzoyl moiety of the inhibitor molecules are associated with the most dramatic differential binding between the altered and wild type dihydrofolate reductases.