Purification, Properties, and Synthesis of Dihydrofolate Reductase from Wild Type and Methotrexate-resistant Hamster Cells

Dihydrofolate reductase from a methotrexate-resistant hamster cell culture was purified to apparent homogeneity by affinity chromatography. A similar procedure was used to prepare enzyme from wild type cells, although the final enzyme protein concentration was too small to be measured. The purified enzymes were not significantly different with regard to substrate specificity, Km values for folate and NADPH, sedimentation coefficient, electrophoretic mobilities at pH values 8.3 and 7.0, turnover number per methotrexate-binding site, and degree of inactivation by antiserum. The rate of radioactive leucine incorporation into the reductase in wild type and resistant cells was compared by specific immunoprecipitation. By this method resistant cells showed a 20-fold increase in the rate of enzyme synthesis. These results indicate that methotrexate-resistant cells accumulate large quantities of a very similar, if not identical, reductase, due at least in part to an increased rate of enzyme synthesis.