Abstract

Folate reductase has been purified from an amethopterin-resistant subline of cultured Sarcoma-180 cells. The molecular weight of this enzyme was estimated to be 21,000. The specific activity of the purified reductase was 3.1 x 10^-8 mole of the enzyme per milligram of protein, and on this basis the preparation was calculated to be 65% pure. During free boundary electrophoresis, however, the enzyme migrates as a single peak toward the anode at pH 8.7. Both folate and dihydrofolate are substrates for this reductase, dihydrofolate being reduced much faster than folate at 30°. The Michaelis constant (K_m) and the turnover number with dihydrofolate as substrate are independent of pH between pH 4.5 and 6.3. When folate is the substrate, both K_m and the turnover number decrease with increasing pH. The enthalpy of activation is +14.7 kcal for dihydrofolate and +5.7 kcal for folate. The properties of the reductase of Sarcoma-180 are compared with those of other preparations of this enzyme from different sources.