The occurrence and properties of dihydrofolate reductase in pea seedlings

Dihydrofolate reductase (E.C. 1.5.1.3[EC]) was found in pea seedlingsand was partially purified by treatments with ammonium sulfate,protamine sulfate and by DEAE-cellulose column chromatography.Some properties of the enzyme were investigated. Optimum pHfor the reaction was 6.5. In the enzyme reaction, FAH₂ and NADPH₂were specifically required. MICHAELIS constants for FAH₂ andNADPH₂ were 4.3x10^–6 M and 4.0x10^–5 M, respectively.Folate antagonists such as aminopterin, methotrexate and pyrimethaminewere potent inhibitors of this enzyme. Enzyme activity was almostcompletely inhibited at a concentration of 10^–7 M of aminopterinand methotrexate and 10^–6 M of pyrimethamine. Growth of germinating pea seeds was inhibited by aminopterin,methotrexate and pyrimethamine, and it recovered significantlywith a tetrahydro-derivative of folate, CF, but not with dihydrofolicor folic acid. These results suggest that growth inhibitionof pea seedlings by these antagonists is due to inhibition ofdihydrofolate reductase in seedlings. ¹Studies on the enzymatic synthesis and metabolism of folatecoenzymes in plants IV. (For the previous paper, Part III, seeReference (21)) . Part of this paper was presented at the AnnualMeeting of the Agricultural Chemical Society of Japan held atTokyo on April 4, 1967 (Received October 8, 1969; )