Dihydrofolate reductase was purified quickly and simply from small quantities of cultured mammalian cells by affinity chromatography. On gel electrophoresis of the purified enzyme, multiple bands of activity resulted from enzyme-buffer interaction at low but not high buffer concentration. A Ferguson plot (Ferguson, 1964) showed that this heterogeneity was due to a charge difference with no alteration in the size of the enzyme. Stimulation of enzyme activity by KCl, urea and p-hydroxymercuribenzoate, and inhibition by methotrexate and trimethoprim, showed only minor differences between the various enzymes.
Folate and methotrexate markedly increased the activity of dihydrofolate reductase in cultured cells of human origin. Folate did not have this effect in the cells of other species tested, whereas methotrexate increased enzyme activity in some other species. Neither compound increased enzyme activity in a somatic hybrid, although such an increase occurred in one of the parent cell lines. Active enzyme synthesis appeared necessary for the effect of folate to occur. Degradation of the increased enzyme after cycloheximide treatment of cells proceeded at least as rapidly as in control cultures and was associated with a decrease in the intracellular folate pool, whereas folate added after 48 h of culture stabilized the enzyme at this same intracellular concentration. Folate, added at 24 h of culture, produced the greatest increase in activity, but the subsequent rate of decrease was greater than that when folate was added at 48 h, although the intracellular concentration of folate in the latter case was only half that in the former.
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