Activation of mammalian folylpolyglutamate synthetase by sodium bicarbonate

Abstract

NaHCO 3 activated the folylpolyglutamate synthetase (FPGS) from rat liver and the human leukemia cell lines K562 and CCRF-CEM by 1.7- to 2.0-fold. Optimal activation was achieved by 10 m m NaHCO 3 in all cases; NaCl, sodium formate, sodium acetate, NaN 3, and Na 2SO 3 at 10 m m did not cause activation. Activation could be masked if assay solutions which had extensively absorbed atmospheric CO 2 were used. Activation of the human CCRF-CEM FPGS was examined in detail. K m and V max values for pteroyl substrates (aminopterin or methotrexate) and l-glutamate increased proportionally in the presence of NaHCO 3; there was thus no apparent change in the catalytic efficiency ( V max K m ) of the FPGS reaction with these substrates. However, NaHCO 3 increased the efficiency of the reaction with respect to ATP by decreasing its apparent K m while increasing the V max of the reaction. NaHCO 3 also activated FPGS activity when folic acid, dihydrofolic acid and tetrahydrofolic acid were substrates. The relative distribution of products synthesized from methotrexate or tetrahydrofolate by FPGS was not altered by addition of NaHCO 3. The potency of 5,8-dideazapteroylornithine, an FPGS-specific inhibitor, was not changed by the presence of NaHCO 3 (IC 50 = 0.4 μ m). These results suggest that FPGS activity with folates and classical antifolates may be activated at physiological concentrations of NaHCO 3. In addition, inadvertent contamination of assay solutions with bicarbonate from atmospheric CO 2 may cause artifacts in the determination of activity levels and kinetic constants of FPGS.