Bolanowska W E, Russell C A, McGuire J J
Grace Cancer Drug Center, Roswell Park Memorial Institute, Buffalo, New York 14263.
Arch Biochem Biophys. 1990 Sep;281(2):198-203. doi: 10.1016/0003-9861(90)90432-x.
NaHCO3 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 mM NaHCO3 in all cases; NaCl, sodium formate, sodium acetate, NaN3, and Na2SO3 at 10 mM did not cause activation. Activation could be masked if assay solutions which had extensively absorbed atmospheric CO2 were used. Activation of the human CCRF-CEM FPGS was examined in detail. Km and Vmax values for pteroyl substrates (aminopterin or methotrexate) and L-glutamate increased proportionally in the presence of NaHCO3; there was thus no apparent change in the catalytic efficiency (Vmax/Km) of the FPGS reaction with these substrates. However, NaHCO3 increased the efficiency of the reaction with respect to ATP by decreasing its apparent Km while increasing the Vmax of the reaction. NaHCO3 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 NaHCO3. The potency of 5,8-dideazapteroylornithine, an FPGS-specific inhibitor, was not changed by the presence of NaHCO3 (IC50 = 0.4 microM). These results suggest that FPGS activity with folates and classical antifolates may be activated at physiological concentrations of NaHCO3. In addition, inadvertent contamination of assay solutions with bicarbonate from atmospheric CO2 may cause artifacts in the determination of activity levels and kinetic constants of FPGS.
碳酸氢钠使大鼠肝脏以及人白血病细胞系K562和CCRF - CEM中的叶酰聚谷氨酸合成酶（FPGS）的活性提高了1.7至2.0倍。在所有情况下，10 mM的碳酸氢钠均可实现最佳激活效果；10 mM的氯化钠、甲酸钠、乙酸钠、叠氮化钠和亚硫酸钠均未引起激活。如果使用大量吸收了大气中二氧化碳的测定溶液，激活作用可能会被掩盖。对人CCRF - CEM FPGS的激活作用进行了详细研究。在存在碳酸氢钠的情况下，蝶酰底物（氨基蝶呤或甲氨蝶呤）和L - 谷氨酸的米氏常数（Km）和最大反应速度（Vmax）值成比例增加；因此，FPGS与这些底物反应的催化效率（Vmax/Km）没有明显变化。然而，碳酸氢钠通过降低ATP的表观Km同时增加反应的Vmax，提高了与ATP反应的效率。当叶酸、二氢叶酸和四氢叶酸作为底物时，碳酸氢钠也能激活FPGS活性。添加碳酸氢钠不会改变FPGS由甲氨蝶呤或四氢叶酸合成的产物的相对分布。FPGS特异性抑制剂5,8 - 二去氮蝶酰鸟氨酸的效力不受碳酸氢钠的影响（半数抑制浓度IC50 = 0.4 microM）。这些结果表明，在生理浓度的碳酸氢钠条件下，FPGS对叶酸和经典抗叶酸药物的活性可能会被激活。此外，测定溶液意外被大气中二氧化碳的碳酸氢盐污染可能会在FPGS活性水平和动力学常数的测定中造成假象。
