Activation of mammalian folylpolyglutamate synthetase by sodium bicarbonate.
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.
Bolanowska
W E
WE
Grace Cancer Drug Center, Roswell Park Memorial Institute, Buffalo, New York 14263.
Russell
C A
CA
McGuire
J J
JJ
D000818:Animals; D001639:Bicarbonates / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D009952:Ornithine / Q000031:analogs & derivatives / Q000494:pharmacology; D010453:Peptide Synthases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000201:enzymology*; D051381:Rats; D012964:Sodium / Q000494:pharmacology*; D017693:Sodium Bicarbonate; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000201:enzymology
D001639:Bicarbonates; C065546:5,8-dideazapteroylornithine; D017693:Sodium Bicarbonate; D012964:Sodium; D009952:Ornithine; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase