Thymidylate synthetase activity in normal and Brugia pahangi-infected Aedes aegypti

Abstract

Thymidylate synthetase (5,10-methylenetetrahydrofolate: dUMP C-methyltransferase; EC 2.1.2.45; TMPS) activity was detected in crude extracts of 4 to 5 day-old adult male and uninfected female Aedes aegypti. Mosquito TMPS was strongly inhibited by Mg 2+ and Ca 2+ ( id 50 values of 0.25 and 0.12 μM, respectively) but not by other divalent cations. The K m values for dUMP and 5, 10-methylene-tetrahydrofolate were 55 and 100 μM, respectively. It appeared that mosquito TMPS would not be subject to regulation by physiological concentrations of thymidine mono-, di- and triphosphates or di- and triphosphates of cytidine, uridine, adenosine or guanosine. When subjected to Sephadex G-200 gel filtration, TMPS activity occurred in three peaks, corresponding to molecular weights of 210,000, 310,000 and 450,000. Among a variety of compounds tested as inhibitors of the enzyme, FdUMP was the most potent, with an id 50 value of 0.5 nM; by contrast, FUdR was inactive at 1.0 mM. The polycyclic sulfonated anionic drug, suramin, was also a strong inhibitor of mosquito TMPS, with an id 50 value of 0.3 μM. Various 2,4-diaminoheterocyclic antifols, including methotrexate, either were inactive or were relatively weak inhibitors of this enzyme. The total activity of TMPS did not change in extracts of female mosquitoes prepared 7 and 12 days after they fed upon either normal or Brugia pahangi-infected jirds.