Dihydrofolate Reductase - Thymidine Synthase Extraction And Inhibitor Screening Study

Dihydrofolate reductase (DHFR) and Thymidylate synthase (TS), essential enzymes for the biosynthesis of DNA, were critical targets of anticancer, antimicrobial, antifungal, antiviral and antimalarial drugs. In this paper, the separations of DHFR and TS and the application of High Performance Capillary Electrophoresis (HPCE) method in the research of kinetics of enzyme reactions of DHFR were introduced, including the measurement of kinetic parameters of enzyme reactions and the research of kinetics of inhibitory activities.Through the established analysis model for screening of Dihydrofolate reductase inhibitors (DHFRIs) by HPCE, the inhibitions of DHFR activity by Catechins, Luteolin, Paclitaxel, Baicalin, Ginsenoside Rg3 and Total saponins of panax notoginseseng were studied. It was found Catechins, Luteolin and Paclitaxel could markedly inhibit DHFR.DHFR was separated from chicken liver by Affinity chromatography. The inhibitions of DHFR activity by Methotrexate and (-)-Epigallocatechin gallate were studied by Ultraviolet Spectrophotometry.A new assay of chicken liver DHFR using HPCE has been established. The reactants and resultants were separated in above conditions. A method was developed to determine kinetics parameters of chicken liver DHFR by monitoring its product, NADP. By determining the activity of DHFRIs, it was found that the IC50 of Methotrexate matched the values had been published, and this method could be used for the screening of anticancer drugs.By gradually increasing the concentration of Methotrexate in the culture medium, Methotrexate-resistant strains of Lactobacillus casei var. rhamnosus was obtained. Extracts of Methotrexate-resistant L.casei provide a rich source of TS, showing higher activity than which found in extracts of the sensitive strain.