Porownanie wlasciwosci syntazy tymidylanowej oczyszczonej z tasiemca szczurzego, Hymenolepis diminuta, z wlasciwosciami enzymu zywiciela wyizolowanego z regenerujacej watraby szczura

Abstract

Thymidylate synthases (TS) from the tapeworm, Hymenolepis diminuta, and regenerating rat liver have been purified by means of affinity chromatography on immobilized 10-formyl-5,8-dideazafolate and concentrated on immobilized p-aminophenyl-5-fluoro-2'-deoxyuridine monophosphate. Molecular weights of native TS from the tapeworm and regenerating rat liver were 62 kD and 81.5 kD, respectively, and molecular weights of the monomers were 34.4 kD and 34.9 kD, respectively, painting to dimeric structures of both enzymes. The dependence of TS activity on temperature (ARRHENIUS plot) was biphasic for the parasite enzyme, with lower activation energy above 32°C, and monophasic for the host enzyme. 2'-deoxyuridine-5'-monophosphate (dUMP) analogues, 5-fluoro-2'-deoxyuridine-5' -monophosphate (5-FdUMP), 2-tio-5-FdUMP, N⁴-hydroxy-2'-deoxycytidine-5'-monophosphate (N⁴-hydroxy-dCMP) and N⁴-hydroxy-5-FdCMP, were competitive with respect to dUMP, slow-binding inhibitors of TS from both sources, with K₁ values in 10⁻⁶ - 10⁻⁹ M range. 5-FdUMP was distinctly stronger inhibitor of the host than the tapeworm TS, whereas N⁴-hydroksy-5-FdCMP inhibited stronger the parasite enzyme. Interactions of 5,10-methylenetetrahydrofolate (CH₂H₄PteGlu) analogue, 10-propargyl-5,8-dideazafolate (pddPteGlu), and its di- and triglutamates with both enzymes were studied. Inhibition of the parasite and host enzymes by pddPteGlu was of mixed-type with respect to CH₂H₄PteGlu, with K₁ values in 10⁻⁸ M range. Introduction of additional glutamate residues changed inhibition type to noncompetitive with respect to CH₂H₄PteGlu and lowered K₁ values (pddPteGlu₃ < pddPteGlu₂ < pddPteGlu₁). The latter potentiation of inhibitory properties was distinctly stronger in case of the tapeworm than regenerating rat liver TS