Purification and Some Properties of Folate-hydrolyzing Enzyme from Crithidia fasciculata

抄録

The folate-hydrolyzing enzyme was purified 49-fold from the crude extract of Crithidia fasciculata ATCC12857 by heat treatment, column chromatographies on DEAE-cellulose and Sephadex G-200, and preparative polyacrylamide gel electrophoresis. The final preparation was electrophoretically homogeneous. The enzymehad a molecular weight of 200, 000 daltons and consisted of 4 identical subunits of which the molecular weight was about 51, 000 daltons. The enzyme hydrolyzed aminopterin, methotrexate, and PABGmore effectively than folate. The enzyme hydrolyzed the reduced folates, dihydrofolate and 10-formyltetrahydrofolate, more weakly than folate. The enzyme did not act on pteroly-γ, γ-diglutamylglutamate. The optimum pH for the reaction with each substrate described above was 7.0. Km values for folate, methotrexate, aminopterin, and PABGwere 0.13, 0.46, 0.40, and 0.43mM, respectively. The enzyme activity was inhibited by 2-mercaptoethanol, PCMB, chelating reagents such as α, α', α''-tripyridyl and bathophenanthroline, divalent cations such as Hg²⁺, Cu²⁺, Cd²⁺, Pb²⁺, and Zn²⁺, and by pyrophosphate and orthophosphate.