Thymidylate Synthase in Plant Cells: Kinetic and Molecular Properties of the Enzyme from Daucus carota L. Cell Cultures |
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Authors: | Nielsen, Erik Cella, Rino |
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Affiliation: | Departimento di Genetica e Microbiologia "A. Buzzati Traverso" Sezione di Microbiologia e Fisiologia Vegetale, Universit? di Pavia Via S. Epifanio, 14, 27100 Pavia, Italia |
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Abstract: | Thymidylate synthase activity was assayed in cell homogenatesfrom wild or domestic carrot (Daucus carota L.) suspension culturesusing the tritium-release method. The specific activity measuredwas lower than that previously reported for bacterial extracts,and its level did not change a great deal during a 14 day growthcycle. The enzyme was partially purified (about 9 fold) by ammoniumsulfate fractionation and ion-exchange chromatography. Kineticparameters such as pH optimum, Km values for substrate and cofactor,and degree of inhibition by 5-fluoro-2'-deoxyuridine monophosphatewere characterized. The apparent mol wt, evaluated by gel-filtration chromatography,was 185 kDa, which was much higher than that reported for enzymesof bacterial and vertebrate origins and somewhat higher thanthat reported for the thymidylate synthase-dihydrofolate reductasebifunctional polypeptide of some protozoa. (Received April 20, 1987; Accepted February 15, 1988) |
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