Abstract
Dihydrofolate reductase (DHFR) and thymidylate synthase (TS) activities from cell suspension cultures of Daucus carota were shown to copurify on (NH4)2SO4 fractionation, DEAE Sephadex and methotrexate-Sepharose affinity chromatography and to share approximately the same Mr(183 kDa and 185 kDa respectively) as judged by gel filtration on Sephacryl S-200.
The copurified protein migrated as a single band on polyacrylamide gel electrophoresis under denaturing conditions.
Both activities could be eluted from the same position of the native gel.
Moreover, methotrexate-resistant cell lines which overproduce DHFR revealed to have a parallel higher level of TS. It is therefore proposed and discussed that in carrot, similarly to protozoa, TS and DHFR are present on a single bifunctional polypeptide of 58 kDa.
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References
Albani D, Parisi B, Carbonera D, Cella R: Dihydrofolate reductase from Daucus carota cell suspension cultures: purification and molecular and kinetic characterization. Plant Mol Biol 5: 363–372 (1985).
Bollini R, Vitale A: Genetic variability in charge microheterogeneity and polypeptide composition of phaseolin, the major storage protein of Phaseolus vulgaris, and peptide maps of its three major subunits. Physiol Plant 52: 96–100 (1981).
Beverley SM, Ellenberger TE, Cordingley JS: Primary structure of the gene encoding the bifunctional dihydrofolate reductase thymidylate synthase of Leishmania major. Proc Natl Acad Sci USA 83: 2589–2599 (1986).
Cella R, Albani D, Carbonera D, Etteri L, Maestri E, Parisi B: Selection of methotrexate resistant cell lines in Daucus carota: biochemical analysis and genetic characterization by protoplast fusion. J Plant Physiol 127: 135–146 (1987).
Cella R, Crosti P, Parisi B, Nielsen E: Biochemical basis of different sensitivity to methotrexate in Daucus carota and Oryza sativa cell cultures. J Exp Botany 34: 1189–1195 (1983).
Coderre JA, Beverley SM, Schimke RT, Santi DV: Overproduction of a bifunctional thymidylate synthetase-dihydrofolate reductase and DNA amplification in methotrexate-resistant Leishmania tropica. Proc Natl Acad Sci USA 80: 2132–2136 (1983).
De Wayne R, Loehr EV: Elevation of thymidylate synthase activity in CCRF-CEM cells. Cancer Research 31: 1181–1187 (1971).
Dunlap RB, Harding NG, Huennekens FM: Thymidylate synthetase from Amethopterin-resistant Lactobacillus casei. Biochemistry 10: 88–97 (1971).
Ferone R, Roland S: Dihydrofolate reductase: thymidylate synthase, a bifunctional polypeptide from Crithidia fasciculata. Proc Natl Acad Sci USA 77: 5802–5806 (1980).
La Verne Schirch: Folates in serine and glycine metabolism. In: Blackley RL, Benkovic SJ (eds) Folates and Pterins. John Wiley and Sons, New York (1984).
Lomax MIS, Greenberg GR: A new assay of thymidylate synthase activity based on the release of tritium from deoxyuridilate-5-3H. J Biol Chem 242: 109–113 (1967).
Meek TD, Garvey EP, Santi DV: Purification and characterization of the bifunctional thymidylate synthase-dihydrofolic reductase from methotrexate resistant Leishmania tropica. Biochemistry 24: 678–686 (1985).
Nielsen E, Cella R: Thymidylate synthase in plant cells: kinetic and molecular properties of the enzyme from Daucus carota L. Cell cultures. Plant and Cell Physiology (1988) in press.
Noguchi H, Reddy GPV, Pardee AB: Rapid incorporation of label from ribonucleoside diphosphate into DNA by a cell-free high molecular weight fraction from animal cell nuclei. Cell 32: 443–451 (1983).
O'Farrel PH: High resolution of two-dimensional electrophoresis of proteins. J Biol Chem 250: 4007–4021 (1975).
Osborn MJ, Huennekens FM: Enzymatic reduction of dihydrofolic acid. J Biol Chem 233: 969–974 (1958).
Santi DV, Danenberg PV: Folates in pyrimidine nucleotide biosynthesis. In: Blackley RL, Benkovic SJ (eds) Folates and Pterins. John Wiley and Sons, New York (1984).
Srivastava DK, Bernhard SA: Metabolite transfer in enzyme-enzyme complexes. Science 234: 1081–1086 (1986).
Stanley BG, Neal GE, Williams DC: Dihydrofolic reductase. In: Methods in Enzymology, Vol. 18B. Academic Press, New York (1971) pp 775–779.
Woese CR: Bacterial evolution. Microbial Rev 51: 221–271 (1987).
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Cella, R., Nielsen, E. & Parisi, B. Daucus carota cells contain a dihydrofolate reductase: thymidylate synthase bifunctional polypeptide. Plant Mol Biol 10, 331–338 (1988). https://doi.org/10.1007/BF00029883
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DOI: https://doi.org/10.1007/BF00029883