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Dihydrofolate reductase from Daucus carota cell suspension cultures: purification, molecular and kinetic characterization

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Summary

The purification of dihydrofolate reductase (5, 6, 7, 8 tetrahydrofolate: NADP+ oxidoreductase, E.C.: 1.5.1.3) from Daucus carota to apparent homogeneity, is described. The enzyme is a soluble protein with a molecular weight of 183 000±2 500, composed of identical subunits of 58 400±1 000. The enzyme is only weakly recognized by antibodies against human DHFR. The carrot DHFR is characterized by a pH optimum of 5.9, Km values for dihydrofolate and NADPH of 3.7 μM and 2.2 μM, respectively and a turnover number of 4 750 or 1 500 when referring to the 183 K form or the 58 K monomer, respectively. Molecular and kinetic properties are remarkably different from those reported for the soybean enzyme. Sensitivity to methotrexate is similar to that of bacterial and mammalian enzymes while sensitivity to trimethoprim and dihydrotriazine is intermediate between the two groups of organisms.

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References

  • Albrecht AM, Bielder JL, Hutchinson DJ: Two different species of dihydrofolate reductase in mammalian cells differentially resistant to amethopterin and methasquin. Cancer Research 32:1539–1546, 1972.

    Google Scholar 

  • Alt FW, Kellems RE, Bertino JR, Schimke RT: Selective multiplication of dihydrofolate reductase genes in methotrexate-resistant variant of cultured murine cells. J Biol Chem 253:1357–1370, 1978.

    Google Scholar 

  • Barg R, Peleg N, Perl M, Beckmann JS: Isolation of methotrexate-resistant cell lines in Petunia hybrida upon stepwise selection procedure. Plant Mol Biol 3:303–311, 1984.

    Google Scholar 

  • Blakley RL: The Biochemistry of Folic Acid and Related Pteridines. John Wiley and Sons, New York, 1969.

    Google Scholar 

  • Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt Biochem 72:248–254, 1976.

    Google Scholar 

  • Burchall JJ: Dihydrofolate reductase. In: Handbook of Experimental Pharmacology. Springer-Verlag, Berlin, Vol. 64, 1983, pp 55–73.

    Google Scholar 

  • Bushby SRM: Antibacterial activity. In: Handbook of Experimental Pharmacology. Springer-Verlag, Berlin, Vol. 64, 1983, pp 75–105.

    Google Scholar 

  • Cella R, Albani D, Biasini MG, Carbonera D, Parisi B: Isolation and characterization of a carrot cell line resistant to methotrexate. J Exp Bot 35:1390–1397, 1984.

    Google Scholar 

  • Cella R, Crosti P, Nielsen E, Parisi B: Biochemical basis of different sensitivity to methotrexate in Daucus carota and Oryza sativa cell cultures. J Exp Bot 34:1189–1195, 1983.

    Google Scholar 

  • Coderre JA, Beverly SM, Scimke RT, Santi D: overproduction of a bifunctional thymidylate synthetase-dihydrofolate reductase and DNA amplification in methotrexate-resistant Leishmania tropica. Proc Natl Acad Sci 80:2132–2136, 1983.

    Google Scholar 

  • Chang ACY, Nunberg JH, Kaufman RJ, Erlich HA, Shimke RT, Cohen SN: Phenotipic expression in E. coli of a DNA sequence coding for mouse dihydrofolate reductase. Nature 275:617–624, 1978.

    Google Scholar 

  • Crosti P: Effect of folate analogues on the activity of dihydrofolate reductase and on the growth of plant organism. J Exp Bot 32:717–723, 1981.

    Google Scholar 

  • Dixon M, Webb EC: Enzymes. Longmans, 1964.

  • Ferone R, Roland S: Dihydrofolate reductase: thymidylate synthetase, a bifunctional polypeptide from Crithidia fasciculata. Proc Natl Acad Sci US 77:5802–5806, 1980.

    Google Scholar 

  • Hudson L, Hay FC: Practical Immunology. 2nd edn, Blackwell Scientific Publications, Oxford, 1980.

    Google Scholar 

  • Kaufman BT: Methotrexate-Agarose in the purification of Dihydrofolate reductase. In: Jakoby WB, Wilchek M (eds) Methods in Enzymology, Vol. XXXIV, 1974, pp 272–281.

  • Laemmli K: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685, 1970.

    Google Scholar 

  • Mastrangelo IA, Smith HH: Selection and differentiation of aminopterin resistant cells of Datura innoxia. Plant Sci Letters 10:171–179, 1977.

    Google Scholar 

  • Merril CR, Goldman D, Sedman SA, Ebert MH: Ultrasensitive stain for proteins in poluacrilamide gels shows regional variation in cerebrospinal fluid proteins. Science 211:1437–1438, 1981.

    Google Scholar 

  • Misra K, Humphreys SR, Friedkin M, Goldin A, Crawford EJ: Increased dihydrofolate reductase activity as a possible basis of drug resistance in leukemia. Nature 189:39–42, 1961.

    Google Scholar 

  • Nielsen E, Rollo F, Parisi B, Cella R, Sala F: Genetic markers in cultured plant cells: differential sensitivities to amethopterin, azetidine-2-carboxylic acid and hydroxyurea. Plant Sci Letters 15:113–125, 1979.

    Google Scholar 

  • Novak P, Stone D, Burchall JJ: R plasmid dihydrofolate reductase with dimeric subunit structure. J Biol Chem 258:10956–10959, 1983.

    Google Scholar 

  • Osborn MJ, Huennekens FM Enzymatic reduction of dihydrofolic acid. J Biol Chem 233:969–974, 1958.

    Google Scholar 

  • Reddy VA, Rao NA: Dihydrofolate reductase from soybean seedlings: characterization of the enzyme purified by affinity chromatography. Archives Biochem Biophys 174:675–683, 1976.

    Google Scholar 

  • Reddy VA, Rao NA: Dihydrofolate reductase from soybean seedlings: a fluorescenze and circular dichroism study. Archives Biochem Biophys 183:90–97, 1977.

    Google Scholar 

  • Shimamoto K, Nelson OR: Isolation and characterization of aminopterin-resistant cell lines in maize. Planta 153:436–442, 1981.

    Google Scholar 

  • Schimke RT: Gene amplification, drug resistance and cancer. Cancer research 44:1735–1742, 1984.

    Google Scholar 

  • Smith SL, Stone D, Novak P, Baccanari DP, Burchall JJ: R plasmid dihydrofolate reductase with subunits structure. J Biol Chem 254:6222–6225, 1979.

    Google Scholar 

  • Stanley BG, Neal GE, Williams DC: Dihydrofolic reductase. In: Methods in Enzymology, Vol. 18B, Academic Press, 1971, pp 775–779.

  • Sung ZR: Turbidimetric measurement of plant cell culture growth. Plant Physiol 57:460–462, 1976.

    Google Scholar 

  • Suzuki N, Iwai K: The occurence and properties of dihydrofolate reductase in pea seedlings. Plant Cell Physiol, Tokio 11:199–208, 1970.

    Google Scholar 

  • White DWR: Isolation and characterization of methotrexate-resistant mutants of Nicotiana tabacum. In: Fujiwara A (ed) Plant tissue culture 1982, Japanese Association for Plant Tissue Culture.

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Authors and Affiliations

  1. Dipartimento di Genetica e Microbiologia, Via S. Epifanio, 14, 27100, Pavia, Italy

    Diego Albani, Bruno Parisi, Daniela Carbonera & Rino Cella

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  1. Diego Albani
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  2. Bruno Parisi
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  3. Daniela Carbonera
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  4. Rino Cella
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Albani, D., Parisi, B., Carbonera, D. et al. Dihydrofolate reductase from Daucus carota cell suspension cultures: purification, molecular and kinetic characterization. Plant Mol Biol 5, 363–372 (1985). https://doi.org/10.1007/BF00037557

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  • DOI: https://doi.org/10.1007/BF00037557

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