Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1987 Jul;6(7):1853–1858. doi: 10.1002/j.1460-2075.1987.tb02443.x

Purification and immunochemical characterization of a dihydrofolate reductase—thymidylate synthase enzyme complex from wild-carrot cells

Ildiko Toth 1,2, Gabor Lazar 1,2, Howard M Goodman 1,2
PMCID: PMC553568  PMID: 16453780

Abstract

An enzyme complex with dihydrofolate reductase (DHFR, E.C.1.5.1.3.) activity was purified to apparent homogenity from wild-carrot cells. The complex has a mol. wt of 286 kd and contains five polypeptide chains of 95, 70, 50, 45 and 26 kd. The DHFR enzyme activity and methotrexate-binding site are on the 45-kd subunit. Folate analogs (methotrexate, aminopterin and formylaminopterin) as well as SH-group inhibitors [p-hydroxymercuribenzoate, 5,5' -dithiobis(2-nitrobenzoic acid), or N-ethylmaleimide] inhibit DHFR. Thymidylate synthase (TS, E.C.2.1.1.45) activity co-purified with the enzyme complex through each of seven steps and co-eluted from gel filtration columns with the DHFR activity at the mol. wt of the enzyme complex. Further identification of TS within the complex was achieved using a Leishmania DHFR-TS antisera which specifically inhibited the carrot TS, although it immunoprecipitated both TS and DHFR. Polyclonal antisera, raised against and specific for the complex as judged by Ouchterlony double diffusion tests and Western blot analysis, inhibited and immunoprecipitated both DHFR and TS. The Leishmania antisera also identified the 70-kd polypeptide within the purified complex as TS in a Western blot experiment. The functions of the other three polypeptides have not yet been established.

Keywords: higher plants, multimeric protein, thymidylate synthesis

Full text

PDF
1853

Images in this article

1854Fig. 1.
on p.1854
1855Fig. 2.
on p.1855
1855Fig. 3.
on p.1855
1855Fig. 4.
on p.1855
1856Fig. 6.
on p.1856
1856Fig. 7.
on p.1856
1857Fig. 9.
on p.1857
1857Fig. 10.
on p.1857

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Allen J. R., Lasser G. W., Goldman D. A., Booth J. W., Mathews C. K. T4 phage deoxyribonucleotide-synthesizing enzyme complex. Further studies on enzyme composition and regulation. J Biol Chem. 1983 May 10;258(9):5746–5753. [PubMed] [Google Scholar]
  2. Baccanari D. P., Tansik R. L., Paterson S. J., Stone D. Characterization and amino acid sequence of Neisseria gonorrhoeae dihydrofolate reductase. J Biol Chem. 1984 Oct 10;259(19):12291–12298. [PubMed] [Google Scholar]
  3. Chen M. J., Shimada T., Moulton A. D., Cline A., Humphries R. K., Maizel J., Nienhuis A. W. The functional human dihydrofolate reductase gene. J Biol Chem. 1984 Mar 25;259(6):3933–3943. [PubMed] [Google Scholar]
  4. Erickson J. S., Mathews C. K. T4 bacteriophage-specific dihydrofolate reductase: purification to homogeneity by affinity chromatography. Biochem Biophys Res Commun. 1971 Jun 4;43(5):1164–1170. doi: 10.1016/0006-291x(71)90585-7. [DOI] [PubMed] [Google Scholar]
  5. Fisher P. A., Berrios M., Blobel G. Isolation and characterization of a proteinaceous subnuclear fraction composed of nuclear matrix, peripheral lamina, and nuclear pore complexes from embryos of Drosophila melanogaster. J Cell Biol. 1982 Mar;92(3):674–686. doi: 10.1083/jcb.92.3.674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Henikoff S., Keene M. A., Sloan J. S., Bleskan J., Hards R., Patterson D. Multiple purine pathway enzyme activities are encoded at a single genetic locus in Drosophila. Proc Natl Acad Sci U S A. 1986 Feb;83(3):720–724. doi: 10.1073/pnas.83.3.720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Horigome T., Sugano H. A rapid method for removal of detergents from protein solution. Anal Biochem. 1983 Apr 15;130(2):393–396. doi: 10.1016/0003-2697(83)90605-x. [DOI] [PubMed] [Google Scholar]
  8. Kumar A. A., Blankenship D. T., Kaufman B. T., Freisheim J. H. Primary structure of chicken liver dihydrofolate reductase. Biochemistry. 1980 Feb 19;19(4):667–678. doi: 10.1021/bi00545a010. [DOI] [PubMed] [Google Scholar]
  9. Lacks S. A., Springhorn S. S., Rosenthal A. L. Effect of the composition of sodium dodecyl sulfate preparations on the renaturation of enzymes after polyacrylamide gel electrophoresis. Anal Biochem. 1979 Dec;100(2):357–363. doi: 10.1016/0003-2697(79)90241-0. [DOI] [PubMed] [Google Scholar]
  10. Mackenzie R. E., Baugh C. M. Tetrahydropterolypolyglutamate derivatives as substrates of two multifunctional proteins with folate-dependent enzyme activities. Biochim Biophys Acta. 1980 Jan 11;611(1):187–195. doi: 10.1016/0005-2744(80)90054-6. [DOI] [PubMed] [Google Scholar]
  11. Meek T. D., Garvey E. P., Santi D. V. Purification and characterization of the bifunctional thymidylate synthetase-dihydrofolate reductase from methotrexate-resistant Leishmania tropica. Biochemistry. 1985 Jan 29;24(3):678–686. doi: 10.1021/bi00324a021. [DOI] [PubMed] [Google Scholar]
  12. Mell G. P., Martelli M., Kirchner J., Huennekens F. M. Multiple forms of dihydrofolate reductase. Biochem Biophys Res Commun. 1968 Oct 10;33(1):74–79. doi: 10.1016/0006-291x(68)90257-x. [DOI] [PubMed] [Google Scholar]
  13. Myoda T. T., Funanage V. L. Coregulation of dihydrofolate reductase and thymidylate synthase B in bacillus subtilis. Biochim Biophys Acta. 1985 Feb 20;824(2):99–103. doi: 10.1016/0167-4781(85)90085-5. [DOI] [PubMed] [Google Scholar]
  14. Nielsen B. L., Brown L. R. The basis for colored silver-protein complex formation in stained polyacrylamide gels. Anal Biochem. 1984 Sep;141(2):311–315. doi: 10.1016/0003-2697(84)90047-2. [DOI] [PubMed] [Google Scholar]
  15. Noguchi H., Prem veer Reddy G., Pardee A. B. Rapid incorporation of label from ribonucleoside disphosphates into DNA by a cell-free high molecular weight fraction from animal cell nuclei. Cell. 1983 Feb;32(2):443–451. doi: 10.1016/0092-8674(83)90464-6. [DOI] [PubMed] [Google Scholar]
  16. Prem veer Reddy G., Pardee A. B. Multienzyme complex for metabolic channeling in mammalian DNA replication. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3312–3316. doi: 10.1073/pnas.77.6.3312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Reddy V. A., Rao N. A. Dihydrofolate reductase from soybean seedlings. Characterization of the enzyme purified by affinity chromatography. Arch Biochem Biophys. 1976 Jun;174(2):675–683. doi: 10.1016/0003-9861(76)90398-2. [DOI] [PubMed] [Google Scholar]
  18. Rowe P. B., McCairns E., Madsen G., Sauer D., Elliott H. De novo purine synthesis in avian liver. Co-purification of the enzymes and properties of the pathway. J Biol Chem. 1978 Nov 10;253(21):7711–7721. [PubMed] [Google Scholar]
  19. Server A. C., Smith J. A., Waxham M. N., Wolinsky J. S., Goodman H. M. Purification and amino-terminal protein sequence analysis of the mumps virus fusion protein. Virology. 1985 Jul 30;144(2):373–383. doi: 10.1016/0042-6822(85)90279-x. [DOI] [PubMed] [Google Scholar]
  20. Smith G. K., Mueller W. T., Wasserman G. F., Taylor W. D., Benkovic S. J. Characterization of the enzyme complex involving the folate-requiring enzymes of de novo purine biosynthesis. Biochemistry. 1980 Sep 2;19(18):4313–4321. doi: 10.1021/bi00559a026. [DOI] [PubMed] [Google Scholar]
  21. Staehelin T., Hobbs D. S., Kung H., Lai C. Y., Pestka S. Purification and characterization of recombinant human leukocyte interferon (IFLrA) with monoclonal antibodies. J Biol Chem. 1981 Sep 25;256(18):9750–9754. [PubMed] [Google Scholar]
  22. Takeishi K., Kaneda S., Ayusawa D., Shimizu K., Gotoh O., Seno T. Nucleotide sequence of a functional cDNA for human thymidylate synthase. Nucleic Acids Res. 1985 Mar 25;13(6):2035–2043. doi: 10.1093/nar/13.6.2035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Vandekerckhove J., Bauw G., Puype M., Van Damme J., Van Montagu M. Protein-blotting on Polybrene-coated glass-fiber sheets. A basis for acid hydrolysis and gas-phase sequencing of picomole quantities of protein previously separated on sodium dodecyl sulfate/polyacrylamide gel. Eur J Biochem. 1985 Oct 1;152(1):9–19. doi: 10.1111/j.1432-1033.1985.tb09157.x. [DOI] [PubMed] [Google Scholar]
  25. Wasserman G. F., Benkovic P. A., Young M., Benkovic S. J. Kinetic relationships between the various activities of the formyl-methenyl-methylenetetrahydrofolate synthetase. Biochemistry. 1983 Mar 1;22(5):1005–1013. doi: 10.1021/bi00274a002. [DOI] [PubMed] [Google Scholar]
  26. Zelikson R., Luzzati M. Mitochondrial and cytoplasmic distribution in Saccharmoyces cerevisiae of enzymes involved in folate-coenzyme-mediated one-carbon-group transfer. A genetic and biochemical study of the enzyme deficiencies in mutants tmp3 and ade3. Eur J Biochem. 1977 Sep 15;79(1):285–292. doi: 10.1111/j.1432-1033.1977.tb11808.x. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES