Purification and Immunochemical Characterization of a Dihydrofolate Reductase-thymidylate Synthase Enzyme Complex from Wild-carrot Cells

Overview

Journal EMBO J

Specialties Biotechnology
Molecular Biology

Date 1987 Jul 1

PMID 16453780

Citations 9

Authors

I Toth

G Lazar

H M Goodman

Affiliations

Soon will be listed here.

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.

Citing Articles

Distinctive features and differential regulation of the DRTS genes of Arabidopsis thaliana.

Maniga A, Ghisaura S, Perrotta L, Marche M, Cella R, Albani D PLoS One. 2017; 12(6):e0179338.

PMID: 28594957 PMC: 5464667. DOI: 10.1371/journal.pone.0179338.

Folates in Plants: Research Advances and Progress in Crop Biofortification.

Gorelova V, Ambach L, Rebeille F, Stove C, Van Der Straeten D Front Chem. 2017; 5:21.

PMID: 28424769 PMC: 5372827. DOI: 10.3389/fchem.2017.00021.

Coregulation of dihydrofolate reductase and thymidylate synthase in overproducer cell lines of wild carrot.

Lazar G, Toth I, Haakonsen L, Goodman H Plant Physiol. 1989; 91(3):1168-73.

PMID: 16667128 PMC: 1062136. DOI: 10.1104/pp.91.3.1168.

Mapping and expression of a bifunctional thymidylate synthase, dihydrofolate reductase gene from maize.

Cox K, Robertson D, Fites R Plant Mol Biol. 2000; 41(6):733-9.

PMID: 10737138 DOI: 10.1023/a:1006324328355.

Determination of copy number of rRNA genes in Pneumocystis carinii f. sp. hominis.

Tang X, Bartlett M, Smith J, Lu J, Lee C J Clin Microbiol. 1998; 36(9):2491-4.

PMID: 9705380 PMC: 105150. DOI: 10.1128/JCM.36.9.2491-2494.1998.

References

Chen M, Shimada T, Moulton A, Cline A, Humphries R, Maizel J . The functional human dihydrofolate reductase gene. J Biol Chem. 1984; 259(6):3933-43. View

Nielsen B, Brown L . The basis for colored silver-protein complex formation in stained polyacrylamide gels. Anal Biochem. 1984; 141(2):311-5. DOI: 10.1016/0003-2697(84)90047-2. View

Allen J, Lasser G, Goldman D, Booth J, Mathews C . T4 phage deoxyribonucleotide-synthesizing enzyme complex. Further studies on enzyme composition and regulation. J Biol Chem. 1983; 258(9):5746-53. View

Smith G, Mueller W, Wasserman G, Taylor W, Benkovic S . Characterization of the enzyme complex involving the folate-requiring enzymes of de novo purine biosynthesis. Biochemistry. 1980; 19(18):4313-21. DOI: 10.1021/bi00559a026. View

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; 76(9):4350-4. PMC: 411572. DOI: 10.1073/pnas.76.9.4350. View

Lacks S, SPRINGHORN S, ROSENTHAL A . Effect of the composition of sodium dodecyl sulfate preparations on the renaturation of enzymes after polyacrylamide gel electrophoresis. Anal Biochem. 1979; 100(2):357-63. DOI: 10.1016/0003-2697(79)90241-0. View

Reddy V, RAO N . Dihydrofolate reductase from soybean seedlings. Characterization of the enzyme purified by affinity chromatography. Arch Biochem Biophys. 1975; 174(2):675-83. DOI: 10.1016/0003-9861(76)90398-2. View

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; 13(6):2035-43. PMC: 341133. DOI: 10.1093/nar/13.6.2035. View

Server A, Smith J, Waxham M, Wolinsky J, Goodman H . Purification and amino-terminal protein sequence analysis of the mumps virus fusion protein. Virology. 1985; 144(2):373-83. DOI: 10.1016/0042-6822(85)90279-x. View

10.

Myoda T, Funanage V . Coregulation of dihydrofolate reductase and thymidylate synthase B in bacillus subtilis. Biochim Biophys Acta. 1985; 824(2):99-103. DOI: 10.1016/0167-4781(85)90085-5. View

11.

Erickson J, Mathews C . T4 bacteriophage-specific dihydrofolate reductase: purification to homogeneity by affinity chromatography. Biochem Biophys Res Commun. 1971; 43(5):1164-70. DOI: 10.1016/0006-291x(71)90585-7. View

12.

Mell G, Martelli M, Kirchner J, Huennekens F . Multiple forms of dihydrofolate reductase. Biochem Biophys Res Commun. 1968; 33(1):74-9. DOI: 10.1016/0006-291x(68)90257-x. View

13.

Fisher P, 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; 92(3):674-86. PMC: 2112026. DOI: 10.1083/jcb.92.3.674. View

14.

Reddy G, Pardee A . Multienzyme complex for metabolic channeling in mammalian DNA replication. Proc Natl Acad Sci U S A. 1980; 77(6):3312-16. PMC: 349605. DOI: 10.1073/pnas.77.6.3312. View

15.

Kumar A, Blankenship D, KAUFMAN B, Freisheim J . Primary structure of chicken liver dihydrofolate reductase. Biochemistry. 1980; 19(4):667-78. DOI: 10.1021/bi00545a010. View

16.

Baccanari D, Tansik R, Paterson S, Stone D . Characterization and amino acid sequence of Neisseria gonorrhoeae dihydrofolate reductase. J Biol Chem. 1984; 259(19):12291-8. View

17.

Noguchi H, Reddy G, Pardee A . Rapid incorporation of label from ribonucleoside disphosphates into DNA by a cell-free high molecular weight fraction from animal cell nuclei. Cell. 1983; 32(2):443-51. DOI: 10.1016/0092-8674(83)90464-6. View

18.

Horigome T, Sugano H . A rapid method for removal of detergents from protein solution. Anal Biochem. 1983; 130(2):393-6. DOI: 10.1016/0003-2697(83)90605-x. View

19.

MacKenzie R, Baugh C . Tetrahydropterolypolyglutamate derivatives as substrates of two multifunctional proteins with folate-dependent enzyme activities. Biochim Biophys Acta. 1980; 611(1):187-95. DOI: 10.1016/0005-2744(80)90054-6. View

20.

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; 79(1):285-92. DOI: 10.1111/j.1432-1033.1977.tb11808.x. View