Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1998 Aug 1;333(Pt 3):839–845. doi: 10.1042/bj3330839

Glutathione S-transferases of the yeast Yarrowia lipolytica have unusually large molecular mass.

V Foley 1, D Sheehan 1
PMCID: PMC1219652  PMID: 9677348

Abstract

Two similar glutathione S-transferases (GSTs), which do not bind to glutathione- or S-hexylglutathione-agarose affinity resins, have been purified from the yeast Yarrowia lipolytica. An approx. 400-fold purification was obtained by a combination of DEAE-Sephadex, phenyl-Sepharose, hydroxyapatite and Mono-Q anion-exchange chromatography. The native molecular mass of both proteins was estimated as approx. 110 kDa by both Superose-12 gel-filtration chromatography and non-denaturing electrophoresis. SDS/PAGE indicated a subunit mass of 50 kDa. Reverse-phase HPLC of purified proteins gave a single, well-resolved, peak, suggesting that the proteins are homodimers. Identical behaviour on HPLC, native electrophoresis and SDS/PAGE, N-terminal sequencing, sensitivity to a panel of inhibitors and identical specific activities with 1-chloro-2,4-dinitrobenzene as substrate suggest that the two isoenzymes are very similar. The enzymes do not immunoblot with antisera to any of the main GST classes, and N-terminal sequencing suggests no clear relationship with previously characterized enzymes, such as that of the fungus, Phanerochaete chrysosporium [Dowd, Buckley and Sheehan (1997) Biochem. J. 324, 243-248]. It is possible that the two isoenzymes arise as a result of post-translational modification of a single GST isoenzyme.

Full Text

The Full Text of this article is available as a PDF (314.5 KB).

Selected References

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

  1. Arca P., Hardisson C., Suárez J. E. Purification of a glutathione S-transferase that mediates fosfomycin resistance in bacteria. Antimicrob Agents Chemother. 1990 May;34(5):844–848. doi: 10.1128/aac.34.5.844. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Armstrong R. N. Structure, catalytic mechanism, and evolution of the glutathione transferases. Chem Res Toxicol. 1997 Jan;10(1):2–18. doi: 10.1021/tx960072x. [DOI] [PubMed] [Google Scholar]
  3. Bartling D., Radzio R., Steiner U., Weiler E. W. A glutathione S-transferase with glutathione-peroxidase activity from Arabidopsis thaliana. Molecular cloning and functional characterization. Eur J Biochem. 1993 Sep 1;216(2):579–586. doi: 10.1111/j.1432-1033.1993.tb18177.x. [DOI] [PubMed] [Google Scholar]
  4. Bilang J., Sturm A. Cloning and characterization of a glutathione S-transferase that can be photolabeled with 5-azido-indole-3-acetic acid. Plant Physiol. 1995 Sep;109(1):253–260. doi: 10.1104/pp.109.1.253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  6. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  7. Carlberg I., Mannervik B. Purification and characterization of glutathione reductase from calf liver. An improved procedure for affinity chromatography on 2',5'-ADP-Sepharose 4B. Anal Biochem. 1981 Sep 15;116(2):531–536. doi: 10.1016/0003-2697(81)90398-5. [DOI] [PubMed] [Google Scholar]
  8. Coschigano P. W., Magasanik B. The URE2 gene product of Saccharomyces cerevisiae plays an important role in the cellular response to the nitrogen source and has homology to glutathione s-transferases. Mol Cell Biol. 1991 Feb;11(2):822–832. doi: 10.1128/mcb.11.2.822. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Di Ilio C., Aceto A., Piccolomini R., Allocati N., Faraone A., Bucciarelli T., Barra D., Federici G. Purification and characterization of a novel glutathione transferase from Serratia marcescens. Biochim Biophys Acta. 1991 Apr 8;1077(2):141–146. doi: 10.1016/0167-4838(91)90050-a. [DOI] [PubMed] [Google Scholar]
  10. Dirr H. W., Reinemer P. Equilibrium unfolding of class pi glutathione S-transferase. Biochem Biophys Res Commun. 1991 Oct 15;180(1):294–300. doi: 10.1016/s0006-291x(05)81291-4. [DOI] [PubMed] [Google Scholar]
  11. Dirr H., Reinemer P., Huber R. X-ray crystal structures of cytosolic glutathione S-transferases. Implications for protein architecture, substrate recognition and catalytic function. Eur J Biochem. 1994 Mar 15;220(3):645–661. doi: 10.1111/j.1432-1033.1994.tb18666.x. [DOI] [PubMed] [Google Scholar]
  12. Dowd C. A., Buckley C. M., Sheehan D. Glutathione S-transferases from the white-rot fungus, Phanerochaete chrysosporium. Biochem J. 1997 May 15;324(Pt 1):243–248. doi: 10.1042/bj3240243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fitzpatrick P. J., Krag T. O., Højrup P., Sheehan D. Characterization of a glutathione S-transferase and a related glutathione-binding protein from gill of the blue mussel, Mytilus edulis. Biochem J. 1995 Jan 1;305(Pt 1):145–150. doi: 10.1042/bj3050145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fournier D., Bride J. M., Poirie M., Bergé J. B., Plapp F. W., Jr Insect glutathione S-transferases. Biochemical characteristics of the major forms from houseflies susceptible and resistant to insecticides. J Biol Chem. 1992 Jan 25;267(3):1840–1845. [PubMed] [Google Scholar]
  15. Grove G., Zarlengo R. P., Timmerman K. P., Li N. Q., Tam M. F., Tu C. P. Characterization and heterospecific expression of cDNA clones of genes in the maize GSH S-transferase multigene family. Nucleic Acids Res. 1988 Jan 25;16(2):425–438. doi: 10.1093/nar/16.2.425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Habig W. H., Pabst M. J., Jakoby W. B. Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J Biol Chem. 1974 Nov 25;249(22):7130–7139. [PubMed] [Google Scholar]
  17. Hathout Y., Ellis T., Fabris D., Fenselau C. Cross-linking of human placenta pi class glutathione S-transferase dimer by chlorambucil. Chem Res Toxicol. 1996 Sep;9(6):1044–1049. doi: 10.1021/tx950193h. [DOI] [PubMed] [Google Scholar]
  18. Hayes J. D., Pulford D. J. The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit Rev Biochem Mol Biol. 1995;30(6):445–600. doi: 10.3109/10409239509083491. [DOI] [PubMed] [Google Scholar]
  19. Hussey A. J., Hayes J. D. Characterization of a human class-Theta glutathione S-transferase with activity towards 1-menaphthyl sulphate. Biochem J. 1992 Sep 15;286(Pt 3):929–935. doi: 10.1042/bj2860929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ji X., von Rosenvinge E. C., Johnson W. W., Tomarev S. I., Piatigorsky J., Armstrong R. N., Gilliland G. L. Three-dimensional structure, catalytic properties, and evolution of a sigma class glutathione transferase from squid, a progenitor of the lens S-crystallins of cephalopods. Biochemistry. 1995 Apr 25;34(16):5317–5328. doi: 10.1021/bi00016a003. [DOI] [PubMed] [Google Scholar]
  21. Ketley J. N., Habig W. H., Jakoby W. B. Binding of nonsubstrate ligands to the glutathione S-transferases. J Biol Chem. 1975 Nov 25;250(22):8670–8673. [PubMed] [Google Scholar]
  22. Koonin E. V., Mushegian A. R., Tatusov R. L., Altschul S. F., Bryant S. H., Bork P., Valencia A. Eukaryotic translation elongation factor 1 gamma contains a glutathione transferase domain--study of a diverse, ancient protein superfamily using motif search and structural modeling. Protein Sci. 1994 Nov;3(11):2045–2054. doi: 10.1002/pro.5560031117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kutchan T. M., Hochberger A. Nucleotide Sequence of a cDNA Encoding a Constitutively Expressed Glutathione S-Transferase from Cell Suspension Cultures of Silene cucubalus. Plant Physiol. 1992 Jun;99(2):789–790. doi: 10.1104/pp.99.2.789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  25. Lawrence R. A., Burk R. F. Glutathione peroxidase activity in selenium-deficient rat liver. Biochem Biophys Res Commun. 1976 Aug 23;71(4):952–958. doi: 10.1016/0006-291x(76)90747-6. [DOI] [PubMed] [Google Scholar]
  26. Leaver M. J., Scott K., George S. G. Cloning and characterization of the major hepatic glutathione S-transferase from a marine teleost flatfish, the plaice (Pleuronectes platessa), with structural similarities to plant, insect and mammalian Theta class isoenzymes. Biochem J. 1993 May 15;292(Pt 1):189–195. doi: 10.1042/bj2920189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mannervik B., Alin P., Guthenberg C., Jensson H., Tahir M. K., Warholm M., Jörnvall H. Identification of three classes of cytosolic glutathione transferase common to several mammalian species: correlation between structural data and enzymatic properties. Proc Natl Acad Sci U S A. 1985 Nov;82(21):7202–7206. doi: 10.1073/pnas.82.21.7202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mannervik B., Lindström L., Bártfai T. Partial purification and characterization of glyoxalase I from porcine erythrocytes. Eur J Biochem. 1972 Sep 18;29(2):276–281. doi: 10.1111/j.1432-1033.1972.tb01985.x. [DOI] [PubMed] [Google Scholar]
  29. McCarthy R. M., Farmer P., Sheehan D. Binding of 2-hydroxy-5-nitrobenzyl alcohol to rat alpha class glutathione S-transferases; evidence for binding at tryptophan 21. Biochim Biophys Acta. 1996 Apr 16;1293(2):185–190. doi: 10.1016/0167-4838(95)00221-9. [DOI] [PubMed] [Google Scholar]
  30. Meyer D. J., Coles B., Pemble S. E., Gilmore K. S., Fraser G. M., Ketterer B. Theta, a new class of glutathione transferases purified from rat and man. Biochem J. 1991 Mar 1;274(Pt 2):409–414. doi: 10.1042/bj2740409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Meyer D. J. Significance of an unusually low Km for glutathione in glutathione transferases of the alpha, mu and pi classes. Xenobiotica. 1993 Aug;23(8):823–834. doi: 10.3109/00498259309059411. [DOI] [PubMed] [Google Scholar]
  32. Meyer R. C., Jr, Goldsbrough P. B., Woodson W. R. An ethylene-responsive flower senescence-related gene from carnation encodes a protein homologous to glutathione S-transferases. Plant Mol Biol. 1991 Aug;17(2):277–281. doi: 10.1007/BF00039505. [DOI] [PubMed] [Google Scholar]
  33. Moutiez M., Aumercier M., Schöneck R., Meziane-Cherif D., Lucas V., Aumercier P., Ouaissi A., Sergheraert C., Tartar A. Purification and characterization of a trypanothione-glutathione thioltransferase from Trypanosoma cruzi. Biochem J. 1995 Sep 1;310(Pt 2):433–437. doi: 10.1042/bj3100433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nishida M., Kong K. H., Inoue H., Takahashi K. Molecular cloning and site-directed mutagenesis of glutathione S-transferase from Escherichia coli. The conserved tyrosyl residue near the N terminus is not essential for catalysis. J Biol Chem. 1994 Dec 23;269(51):32536–32541. [PubMed] [Google Scholar]
  35. Ostlund Farrants A. K., Meyer D. J., Coles B., Southan C., Aitken A., Johnson P. J., Ketterer B. The separation of glutathione transferase subunits by using reverse-phase high-pressure liquid chromatography. Biochem J. 1987 Jul 15;245(2):423–428. doi: 10.1042/bj2450423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Pemble S. E., Taylor J. B. An evolutionary perspective on glutathione transferases inferred from class-theta glutathione transferase cDNA sequences. Biochem J. 1992 Nov 1;287(Pt 3):957–963. doi: 10.1042/bj2870957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Perito B., Allocati N., Casalone E., Masulli M., Dragani B., Polsinelli M., Aceto A., Di Ilio C. Molecular cloning and overexpression of a glutathione transferase gene from Proteus mirabilis. Biochem J. 1996 Aug 15;318(Pt 1):157–162. doi: 10.1042/bj3180157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Phillips M. F., Mantle T. J. Inactivation of mouse liver glutathione S-transferase YfYf (Pi class) by ethacrynic acid and 5,5'-dithiobis-(2-nitrobenzoic acid). Biochem J. 1993 Aug 15;294(Pt 1):57–62. doi: 10.1042/bj2940057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Radola B. J. Thin-layer isoelectric focusing of proteins. Biochim Biophys Acta. 1969 Nov 11;194(1):335–338. doi: 10.1016/0005-2795(69)90214-1. [DOI] [PubMed] [Google Scholar]
  40. Reinemer P., Prade L., Hof P., Neuefeind T., Huber R., Zettl R., Palme K., Schell J., Koelln I., Bartunik H. D. Three-dimensional structure of glutathione S-transferase from Arabidopsis thaliana at 2.2 A resolution: structural characterization of herbicide-conjugating plant glutathione S-transferases and a novel active site architecture. J Mol Biol. 1996 Jan 19;255(2):289–309. doi: 10.1006/jmbi.1996.0024. [DOI] [PubMed] [Google Scholar]
  41. Ricci G., Caccuri A. M., Lo Bello M., Rosato N., Mei G., Nicotra M., Chiessi E., Mazzetti A. P., Federici G. Structural flexibility modulates the activity of human glutathione transferase P1-1. Role of helix 2 flexibility in the catalytic mechanism. J Biol Chem. 1996 Jul 5;271(27):16187–16192. doi: 10.1074/jbc.271.27.16187. [DOI] [PubMed] [Google Scholar]
  42. Sheehan D., Casey J. P. Microbial glutathione S-transferases. Comp Biochem Physiol B. 1993 Jan;104(1):1–6. doi: 10.1016/0305-0491(93)90330-8. [DOI] [PubMed] [Google Scholar]
  43. Sánchez M., Prado M., Iglesias F. J., Domínguez A. Cloning and sequencing of the URA5 gene from the yeast Yarrowia lipolytica. Yeast. 1995 Apr 30;11(5):425–433. doi: 10.1002/yea.320110505. [DOI] [PubMed] [Google Scholar]
  44. Takahashi Y., Nagata T. parB: an auxin-regulated gene encoding glutathione S-transferase. Proc Natl Acad Sci U S A. 1992 Jan 1;89(1):56–59. doi: 10.1073/pnas.89.1.56. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tomarev S. I., Zinovieva R. D., Guo K., Piatigorsky J. Squid glutathione S-transferase. Relationships with other glutathione S-transferases and S-crystallins of cephalopods. J Biol Chem. 1993 Feb 25;268(6):4534–4542. [PubMed] [Google Scholar]
  46. Vuilleumier S., Leisinger T. Protein engineering studies of dichloromethane dehalogenase/glutathione S-transferase from Methylophilus sp. strain DM11. Ser12 but not Tyr6 is required for enzyme activity. Eur J Biochem. 1996 Jul 15;239(2):410–417. doi: 10.1111/j.1432-1033.1996.0410u.x. [DOI] [PubMed] [Google Scholar]
  47. Wilce M. C., Board P. G., Feil S. C., Parker M. W. Crystal structure of a theta-class glutathione transferase. EMBO J. 1995 May 15;14(10):2133–2143. doi: 10.1002/j.1460-2075.1995.tb07207.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wilce M. C., Parker M. W. Structure and function of glutathione S-transferases. Biochim Biophys Acta. 1994 Mar 16;1205(1):1–18. doi: 10.1016/0167-4838(94)90086-8. [DOI] [PubMed] [Google Scholar]
  49. Zettl R., Schell J., Palme K. Photoaffinity labeling of Arabidopsis thaliana plasma membrane vesicles by 5-azido-[7-3H]indole-3-acetic acid: identification of a glutathione S-transferase. Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):689–693. doi: 10.1073/pnas.91.2.689. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES