Measurement of Glutathione Transferases

Bengt Mannervik1, Per Jemth1

1 Uppsala University, Uppsala
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 6.4
DOI:  10.1002/0471140856.tx0604s01
Online Posting Date:  May, 2001
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Abstract

There are multiple glutathione transferase genes, the proteins for which have different substrate specificities. The various genes are differentially expressed such that species and organs and tissues differ qualitatively and quantitatively for cytosolic and membraneā€bound forms. This unit provides protocols for analysis of transferase activity in a continuous spectrophotometric assay and an assay with dichloromethane as the substrate.

     
 
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Table of Contents

  • Basic Protocol 1: Continuous Spectrophotometric Assays for Glutathione Transferase Activity
  • Alternate Protocol 1: Measurement of Glutathione Transferase T1‐1 Activity with Dichloromethane as the Substrate
  • Reagents and Solutions
  • Commentary
  • Tables
     
 
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Materials

Basic Protocol 1: Continuous Spectrophotometric Assays for Glutathione Transferase Activity

  Materials
  • Assay buffer (see recipe and Table 6.4.1)
  • Glutathione (GSH) stock solution (see recipe and Table 6.4.1; make 20 times the desired final concentration indicated in the table)
  • Additional reagents for measurements with cumene hydroperoxide (prepare fresh):
  •  Cumene hydroperoxide stock solution (see Table 6.4.1)
  •  6 U/ml glutathione reductase in assay buffer
  •  2 mM NADPH in 10 mM Tris⋅Cl, pH 7.0
  • Cell lysate or purified enzyme solution (amount depends on the activity of the sample)
  • Electrophilic substrate (see Table 6.4.1; also see recipe for stock solution)
  • Spectrophotometer, thermostatically controlled
  • 30°C water bath

Alternate Protocol 1: Measurement of Glutathione Transferase T1‐1 Activity with Dichloromethane as the Substrate

  • Assay buffer: 0.1 M Tris⋅Cl, pH 8.5 ( appendix 2A)
  • 0.5 M (3.2% v/v) dichloromethane in 95% ethanol
  • 25% (w/v) trichloroacetic acid (TCA)
  • Nash reagent (see recipe)
  • 5‐ml vials, sealable
  • Water bath, 42°C
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Figures

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Literature Cited

Literature Cited
   Ålin, P., Jensson, H., Guthenberg, C., Danielson, U.H., Tahir, M.K., and Mannervik, B. 1985. Purification of major basic glutathione transferase isoenzymes from rat liver by use of affinity chromatography and fast protein liquid chromatofocusing. Anal. Biochem. 146:313‐320.
   Guthenberg, C., Jensson, H., Nyström, L., Österlund, E., Tahir, M.K., and Mannervik, B. 1985a. Isoenzymes of glutathione transferase in rat kidney cytosol. Biochem. J. 230:609‐615.
   Guthenberg, C., Ålin, P., and Mannervik, B. 1985b. Glutathione transferase from rat testis. Methods Enzymol. 113:507‐510.
   Guthenberg, C., Warholm, M., Rane, A., and Mannervik, B. 1986. Two distinct forms of glutathione transferase from human foetal liver: Purification and comparison with isoenzymes isolated from adult liver and placenta. Biochem. J. 235:741‐745.
   Hao, X.‐Y., Castro, V.M., Bergh, J., Sundström, B., and Mannervik, B. 1994. Isoenzyme‐specific quantitative immunoassays for cytosolic glutathione transferases and measurement of the enzymes in blood plasma from cancer patients and in tumor cell lines. Biochim. Biophys. Acta 1225:223‐230.
   Hayes, J.D. and Pulford, D.J. 1995. 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. 30:445‐600.
   Hubatsch, I., Ridderström, M., and Mannervik, B. 1998. Human glutathione transferase A4‐4: An Alpha class enzyme with high catalytic efficiency in the conjugation of 4‐hydroxynonenal and other genotoxic products of lipid peroxidation. Biochem. J. 330:175‐179.
   Mannervik, B. and Guthenberg, C. 1981. Glutathione transferase (human placenta). Methods Enzymol. 77:231‐235.
   Mannervik, B. and Danielson, U.H. 1988. Glutathione transferases: Structure and catalytic activity. CRC Crit. Rev. Biochem. 23:283‐337.
   Mannervik, B. and Widersten, M. 1995. Human glutathione transferases: Classification, tissue distribution, structure, and functional properties. In Advances in Drug Metabolism in Man (G.M. Pacifici and G.N. Fracchia, eds.) pp. 408‐459. European Commission, Luxembourg.
   Mannervik, B., Awasthi, Y.C., Board, P.G., Hayes, J.D., Di Ilio, C., Ketterer, B., Listowsky, I., Morgenstern, R., Muramatsu, M., Pearson, W.R., Pickett, C.B., Sato, K., Widersten, M., and Wolf, C. R. 1992. Nomenclature for human glutathione transferases. Biochem. J. 282:305‐306.
   Marcus, C.J., Habig, W.H., and Jakoby, W.B. 1978. Glutathione transferase from human erythrocytes: Nonidentity with the enzymes from liver. Arch. Biochem. Biophys. 188:287‐293.
   Morgenstern, R., Guthenberg, C., and DePierre, J.W. 1982. Microsomal glutathione transferase: Purification, initial characterization and demonstration that it is not identical to the cytosolic glutathione transferases A, B and C. Eur. J. Biochem. 128:243‐248.
   Ostlund Farrants, A.‐K., Meyer, D.J., Coles, B., Southan, C., Aitken, A., Johnson, P.J., and Ketterer, B. 1987. The separation of glutathione transferase subunits by using reverse‐phase high‐pressure liquid chromatography. Biochem. J. 245:423‐428.
   Robertson, I.G.C., Jensson, H., Guthenberg, C., Tahir, M.K., Jernström, B., and Mannervik, B. 1985. Differences in the occurrence of glutathione transferase isoenzymes in rat lung and liver. Biochem. Biophys. Res. Commun. 127:80‐86.
   Seidegård, J., DePierre, J.W., and Pero, R.W. 1985. Hereditary interindividual differences in the glutathione transferase activity towards trans‐stilbene oxide in resting human mononuclear leukocytes are due to a particular isozyme(s). Carcinogenesis 6:1211‐1216.
   Sherratt, P.J., Pulford, D.J., Harrison, D.J., Green, T., and Hayes, J.D. 1997. Evidence that human class Theta glutathione transferase T1‐1 can catalyse the activation of dichloromethane, a liver and lung carcinogen in the mouse: Comparison of the tissue distribution of GST T1‐1 with that of classes Alpha, Mu and Pi GST in human. Biochem. J. 326:837‐846.
   Tahir, M.K., Özer, N., and Mannervik, B. 1988. Isoenzymes of glutathione transferase in rat small intestine. Biochem. J. 253:759‐764.
   Tan, K.‐L. and Board, P.G. 1996. Purification and characterization of a recombinant human Theta‐class glutathione transferase (GST T2‐2). Biochem. J. 315:727‐732.
   Warholm, M., Guthenberg, C., von Bahr, C., and Mannervik, B. 1985. Glutathione transferases from human liver. Methods Enzymol. 113:499‐504.
   Warholm, M., Jensson, H., Tahir, M.K., and Mannervik, B. 1986. Purification and characterization of three distinct glutathione transferases from mouse liver. Biochemistry 25:4119‐4125.
Key References
   Hayes and Pulford, 1995. See .
  Recent review article.
   Mannervik and Widersten, 1995. See .
  Recent review articles on human glutathione transferases.
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