γ‐Glutamyl Transpeptidase Activity Assay

Yoshitaka Ikeda1, Naoyuki Taniguchi1

1 Osaka University Medical School, Osaka
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 6.6
DOI:  10.1002/0471140856.tx0606s05
Online Posting Date:  May, 2001
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Abstract

Gamma‐glutamyl transpeptidase catalyzes the transfer of a gamma‐glutamyl bond and is of importance in glutathione metabolism. The most common assay methods for this enzyme, in which gamma‐glutamyl derivatives of p‐nitroanilineare used as a gamma‐glutamyl donor, are described in this unit.

     
 
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Table of Contents

  • γ‐Glutamyl Transpeptidase Activity Assay
  • Basic Protocol 1: Spectrophotometric Assay Using γ‐Glutamyl‐p‐Nitroanilide for GGT Activity
  • Alternate Protocol 1: Use of L‐γ‐Glutamyl‐3‐Carboxy‐4‐Nitroanilide as the Donor Substrate to Measure GGT Activity
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Spectrophotometric Assay Using γ‐Glutamyl‐p‐Nitroanilide for GGT Activity

  Materials
  • L‐γ‐Glutamyl‐p‐nitroanilide (free base or HCl salt; Sigma)
  • 0.2 N HCl
  • Glycylglycine free base
  • 0.5 M Tris⋅Cl, pH 9.0 to 9.5 ( appendix 2A)
  • Sample (e.g., tissue or cell homogenate, membrane preparation, microsome suspension)
  • Homogenate buffer (see recipe)
  • Potter‐Elvehjem homogenizer or Dounce homogenizer
  • Spectrophotometer with temperature‐controlled cuvette holder
  • Semimicro cell (light path = 1 cm)

Alternate Protocol 1: Use of L‐γ‐Glutamyl‐3‐Carboxy‐4‐Nitroanilide as the Donor Substrate to Measure GGT Activity

  • L‐γ‐glutamyl‐3‐carboxy‐4‐nitroanilide (ammonium salt; Sigma)
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Figures

Videos

Literature Cited

Literature Cited
   Anderson, M.E., Allison, R.D., and Meister, A. 1982. Interconversion of leukotrienes catalyzed by purified γ‐glutamyl transpeptidase: Concomitant formation of leukotriene D4 and γ‐glutamyl amino acids. Proc. Natl. Acad. Sci. U.S.A. 79:1088‐1091.
   Carter, B.Z., Wiseman, A.L., Orkiszewski, R., Ballard, K.D., Ou, C.N., and Lieberman, M.W. 1997. Metabolism of leukotriene C4 in γ‐glutamyl transpeptidase‐deficient mice. J. Biol. Chem. 272:12305‐12310.
   Carter, B.Z., Shi, Z.Z., Barrios, R., and Lieberman, M.W. 1998. γ‐Glutamyl leukotrienase, a γ‐glutamyl transpeptidase gene family member, is expressed primarily in spleen. J. Biol. Chem. 273:28277‐28285.
   Heisterkamp, N., Rajpert‐De‐Meyts, E., Uribe, L., Forman, H.J., and Groffen, J. 1991. Identification of a human γ‐glutamyl cleaving enzyme related to, but distinct from, γ‐glutamyl transpeptidase. Proc. Natl. Acad. Sci. U.S.A. 88:6303‐6307.
   Ikeda, Y., Fujii, J., and Taniguchi, N. 1993. Significance of Arg‐107 and Glu‐108 in the catalytic mechanism of human γ‐glutamyl transpeptidase: Identification by site‐directed mutagenesis. J. Biol. Chem. 268:3980‐3985.
   Ikeda, Y., Fujii, J., Anderson, M.E., Taniguchi, N., and Meister, A. 1995. Involvement of Ser‐451 and Ser‐452 in the catalysis of human γ‐glutamyl transpeptidase. J. Biol. Chem. 270:22223‐22228.
   Ikeda, Y., Fujii, J., and Taniguchi, N. 1996. Effects of substitutions of conserved histidine residues in human γ‐glutamyl transpeptidase. J. Biochem. 119:1166‐1170.
   Lieberman, M.W., Wiseman, A.L., Shi, Z.Z., Carter, B.Z., Barrios, R., Ou, C.N., Chevez‐Barrios, P., Wang, Y., Habib, G.M., Goodman, J.C., Huang, S.L., Lebovitz, R.M., and Matzuk, M.M. 1996. Growth retardation and cysteine deficiency in gamma‐glutamyl transpeptidase‐deficient mice. Proc. Natl. Acad. Sci. U.S.A. 93:7923‐7926.
   Meister, A. and Anderson, M.E. 1983. Glutathione. Annu. Rev. Biochem. 52:711‐760.
   Meister, A. and Larsson, A. 1995. Glutathione synthetase deficiency and other disorders of the γ‐glutamyl cycle. In The Metabolic and Molecular Bases of Inherited Diseases, 7th ed., Vol.I (C.R. Scriver, A.L. Beaudet, W.S. Sly, and D. Valle, eds.) pp. 1461‐1477. McGraw‐Hill, New York.
   Meister, A., Tate, S.S., and Griffith, O.W. 1981. γ‐Glutamyl transpeptidase. Methods Enzymol. 77:237‐253.
   Smith, G.D., Ding, J.L., and Peters, J.L. 1979. A sensitive fluorimetric assay for γ‐glutamyl transferase. Anal. Biochem. 100:136‐139.
   Suzuki, Y., Ishizuka, H., Kaneda, H., and Taniguchi, N. 1987. γ‐Glutamyl transpeptidase in rat liver during 3′‐Me‐DAB hepatocarcinogenesis: Immunohistochemical and enzyme histochemical study. J. Histochem. Cytochem. 35:3‐7.
   Taniguchi, N. and Ikeda, Y. 1998. γ‐Glutamyl transpeptidase: Catalytic mechanism and gene expression. Adv. Enzymol. Relat. Areas Mol. Biol. 72:239‐278.
   Tate, S.S. and Meister, A. 1985. γ‐Glutamyl transpeptidase from kidney. Methods Enzymol. 113:400‐419.
   Thompson, G.A. and Meister, A. 1976. Hydrolysis and transfer reactions catalyzed by γ‐glutamyl transpeptidase: Evidence for separate substrate sites and for high affinity of L‐cystine. Biochem. Biophys. Res. Commun. 71:32‐36.
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