Purification and characterization of γ-Glutamyltransferase from rat pancreas

Shizuko Takahashi, Howard M. Steinman, Douglas Ball

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8 Scopus citations

Abstract

γ-Glutamyltransferase ((5-giutamyl)-peptide:amino-acid 5-glutamyltransferase, EC 2.3.2.2.) from rat pancreas has been purified to homogeneity and shown to be a glycoprotein of apparent molecular weight 68 000, composed of one heavy and one light subunit, with respective molecular weights 43 000 and 25 000. At the optimum pH 8.0 the specific activity of the purified enzyme is 630 units/mg protein, with l-γ-glutamyl-pnitroanilide as substrate (Km = 0.9 mM) and 20 mM glycylgiycine as acceptor. The enzyme is inactivated by the active-site modifying agent and glutamine analogue, 6-diazo-5-oxo-l-norleucine, through a specific and stoichiometric reaction with the light subunit (Ki = 1.2 mM); both the inactivation and the modification of the light subunit are accelerated by maleate and prevented by S-methylglutathione. The enzyme is also inactivated by the fluorescent alkylating agent 5-iodoacetamidofluorescein, by specific and stoichiometric incorporation of the fluorescent moiety into the light subunit, which is likewise prevented by S-methylglutathione, but is unaffected by maleate. Antiserum to rat kidney γ-glutamyltransferase cross-reacts with the pancreas enzyme in immunodiffusion and inhibits its activity in the p-nitroanilide assay. Despite structural, enzymological and immunological similarities between the pancreas and kidney enzymes, their amino acid compositions are markedly different. The rat pancreas enzyme shows an interesting ontological development, being present in minimal amounts in the fetus, and increasing dramatically on birth and during the following 2 days.

Original languageEnglish (US)
Pages (from-to)66-73
Number of pages8
JournalBiochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Volume707
Issue number1
DOIs
Publication statusPublished - Sep 22 1982

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Keywords

  • (Rat pancreas)
  • Amino acid composition
  • γ-Glutamyltransferase

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology

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