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Molecular Pharmacology

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Research ArticleArticle

Characterization of Human Cellular γ-Glutamyl Hydrolase

M. S. Rhee, B. Lindau-Shepard, K. J. Chave, J. Galivan and T. J. Ryan
Molecular Pharmacology June 1998, 53 (6) 1040-1046;
M. S. Rhee
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B. Lindau-Shepard
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K. J. Chave
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J. Galivan
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T. J. Ryan
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Abstract

A previously identified cDNA encoding a human γ-glutamyl hydrolase was expressed in a baculovirus system. The expressed protein had molecular mass of 37 kDa. Treatment of the protein with PNGase F produced a protein of molecular mass of 30 kDa, indicating that the protein contained asparagine-linked glycosylation. Sequence analysis of the expressed protein indicated that a 24-amino-acid signal peptide had been removed. A polyclonal antibody to the expressed enzyme was used in Western blot analysis of partially purified lysates of HL-60 promyeloid leukemia cells and MCF-7 breast cancer cells. The HL-60 and MCF-7 enzymes appeared as two closely spaced bands with a molecular mass of 37 kDa. Treatment of the HL-60 enzyme with PNGase F produced a protein with a molecular mass of 30 kDa. The activities of the expressed enzyme and the enzyme from HL-60 cells were similar on methotrexate polyglutamates. Methotrexate-γ-Glu is a poor substrate for the human enzyme relative to methotrexate γ-Glu2–5. During hydrolysis of methotrexate-γ-Glu4, all possible pterin-containing cleavage products (methotrexate and methotrexate-γ-Glu1–3) appear. The results demonstrated that the human enzyme cleaves both the ultimate and penultimate γ-linkages of methotrexate polyglutamates. Glutamate was released as either glutamic acid or γ-Glu2. Longer chain species of γ-Glun>2 were not observed. Inhibition by iodoacetic acid suggested that both the expressed enzyme and the HL-60 enzyme may contain a catalytically essential cysteine. These results indicate that the identified cDNA encodes the intracellular γ-glutamyl hydrolase found in a variety of human tumor cells and that the baculovirus-expressed enzyme is a suitable model for further structural and enzymatic studies.

Footnotes

  • Send reprint requests to: Dr. T. J. Ryan, Wadsworth Center, New York State Department of Health, Empire State Plaza, Albany, NY 12201-0509. E-mail:thomas.ryan{at}wadsworth.org

  • This study was supported in part by Grant CA25933 from the National Cancer Institute/National Institutes of Health/Department of Health and Human Services.

  • Abbreviations:
    GH
    γ-glutamyl hydrolase
    MTX
    methotrexate
    hGH
    human γ-glutamyl hydrolase
    FPGS
    folylpolyglutamate synthetase
    HPLC
    high performance liquid chromatography
    OPA
    o-phthalaldehyde
    OBG
    octyl β-d-glucoside
    PteGlun
    folylpolyglutamate
    PNGase F
    peptide-N4-(N-acetyl-β-glucosaminyl) asparagine amidase
    SDS
    sodium dodecyl sulfate
    PAGE
    polyacrylamide gel electrophoresis
    • Received January 13, 1998.
    • Accepted March 2, 1998.
  • The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 53 (6)
Molecular Pharmacology
Vol. 53, Issue 6
1 Jun 1998
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Research ArticleArticle

Characterization of Human Cellular γ-Glutamyl Hydrolase

M. S. Rhee, B. Lindau-Shepard, K. J. Chave, J. Galivan and T. J. Ryan
Molecular Pharmacology June 1, 1998, 53 (6) 1040-1046;

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Research ArticleArticle

Characterization of Human Cellular γ-Glutamyl Hydrolase

M. S. Rhee, B. Lindau-Shepard, K. J. Chave, J. Galivan and T. J. Ryan
Molecular Pharmacology June 1, 1998, 53 (6) 1040-1046;
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