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
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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
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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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