PT - JOURNAL ARTICLE AU - M. S. Rhee AU - B. Lindau-Shepard AU - K. J. Chave AU - J. Galivan AU - T. J. Ryan TI - Characterization of Human Cellular γ-Glutamyl Hydrolase DP - 1998 Jun 01 TA - Molecular Pharmacology PG - 1040--1046 VI - 53 IP - 6 4099 - http://molpharm.aspetjournals.org/content/53/6/1040.short 4100 - http://molpharm.aspetjournals.org/content/53/6/1040.full SO - Mol Pharmacol1998 Jun 01; 53 AB - 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. The American Society for Pharmacology and Experimental Therapeutics