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Vol. 53, Issue 6, 1040-1046, June 1998
-Glutamyl
Hydrolase
Wadsworth Center, Division of Molecular Medicine, New York State
Department of Health, Albany, New York 12201-0509
A previously identified cDNA encoding a human 
-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--Glu is a poor substrate for the human
enzyme relative to methotrexate -Glu2-5. During
hydrolysis of methotrexate--Glu4, all possible
pterin-containing cleavage products (methotrexate and
methotrexate--Glu1-3) appear. The results demonstrated that the human enzyme cleaves both the ultimate and penultimate -linkages of methotrexate polyglutamates. Glutamate was released as
either glutamic acid or -Glu2. Longer chain species of
-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 -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.
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