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Purification and cloning of a Delta class glutathione S ‐transferase displaying high peroxidase activity isolated from the German cockroach Blattella germanica
Author(s) -
Ma Bennett,
Chang Frank N.
Publication year - 2007
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2007.05728.x
Subject(s) - german cockroach , glutathione , glutathione s transferase , cumene hydroperoxide , biochemistry , microbiology and biotechnology , peptide sequence , edman degradation , peroxidase , isozyme , chemistry , amino acid , biology , enzyme , gpx3 , glutathione peroxidase , cockroach , gene , ecology , catalysis
A highly active glutathione S ‐transferase was purified from adult German cockroaches, Blattella germanica . The purified enzyme appeared as a single band of 24 kDa by SDS/PAGE, and had a different electrophoretic mobility than, a previously identified Sigma class glutathione S ‐transferase (Bla g 5). Kinetic study of 1‐chloro‐2,4‐dinitrobenzene conjugation revealed a high catalytic rate but common substrate‐binding and cosubstrate‐binding affinities, with V max , k cat , K m for 1‐chloro‐2,4‐dinitrobenzene and K m for glutathione estimated to be 664 µmol·mg −1 ·min −1 , 545 s −1 , 0.33 m m and 0.76 m m , respectively. Interestingly, this enzyme possessed the highest activity for cumene hydroperoxide among insect glutathione S ‐transferases reported to date. Along with the ability to metabolize 1,1,1‐trichloro‐2,2‐bis( p ‐chlorophenyl)ethane and 4‐hydroxynonenal, this glutathione S ‐transferase may play a role in defense against insecticides as well as oxidative stress. On the basis of the amino acid sequences obtained from Edman degradation and MS analyses, a 987‐nucleotide cDNA clone encoding a glutathione S ‐transferase ( BggstD1 ) was isolated. The longest ORF encoded a 24 614 Da protein consisting of 216 amino acid residues. The sequence had close similarities (∼ 45–60%) to that of Delta class glutathione S ‐transferases, but had only 14% identity to Bla g 5. The putative amino acid sequence contained matching peptide fragments of the purified glutathione S ‐transferase. ELISA showed that BgGSTD1 bound to serum IgE obtained from patients with cockroach allergy, indicating that the protein may be a cockroach allergen.