Purification and cloning of a Delta class glutathione S-transferase displaying high peroxidase activity isolated from the German cockroach Blattella germanica.
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 micromol x mg(-1) x min(-1), 545 s(-1), 0.33 mm and 0.76 mm, 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 ( approximately 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.
Ma
Bennett
B
Department of Drug Metabolism, Merck Research Laboratories, West Point, PA 19486, USA. bennett_ma@merck.com
Chang
Frank N
FN
D000485:Allergens / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001555:Benzene Derivatives / Q000737:chemistry; D020048:Blattellidae / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D002384:Catalysis; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007073:Immunoglobulin E / Q000097:blood / Q000276:immunology; D019476:Insect Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009195:Peroxidase / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D000485:Allergens; D001555:Benzene Derivatives; D019476:Insect Proteins; D007073:Immunoglobulin E; D009195:Peroxidase; D005982:Glutathione Transferase; C007164:cumene hydroperoxide