Biochemical characterization of a glutathione S-transferase and a sex pheromone isolated from the German cockroach, Blattella germanica

The cytotoxic effects of blattellaquinone (BTQ), a sex pheromone produced by adult female German cockroaches, have been studied using human lung adenocarcinoma cells. 1,4-Benzoquinone (BQ), a toxic chemical implicated in benzene toxicity, was used as a reference compound. Both BQ and BTQ showed comparable cytotoxicity and effectiveness in generating reactive oxygen species. However, BTQ was 4-fold more efficient in depleting cellular glutathione (GSH) content than BQ. Of the five GSH adducts of BTQ isolated, three were identified as mono-GSH conjugates and the other two were di-conjugates. Mass spectrometric and NMR analyses of the di-conjugates showed that the second GSH molecule displaced the isovaleric acid moiety, potentially via a nucleophilic substitution reaction. The ability of BTQ to conjugate a second GSH molecule without quinone regeneration indicated it may be a more effective cross-linking agent than BQ.;A highly active glutathione S-transferase (GST) was purified from adult German cockroaches, Blattella germanica. The purified enzyme appeared as a single band of 24.5 kDa by SDS-gel electrophoresis, and had a different electrophoretic mobility than a previously identified Sigma class GST (Bla g 5). Kinetic study of CDNB conjugation revealed high catalytic rate but common substrate- and co-substrate binding affinities with k cat, Vmax, Km CDNB, and Km GSH values estimated to be 545 s -1, 664 mumol/min/mg protein, 0.33 mM, 0.76 mM, respectively. This GST's ability to metabolize DDT, cumene hydroperoxide, and 4-hydroxynonenal suggested the enzyme may play a role in defense against insecticides as well as oxidative stress. Based on the amino acid sequence obtained from Edman degradation and mass spectrometric analysis, a 987-nucleotide cDNA clone encoding a GST (BggstD1) was isolated. The longest open reading frame encoded a 24,614 Da protein consisting of 216 amino acid residues. The sequence had close similarities (∼45--60%) to that of Delta class GSTs, but had only 14% identity to Bla g 5. The putative amino acid sequence contained matching peptide fragments of the purified GST. ELISA assay showed that BgGSTD1 bound to serum-IgE obtained from patients with cockroach allergy, indicating the protein may be a cockroach allergen.