SEQUENCE, CATALYTIC PROPERTIES AND EXPRESSION OF CHICKEN GLUTATHIONE-DEPENDENT PROSTAGLANDIN D-2 SYNTHASE, A NOVEL CLASS SIGMA-GLUTATHIONE-S-TRANSFERASE

The Expressed Sequence Tag database has been screened for cDNA clones encoding prostaglandin D-2 synthases (PGDSs) by using a BLAST search w ith the N-terminal amino acid sequence of rat GSH-dependent PGDS, a cl ass Sigma glutathione S-transferase (GST). This resulted in the identi fication of a cDNA from chicken spleen containing an insert of approx. 950 bp that encodes a protein of 199 amino acid residues with a predi cted molecular mass of 22 732 Da. The deduced primary structure of the chicken protein was not only found to possess 70% sequence identity w ith rat PGDS but it also demonstrated more than 35% identity with clas s Sigma GSTs from a range of invertebrates. The open reading frame of the chicken cDNA was expressed in Escherichia coli and the purified pr otein was found to display high PGDS activity. It also catalysed the c onjugation of glutathione with a wide range of aryl halides, organic i sothiocyanates and alpha,beta-unsaturated carbonyls, and exhibited glu tathione peroxidase activity towards cumene hydroperoxide. Like other GSTs, chicken PGDS was found to be inhibited by non-substrate ligands such as Cibacron Blue, haematin and organotin compounds. Western blott ing experiments showed that among the organs studied, the expression o f PGDS in the female chicken is highest in liver, kidney and intestine , with only small amounts of the enzyme being found in chicken spleen; in contrast, the rat has highest levels of PGDS in the spleen. Collec tively, these results show that the structure and function, but not th e expression, of the GSH-requiring PGDS is conserved between chicken a nd rat.