Sequence, catalytic properties and expression of chicken glutathione-dependent prostaglandin D2 synthase, a novel class Sigma glutathione S-transferase.
The Expressed Sequence Tag database has been screened for cDNA clones encoding prostaglandin D2 synthases (PGDSs) by using a BLAST search with the N-terminal amino acid sequence of rat GSH-dependent PGDS, a class Sigma glutathione S-transferase (GST). This resulted in the identification of a cDNA from chicken spleen containing an insert of approx. 950 bp that encodes a protein of 199 amino acid residues with a predicted molecular mass of 22732 Da. The deduced primary structure of the chicken protein was not only found to possess 70% sequence identity with rat PGDS but it also demonstrated more than 35% identity with class Sigma GSTs from a range of invertebrates. The open reading frame of the chicken cDNA was expressed in Escherichia coli and the purified protein was found to display high PGDS activity. It also catalysed the conjugation of glutathione with a wide range of aryl halides, organic isothiocyanates and alpha,beta-unsaturated carbonyls, and exhibited glutathione 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 blotting experiments showed that among the organs studied, the expression of 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. Collectively, these results show that the structure and function, but not the expression, of the GSH-requiring PGDS is conserved between chicken and rat.
Thomson
A M
AM
Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, U.K.
Meyer
D J
DJ
Hayes
J D
JD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D002645:Chickens; D018076:DNA, Complementary / Q000378:metabolism; D005260:Female; D005978:Glutathione / Q000378:metabolism*; D019746:Intramolecular Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D054834:Lipocalins; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011487:Protein Conformation; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment
D018076:DNA, Complementary; D054834:Lipocalins; D011994:Recombinant Proteins; D019746:Intramolecular Oxidoreductases; C022466:prostaglandin R2 D-isomerase; D005978:Glutathione