STRUCTURAL AND FUNCTIONAL-ANALYSIS OF A GLUTATHIONE-S-TRANSFERASE FROM ASCARIS-SUUM

A recombinant glutathione S-transferase (GST) (EC 2.5.1.18) from the p arasitic nematode Ascaris suum (AsGST1) displays specific activity wit h a variety of model substrates and secondary products of lipid peroxi dation. The AsGST1 interacts with a range of model inhibitors, haemati n-related compounds, bile acids and anthelminthics. The reported varia tions in biochemical activity correlate with structural differences ob served by homology modelling. Here, differences in the topography of t he proposed substrate binding site between the AsGST1 and the host GST s were identified. A rabbit polyclonal antiserum was raised against th e glutathione-binding proteins of A. suum and specific antibodies agai nst AsGST1 were affinity-purified using the recombinant protein. These antibodies were used to localize the AsGST1 in adult worms by immunoh istochemical staining. The strongest immunostaining for AsGST1 was loc alized in the intestine in all worms examined. This suggests that the enzyme may be responsible for the metabolism of materials that are inc orporated from the environment, as well as for molecules that are excr eted or secreted from the parasite to the environment. It also demonst rates the accessibility of the enzyme to an inhibitor or blocking anti body. In addition, the structure and sequence of the gene encoding AsG ST1 have been determined. Southern-blot analyses of the AscST1 gene su ggests that it is a single-copy gene. The nucleotide sequence analysis revealed that the gene is composed of four exons and three introns, a nd potential regulatory elements were identified in the 5' flanking se quence.