Biochemical analysis of recombinant glutathione S-transferase of Fasciola hepatica
| Autor: | Sonia Mailer, Jennifer L. Sexton, L. Salvatore, Terry W. Spithill, Gene Wijffels, Ian McCauley, Michael Panaccio |
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| Rok vydání: | 1995 |
| Předmět: |
Molecular Sequence Data
medicine.disease_cause Isozyme law.invention Substrate Specificity Sulfobromophthalein chemistry.chemical_compound Hepatica law medicine Organotin Compounds Fasciola hepatica Animals Amino Acid Sequence Molecular Biology Escherichia coli DNA Primers Glutathione Transferase Antiserum biology Base Sequence Glutathione DNA Helminth biology.organism_classification Molecular biology Recombinant Proteins Isoenzymes Glutathione S-transferase Biochemistry chemistry Recombinant DNA biology.protein Parasitology |
| Zdroj: | Molecular and biochemical parasitology. 69(2) |
| ISSN: | 0166-6851 |
| Popis: | Four cDNAs encoding GST (rGST1, rGST7, rGST47 and rGST51) of Fasciola hepatica were expressed in Escherichia coli and the rGST proteins purified for biochemical analyses. The rGST proteins are 95% pure as indicated by Coomassie staining of proteins separated by SDS-PAGE. Molecular sieving by HPLC infers that, like the native protein, the rGST proteins form homodimers under non-denaturing conditions. The rGST proteins are recognised by antisera raised to the native GST of F. hepatica. All four rGST proteins from F. hepatica actively conjugate glutathione to the universal substrate, 1-chloro-2,4-dinitrobenzene. The activity of the rGSTs was also measured for substrates which have been shown to have partial specificity for the Alpha, Mu or Pi classes of mammalian GSTs (trans-4-phenyl-3-buten-2-one, ethacrynic acid), for substrates known to be products of lipid peroxidation (trans-2-nonenal, trans,trans-2,4-decadienal) and for epoxy-3-(p-nitrophenoxy)-propane (EPNP), a known substrate for the theta class of GST. No rGST were active with EPNP. rGST47 and 51 showed activity with the other four substrates. rGST7 was active with three substrates whereas rGST1 showed relatively low activity with all substrates except trans,trans-2,4-decadienal. The sensitivity of the rGST activity to inhibition by the GST inhibitors triphenyltin chloride and bromosulphophthalein also varied among the rGSTs with rGST1 showing a 800-fold difference in sensitivity between the inhibitors. These results show that F. hepatica expresses a family of GST isoenzymes which exhibit unique substrate and inhibitor profiles. |
| Databáze: | OpenAIRE |
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