Salvatore L, Wijffels G, Sexton J L, Panaccio M, Mailer S, McCauley I, Spithill T W
Victorian Institute of Animal Science, Attwood, Australia.
Mol Biochem Parasitol. 1995 Feb;69(2):281-8. doi: 10.1016/0166-6851(94)00205-2.
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.
编码肝片吸虫谷胱甘肽S-转移酶（rGST1、rGST7、rGST47和rGST51）的四个cDNA在大肠杆菌中表达，并纯化rGST蛋白用于生化分析。经SDS-PAGE分离的蛋白质考马斯亮蓝染色显示，rGST蛋白纯度达95%。HPLC分子筛分析表明，与天然蛋白一样，rGST蛋白在非变性条件下形成同型二聚体。rGST蛋白可被针对肝片吸虫天然GST产生的抗血清识别。来自肝片吸虫的所有四种rGST蛋白都能将谷胱甘肽与通用底物1-氯-2,4-二硝基苯有效结合。还测定了rGST对已显示对哺乳动物GST的α、μ或π类具有部分特异性的底物（反式-4-苯基-3-丁烯-2-酮、依他尼酸）、已知为脂质过氧化产物的底物（反式-2-壬烯醛、反式，反式-2,4-癸二烯醛）以及环氧-3-（对硝基苯氧基）丙烷（EPNP，已知为θ类GST的底物）的活性。没有rGST对EPNP有活性。rGST47和51对其他四种底物有活性。rGST7对三种底物有活性，而rGST1除了反式，反式-2,4-癸二烯醛外，对所有底物的活性都相对较低。rGST活性对GST抑制剂三苯基氯化锡和溴磺酞的抑制敏感性在不同rGST之间也有所不同，rGST1对这两种抑制剂的敏感性相差800倍。这些结果表明，肝片吸虫表达了一系列具有独特底物和抑制剂谱的GST同工酶。
