Bohets H H, Nouwen E J, De Broe M E, Dierickx P J
I.H.E., Afdeling Toxicologie, Brussel, Belgium.
Biochim Biophys Acta. 1996 Apr 24;1311(2):93-101. doi: 10.1016/0167-4889(95)00200-6.
Cytosolic glutathione S-transferase (GST) (EC 2.5.1.18) isoenzymes of dog kidney and MDCK (an established dog renal cell line) were purified and studied. Specific GST activity was 248 and 317 nmol/min/mg protein, for dog and MDCK, respectively. Cytosolic GST was only partially purified by glutathione affinity chromatography, a substantial amount (43% and 84% for dog kidney and MDCK, respectively) of the GST activity was found in the flow-through fraction. Affinity bound GST was separated into 6 and 3 isoenzymes by anionic chromatofocusing for dog and MDCK, respectively. Flow-through GST was purified by gel filtration, anion exchange chromatography and anionic chromatofocusing showing only one GST isoenzyme, with distinct features from the affinity bound GST, for both dog and MDCK. The isoenzymes were characterized by their kinetic properties, subunit composition, specific substrates and inhibitors and immunoblot. The major dog GSTs (DII, DIV and DVI) correspond to the MDCK isoenzymes (MI, MII and MIII). Comparable pI values, a comparable affinity towards GSH and comparable sensitivities towards the inhibitors N-ethylmaleimide (NEM), triphenyltin chloride, cibacron blue and hematin were observed for the corresponding isoenzymes: DII and MI, DIV and MII, DVI and MIII. Co-electrophoresis showed that the subunit composition was identical for DII and MI, and for DIV and MII. Inhibitor and substrate sensitivities showed that the affinity bound GSTs belong to class pi and mu, the presence of class pi was confirmed by immunoblot analysis. One homodimeric GST isoenzyme was observed in the dog kidney and MDCK flow-through. Both dog and MDCK isoenzyme have a nearly neutral pI, a high affinity towards CDNB and an equal sensitivity towards triphenyltin chloride, cibacron blue and hematin. However, based on inhibitor studies and immunoblot, this isoenzyme could not be attributed to an identified GST class. The overall isoenzyme pattern of dog and MDCK affinity bound and flow through GST is comparable. The dog and MDCK affinity bound GSTs have similar characteristics and all belong to class mu or pi.
对犬肾和MDCK（一种已建立的犬肾细胞系）的胞质谷胱甘肽S-转移酶（GST）（EC 2.5.1.18）同工酶进行了纯化和研究。犬和MDCK的GST比活性分别为248和317 nmol/分钟/毫克蛋白。胞质GST仅通过谷胱甘肽亲和层析进行了部分纯化，在流出组分中发现了大量的GST活性（犬肾和MDCK分别为43%和84%）。通过阴离子色谱聚焦法，将亲和结合的GST分别分离为犬的6种同工酶和MDCK的3种同工酶。通过凝胶过滤、阴离子交换层析和阴离子色谱聚焦法对流出的GST进行纯化，结果表明犬和MDCK的流出GST均仅有一种同工酶，其特征与亲和结合的GST不同。通过动力学性质、亚基组成、特异性底物和抑制剂以及免疫印迹对这些同工酶进行了表征。犬的主要GST（DII、DIV和DVI）与MDCK的同工酶（MI、MII和MIII）相对应。对于相应的同工酶：DII和MI、DIV和MII、DVI和MIII，观察到了可比的pI值、对谷胱甘肽的可比亲和力以及对抑制剂N-乙基马来酰亚胺（NEM）、三苯基氯化锡、汽巴克隆蓝和血红素的可比敏感性。共电泳表明DII和MI以及DIV和MII的亚基组成相同。抑制剂和底物敏感性表明亲和结合的GST属于π类和μ类，免疫印迹分析证实了π类的存在。在犬肾和MDCK的流出物中观察到一种同二聚体GST同工酶。犬和MDCK的同工酶均具有接近中性的pI、对1-氯-2,4-二硝基苯（CDNB）的高亲和力以及对三苯基氯化锡、汽巴克隆蓝和血红素的同等敏感性。然而，基于抑制剂研究和免疫印迹，这种同工酶不能归为已确定的GST类别。犬和MDCK亲和结合及流出的GST的总体同工酶模式具有可比性。犬和MDCK亲和结合的GST具有相似的特征，均属于μ类或π类。
