Dai H, Edens F W, Roe R M
Department of Entomology, North Carolina State University, Raleigh 27695-7647, USA.
J Biochem Toxicol. 1996;11(2):85-96. doi: 10.1002/(SICI)1522-7146(1996)11:2<85::AID-JBT6>3.0.CO;2-Q.
Cytosolic glutathione S-transferase (GST) activities toward 1-chloro-2,4-dinitro-benzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (EA), 1,2-epoxy-3-(p-nitrophenoxyl)propane (EPNP), trans-4-phenyl-3-buten-2-one(t-PBO), delta 5-androstene-3,17-dione (ASD) and trans-stilbene oxide (t-SO); cytosolic glutathione peroxidase activity toward cumene hydroperoxide (CuOOH); and microsomal GST activity toward CDNB were examined in liver, kidney, brain, and lung of adult male and female Japanese quail. In all cases, the renal specific activity per milligram protein was higher than the hepatic activity and was the highest among the four tissues examined. No consistent sex differences in GST activity were observed. The GSTs were purified from quail liver cytosol by S-hexylglutathione and glutathione affinity chromatography. Total GSTs eluted from the S-hexylglutathione affinity column were further separated by chromatofocusing, and the microheterogeneity of the GST isozymes was shown by high-resolution native isoelectrofocusing (IEF) in polyacrylamide slab gels and by SDS-PAGE. Five subunits were identified: QL1 (30.5 kDa), QL2 (27.2 kDa), QL3a (26.8 kDa), QL3b (26.5 kDa), and QL4 (25.5 kDa). Western blot analysis revealed that QL1 and QL2 reacted with antibodies raised against the rat Mu class GSTs (Yb1 and Yb2), and QL3a and QL3b reacted with those raised against the Alpha class (rat Ya and mouse a). Substrate specific activity of each isoform was determined with CDNB, DCNB, CuOOH, EA, t-PBO, ASD, and t-SO. QL3a and QL3b have high reactivity toward CuOOH, while QL1 and QL2 showed high activity toward t-SO. The N-terminal amino acid sequence of QL2 was identical to that of the chicken Mu class GST subunit CL2. However, no sequence was obtained with QL1 due to possible N-terminal blockage.
检测了成年雄性和雌性日本鹌鹑肝脏、肾脏、大脑和肺中胞质谷胱甘肽S-转移酶（GST）对1-氯-2,4-二硝基苯（CDNB）、1,2-二氯-4-硝基苯（DCNB）、依他尼酸（EA）、1,2-环氧-3-（对硝基苯氧基）丙烷（EPNP）、反式-4-苯基-3-丁烯-2-酮（t-PBO）、δ5-雄甾烯-3,17-二酮（ASD）和反式氧化芪（t-SO）的活性；胞质谷胱甘肽过氧化物酶对氢过氧化异丙苯（CuOOH）的活性；以及微粒体GST对CDNB的活性。在所有情况下，每毫克蛋白质的肾脏比活性均高于肝脏活性，且在检测的四种组织中是最高的。未观察到GST活性存在一致的性别差异。通过S-己基谷胱甘肽和谷胱甘肽亲和色谱从鹌鹑肝脏胞质溶胶中纯化GST。从S-己基谷胱甘肽亲和柱洗脱的总GST通过色谱聚焦进一步分离，并且GST同工酶的微异质性通过聚丙烯酰胺平板凝胶中的高分辨率天然等电聚焦（IEF）和SDS-PAGE显示。鉴定出五个亚基：QL1（30.5 kDa）、QL2（27.2 kDa）、QL3a（26.8 kDa）、QL3b（26.5 kDa）和QL4（25.5 kDa）。蛋白质印迹分析表明，QL1和QL2与针对大鼠Mu类GST（Yb1和Yb2）产生的抗体反应，QL3a和QL3b与针对Alpha类（大鼠Ya和小鼠a）产生的抗体反应。用CDNB、DCNB、CuOOH、EA、t-PBO、ASD和t-SO测定每种同工型的底物比活性。QL3a和QL3b对CuOOH具有高反应性，而QL1和QL2对t-SO显示出高活性。QL2的N端氨基酸序列与鸡Mu类GST亚基CL2的相同。然而，由于可能的N端封闭，未获得QL1的序列。
