Chang L H, Chuang L F, Tsai C P, Tu C P, Tam M F
Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, Republic of China.
Biochemistry. 1990 Jan 23;29(3):744-50. doi: 10.1021/bi00455a022.
Glutathione S-transferases (GSTs, EC 2.5.1.18) were isolated from the liver cytosolic fraction of 1 day old Leghorn chicks by S-hexylglutathione and glutathione affinity columns arranged in tandem. After sample loading, the affinity columns were detached from each other and developed separately. Four groups of GSTs (CL 1, 2, 3, and 4) were eluted from the hexylglutathione column, and an additional group of GSTs (CL 2 and 5) was eluted from the glutathione affinity column. CL 2, CL 3, and CL 5 were further purified to homogeneity by chromatofocusing, and the substrate specificities of each group were determined. Fractions from the chromatofocusing column were analyzed by native IEF electrophoresis. Protein bands were electroblotted onto PVDF membrane for N-terminal sequence analysis or extracted from IEF gel and rerun on SDS-PAGE to determine the subunit composition of each GST dimer. CL 2, CL 3, and CL 5 can form homodimers, whereas CL 1 and CL 4 exist only as CL 1-2 and CL 3-4 heterodimers. CL 2 and CL 5 have N-terminal amino acid sequences homologous to rat liver Yb and Ya GSTs, respectively. CL 1 has a unique N-terminal sequence that is not homologous to any known GSTs.
谷胱甘肽S-转移酶（GSTs，EC 2.5.1.18）通过串联排列的S-己基谷胱甘肽和谷胱甘肽亲和柱，从1日龄来航鸡的肝脏胞质部分中分离得到。上样后，将亲和柱彼此分离并分别展开。从己基谷胱甘肽柱上洗脱得到四组GSTs（CL 1、2、3和4），从谷胱甘肽亲和柱上洗脱得到另外一组GSTs（CL 2和5）。通过色谱聚焦法将CL 2、CL 3和CL 5进一步纯化至均一，并测定每组的底物特异性。通过天然IEF电泳分析色谱聚焦柱的馏分。将蛋白条带电转印到PVDF膜上进行N端序列分析，或从IEF凝胶中提取并在SDS-PAGE上重新运行以确定每个GST二聚体的亚基组成。CL 2、CL 3和CL 5可以形成同二聚体，而CL 1和CL 4仅以CL 1-2和CL 3-4异二聚体形式存在。CL 2和CL 5的N端氨基酸序列分别与大鼠肝脏Yb和Ya GSTs同源。CL 1具有独特的N端序列，与任何已知的GSTs均不同源。
