Mitchell A E, Morin D, Lamé M W, Jones A D
Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis 95616-8597, USA.
Chem Res Toxicol. 1995 Dec;8(8):1054-62. doi: 10.1021/tx00050a009.
Three cytosolic glutathione S-transferase (GST, EC 2.5.1.18) isozymes were purified from livers of male CD-1 mice to homogeneity using affinity chromatography and gradient elution. Isozyme molecular masses and purities were determined using electrospray ionization mass spectrometry (ESI/MS), HPLC, and electrophoretic methods. Isozymes were assigned to alpha, pi, and mu classes based on kinetic and electrophoretic properties. Molecular masses determined by mass spectrometry were 25 271.4 +/- 2, 23 478.8 +/- 2, and 25 838.4 +/- 2 Da for alpha, pi, and mu isozymes, respectively. Molecular masses for pi and mu class isozymes agreed with molecular masses derived from nucleic acid sequences, but the molecular mass of the alpha class isozyme was 42 Da greater than calculated for a reported sequence. Analysis of tryptic digests of GST-alpha using tandem mass spectrometry determined that the molecular weight discrepancy could be attributed to N-terminal acetylation. Effects of covalent attachment of 4-hydroxy-2-nonenal (HNE) on catalytic activities of each GST isozyme were investigated by incubating GSTs with HNE. The extent of HNE-protein adduct formation was determined using ESI/MS. Catalytic activity of HNE-modified GSTs toward CDNB was measured. ESI mass spectra of modified enzymes show that the extent of HNE modification to the isozymes (pi > alpha > mu) coincides with the loss of activity seen for each protein. ESI mass spectra also indicated that glutathione-protein adducts form in isozymes during incubations with HNE even though purified proteins had been dialyzed against water before incubations. Glutathionylation of isozymes increased as the concentration of HNE in the incubations was raised. Dialysis of GSTs against 2 mM S-hexylglutathione (to displace GSH) before incubations eliminated S-glutathionylation. Reactivities of these GSTs toward HNE differed and is related to the number of cysteine residues (alpha < mu < pi) within the amino acid sequence.
使用亲和色谱法和梯度洗脱从雄性CD-1小鼠肝脏中纯化出三种胞质谷胱甘肽S-转移酶（GST，EC 2.5.1.18）同工酶，使其达到同质状态。使用电喷雾电离质谱法（ESI/MS）、高效液相色谱法（HPLC）和电泳方法测定同工酶的分子量和纯度。根据动力学和电泳特性将同工酶分为α、π和μ类。通过质谱法测定的α、π和μ同工酶的分子量分别为25271.4±2、23478.8±2和25838.4±2 Da。π和μ类同工酶的分子量与从核酸序列推导的分子量一致，但α类同工酶的分子量比报道序列计算值大42 Da。使用串联质谱法分析GST-α的胰蛋白酶消化产物，确定分子量差异可归因于N端乙酰化。通过将GST与4-羟基-2-壬烯醛（HNE）孵育，研究了HNE的共价连接对每种GST同工酶催化活性的影响。使用ESI/MS测定HNE-蛋白质加合物形成的程度。测量了HNE修饰的GST对CDNB的催化活性。修饰酶的ESI质谱表明，HNE对同工酶的修饰程度（π>α>μ）与每种蛋白质活性丧失情况一致。ESI质谱还表明，即使纯化的蛋白质在孵育前已用水透析，在与HNE孵育期间同工酶中仍会形成谷胱甘肽-蛋白质加合物。随着孵育中HNE浓度的升高，同工酶的谷胱甘肽化增加。孵育前将GST用2 mM S-己基谷胱甘肽透析（以置换GSH）可消除S-谷胱甘肽化。这些GST对HNE的反应性不同，且与氨基酸序列中半胱氨酸残基的数量有关（α<μ<π）。
