Bartolone J B, Birge R B, Sparks K, Cohen S D, Khairallah E A
Department of Molecular and Cell Biology, University of Connecticut, Storrs 06268.
Biochem Pharmacol. 1988 Dec 15;37(24):4763-74. doi: 10.1016/0006-2952(88)90350-4.
A sensitive immunoassay for detecting acetaminophen (APAP) bound to proteins was developed using an affinity purified antibody directed against the N-acetylated end of the APAP molecule. Western blots of electrophoretically resolved liver proteins taken from mice given an hepatotoxic dose of APAP demonstrated that nearly 85% of the total detectable protein-bound APAP was covalently associated with proteins of 44 and 58 kD. Pretreatment of liver extracts with the sulfhydryl-specific reagent, N-ethylmaleimide (NEM), prior to derivatization with the reactive metabolite of APAP, N-acetyl-p-benzoquinone imine (NAPQI), greatly reduced immunochemically detectable APAP-protein adducts and indicated that the antibody detects protein-thiol conjugates of APAP. To investigate the basis of the binding selectivity in vivo, a variety of systems which yielded APAP-protein adducts were analyzed. Systems which activate APAP enzymatically, as in hepatocyte suspensions or in post-mitochondrial (S9) fractions fortified with an NADPH-regenerating system, resulted in a protein binding profile similar to that produced in vivo. Conversely, when extracts or cells were treated with chemically synthesized NAPQI, an alternative protein binding profile was obtained. Two-dimensional electrophoretic analysis of the reduced protein thiol (PSH) content of liver proteins using [3H]NEM labeling revealed that the 58 kD APAP-binding proteins were rich in PSH, whereas the major 44 kD binding protein had virtually no detectable PSH. Many PSH-rich proteins that were not arylated in vivo did bind NAPQI in vitro. However, the 44 kD proteins were not arylated when chemically synthesized NAPQI was added to homogenates or cell suspensions. The present data further suggest that, in addition to the amount and reactivity of free protein sulfhydryls, the cellular localization with respect to the cytochrome P-450 activation site may influence the susceptibility of proteins to NAPQI binding. These findings signal the need for caution in interpreting studies of APAP mechanisms that rely solely on NAPQI addition.
利用针对对乙酰氨基酚（APAP）分子N - 乙酰化末端的亲和纯化抗体，开发了一种用于检测与蛋白质结合的APAP的灵敏免疫测定法。对给予肝毒性剂量APAP的小鼠肝脏蛋白质进行电泳分离后进行的蛋白质免疫印迹分析表明，在可检测到的与蛋白质结合的APAP总量中，近85%与44 kD和58 kD的蛋白质共价结合。在用APAP的活性代谢产物N - 乙酰 - p - 苯醌亚胺（NAPQI）进行衍生化之前，先用巯基特异性试剂N - 乙基马来酰亚胺（NEM）处理肝脏提取物，可大大减少免疫化学可检测到的APAP - 蛋白质加合物，这表明该抗体可检测APAP的蛋白质 - 硫醇缀合物。为了研究体内结合选择性的基础，分析了多种产生APAP - 蛋白质加合物的系统。像在肝细胞悬液中或用NADPH再生系统强化的线粒体后（S9）组分中那样通过酶促激活APAP的系统，产生的蛋白质结合谱与体内产生的相似。相反，当用化学合成的NAPQI处理提取物或细胞时，会获得另一种蛋白质结合谱。使用[³H]NEM标记对肝脏蛋白质的还原型蛋白质硫醇（PSH）含量进行二维电泳分析表明，58 kD的APAP结合蛋白富含PSH，而主要的44 kD结合蛋白几乎没有可检测到的PSH。许多在体内未被芳基化的富含PSH的蛋白质在体外确实能与NAPQI结合。然而，当将化学合成的NAPQI添加到匀浆或细胞悬液中时，44 kD的蛋白质并未被芳基化。目前的数据进一步表明，除了游离蛋白质巯基的数量和反应性外，相对于细胞色素P - 450激活位点的细胞定位可能会影响蛋白质对NAPQI结合的敏感性。这些发现表明，在解释仅依赖添加NAPQI的APAP机制研究时需要谨慎。
