Otterness D M, Powers S P, Miller L J, Weinshilboum R M
Department of Pharmacology, Mayo Clinic/Mayo Foundation, Rochester, Minnesota 55905.
Mol Pharmacol. 1991 Jan;39(1):34-41.
Sulfation is an important pathway in the biotransformation of many drugs, xenobiotic compounds, neurotransmitters, and hormones. The sulfate donor for these reactions is 3'-phosphoadenosine-5'-phosphosulfate (PAPS). We set out to determine whether PAPS might serve as a photoaffinity ligand for sulfotransferase enzymes. UV irradiation of [35S]PAPS with partially purified human liver thermostable (TS) phenol sulfotransferase (PST) radioactively labeled a protein with a molecular mass of 35 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Photoaffinity labeling of TS PST with [35S] PAPS did not require the presence of a phenolic substrate but rather was inhibited by p-nitrophenol, a sulfate acceptor substrate for TS PST. Inhibitors of TS PST enzymatic activity, including 3'-phosphoadenosine-5'-phosphate, ATP, ADP, and 2,6-dichloro-4-nitrophenol, also inhibited photoaffinity labeling of the 35-kDa protein with [35S]PAPS, in a concentration-dependent fashion, with IC50 values of 14 microM, 2.1 mM, 7.7 mM, and 91 microM, respectively. The 35-kDa protein that was radioactively labeled by [35S]PAPS in the presence of UV light coeluted with TS PST enzymatic activity during gel filtration high performance liquid chromatography. [35S]PAPS was then used to photoaffinity label another sulfotransferase enzyme, the thermolabile (TL) form of PST partially purified from human liver. Therefore, [35S]PAPS appears to be a photoaffinity ligand that could be used to study a variety of PAPS-dependent sulfotransferases. Photoaffinity labeling of TS and TL PST, as well as other PAPS-dependent sulfotransferases, should enhance our ability to purify this important group of enzymes and to determine amino acid sequences at or near their active sites.
硫酸化是许多药物、外源性化合物、神经递质和激素生物转化的重要途径。这些反应的硫酸供体是3'-磷酸腺苷-5'-磷酸硫酸酯（PAPS）。我们着手确定PAPS是否可作为磺基转移酶的光亲和配体。用部分纯化的人肝热稳定（TS）酚磺基转移酶（PST）对[35S]PAPS进行紫外线照射，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳估计，放射性标记了一种分子量为35 kDa的蛋白质。用[35S]PAPS对TS PST进行光亲和标记不需要酚类底物存在，但可被对硝基苯酚抑制，对硝基苯酚是TS PST的硫酸受体底物。TS PST酶活性的抑制剂，包括3'-磷酸腺苷-5'-磷酸、ATP、ADP和2,6-二氯-4-硝基苯酚，也以浓度依赖的方式抑制[35S]PAPS对35 kDa蛋白质的光亲和标记，IC50值分别为14 μM、2.1 mM、7.7 mM和91 μM。在紫外线存在下被[35S]PAPS放射性标记的35 kDa蛋白质在凝胶过滤高效液相色谱过程中与TS PST酶活性共洗脱。然后用[35S]PAPS对另一种磺基转移酶进行光亲和标记，即从人肝中部分纯化的PST的热不稳定（TL）形式。因此，[35S]PAPS似乎是一种可用于研究多种PAPS依赖性磺基转移酶的光亲和配体。对TS和TL PST以及其他PAPS依赖性磺基转移酶进行光亲和标记，应能增强我们纯化这一重要酶类并确定其活性位点或附近氨基酸序列的能力。
