A continuous assay for the spectrophotometric analysis of sulfotransferases using aryl sulfotransferase IV
A continuous assay for the spectrophotometric analysis of sulfotransferases using aryl sulfotransferase IV
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DOI：
10.1006/abio.1999.4264
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发表时间：
1999-10-01
影响因子：
2.9
通讯作者：
Wong, CH
中科院分区：
文献类型：
--
作者：
Burkart, MD;Wong, CH
我们开发了一种用于磺基转移酶活性的连续分光光度偶联酶测定方法。该测定基于使用对硝基苯硫酸盐作为硫酸盐供体和酶转换的可见分光光度指示剂，通过重组芳基磺基转移酶从脱硫的 3'-磷酸腺苷-5'-磷酸 (PAP) 再生 3'-磷酸腺苷-5'-磷酸硫酸盐 (PAPS)。在此表达重组大鼠芳基磺基转移酶 IV (AST-IV)，在纯化过程中解析为纯 β 型，并用于再生。通过毛细管区带电泳证明了 β AST-IV 催化 PAP 和对硝基苯硫酸盐合成 PAPS 的活性，并计算了这种逆生理反应的动力学。然后将 beta AST-IV 应用于偶联酶系统，其中通过酶浓度研究和 N-壳糖底物特异性测定来验证市售 Nod 因子磺基转移酶的稳态活性。该测定的潜在应用包括碳水化合物和蛋白质磺基转移酶的快速动力学测定、潜在磺基转移酶底物和抑制剂的高通量筛选以及血液样本和其他组织的特定磺基转移酶活性和底物浓度的生物医学筛选。 (C) 1999 年学术出版社。
We have developed a continuous spectrophotometric coupled-enzyme assay for sulfotransferase activity. This assay is based on the regeneration of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) from the desulfated 3'-phosphoadenosine-5'-phosphate (PAP) by a recombinant aryl sulfotransferase using p-nitrophenyl sulfate as the sulfate donor and visible spectrophotometric indicator of enzyme turnover. Here recombinant rat aryl sulfotransferase IV (AST-IV) is expressed, resolved to the pure beta-form during purification, and utilized for the regeneration. The activity of beta AST-IV to catalyze the synthesis of PAPS from PAP and p-nitrophenyl sulfate is demonstrated via capillary zone electrophoresis, and the kinetics of this reverse-physiological reaction are calculated. beta AST-IV is then applied to the coupled enzyme system, where the steady-state activity of the commercially available Nod factor sulfotransferase is verified with an enzyme concentration study and substrate-specificity assays of N-chitoses. The potential applications of this assay include rapid kinetic determinations for carbohydrate and protein sulfotransferases, high-throughput screening of potential sulfotransferase substrates and inhibitors, and biomedical screening of blood samples and other tissues for specific sulfotransferase enzyme activity and substrate concentration. (C) 1999 Academic Press.