Sugahara K, Ishii T, Yamashina I
Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Japan.
Anal Biochem. 1987 Nov 1;166(2):404-12. doi: 10.1016/0003-2697(87)90591-4.
A method is described for the assay of sulfotransferases, which transfer sulfate from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to glycosaminoglycan acceptors. Following the sulfation reactions, the [35S]sulfate-labeled products are precipitated and then separated from a sulfate donor ([35S]PAPS) and its degradation products by a paper disk method, and then the radioactivity remaining on the paper disk is subsequently determined by liquid scintillation counting. The rapidity and simplicity of the method are advantageous for multiple assays and have allowed us to establish assay conditions for serum sulfotransferases which introduce sulfate at position 6 of the internal N-acetylgalactosamine units of chondroitin, position 2 (amino group) of the glucosamine units of heparan sulfate and sugar units of keratan sulfate, respectively. The assay method will be applicable with modification to the assay of other glycosaminoglycan sulfotransferases and glycoprotein sulfotransferases.
本文描述了一种硫酸转移酶的测定方法，该酶可将硫酸从3'-磷酸腺苷-5'-磷酸硫酸酯（PAPS）转移至糖胺聚糖受体。硫酸化反应后，将[35S]硫酸标记的产物沉淀，然后通过纸盘法将其与硫酸供体（[35S]PAPS）及其降解产物分离，随后通过液体闪烁计数法测定残留在纸盘上的放射性。该方法的快速性和简便性有利于进行多次测定，并使我们能够建立血清硫酸转移酶的测定条件，这些酶分别在软骨素内部N-乙酰半乳糖胺单元的6位、硫酸乙酰肝素葡萄糖胺单元的2位（氨基）和硫酸角质素糖单元上引入硫酸。该测定方法经修改后可适用于其他糖胺聚糖硫酸转移酶和糖蛋白硫酸转移酶的测定。
