一种简单灵敏的腺苷酰硫酸激酶（APS-激酶）活性荧光测定法。

A simple sensitive fluorimetric assay of APS-kinase activity.

作者信息

Wong K P

机构信息

Department of Biochemistry, Faculty of Medicine, National University of Singapore, Kent Ridge.

出版信息

Biochem Pharmacol. 1990 Jan 1;39(1):173-9. doi: 10.1016/0006-2952(90)90662-5.

DOI:10.1016/0006-2952(90)90662-5

PMID:2153377

Abstract

Adenosine phosphosulphokinase (APS-kinase or ATP:adenylylsulphate 3'-phosphotransferase; EC 2.7.1.25) catalyses the formation of 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Its activity in various tissues was measured by transferring the sulphate from PAPS, a product of APS-kinase reaction, to 4-methylumbelliferone (4-MU) to form 4-MU-sulphate (4-MUS) using phenolsulphotransferase (PST) extracted from rat liver. Desalting with Sephadex G-25, together with the addition of EDTA effectively removed the Mg2+ ions from the rat liver extract and thereby inhibited the APS-kinase activity therein in the subsequent PST reaction. 4-MUS formed was measured indirectly by a decrease in the fluorescence of 4-MU by a continuous fluorimetric assay. Kinetic data showed that the substrate, APS, at concentrations at and above 132 microM inhibited the APS kinase reaction. Pyrophosphate (PP) also inhibited the reaction. The apparent Km for APS was 14 microM. Two apparent Km values of 0.12 mM and 1.06 mM were obtained for ATP, while that for Mg2+ was 0.09 mM.

摘要

腺苷磷酸硫酸激酶（APS激酶或ATP：腺苷硫酸3'-磷酸转移酶；EC 2.7.1.25）催化生成3'-磷酸腺苷-5'-磷酸硫酸酯（PAPS）。通过使用从大鼠肝脏中提取的酚硫酸转移酶（PST），将APS激酶反应产物PAPS中的硫酸转移至4-甲基伞形酮（4-MU）以形成4-甲基伞形酮硫酸酯（4-MUS），来测定其在各种组织中的活性。用葡聚糖凝胶G-25脱盐，同时添加EDTA可有效去除大鼠肝脏提取物中的Mg2+离子，从而在随后的PST反应中抑制其中的APS激酶活性。通过连续荧光测定法间接测量形成的4-MUS，方法是通过4-MU荧光的降低来进行。动力学数据表明，底物APS在浓度达到及高于132 microM时会抑制APS激酶反应。焦磷酸（PP）也会抑制该反应。APS的表观Km为14 microM。ATP的两个表观Km值分别为0.12 mM和1.06 mM，而Mg2+的表观Km值为0.09 mM。