Sulfation of indoxyl by human and rat aryl (phenol) sulfotransferases to form indoxyl sulfate
Sulfation of indoxyl by human and rat aryl (phenol) sulfotransferases to form indoxyl sulfate
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DOI：
10.1007/bf03190428
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发表时间：
2002-04-01
影响因子：
1.9
通讯作者：
King, RS
中科院分区：
文献类型：
--
作者：
Banoglu, E;King, RS
本研究的目的是鉴定由吲哚基（3-羟基吲哚）生成硫酸吲哚基的硫转移酶（SULT）异构体。吲哚酚与共底物3′-磷酸腺苷5′-磷酸硫酸酯（PAPS）和人或大鼠肝细胞质或重组硫转移酶一起孵育。用高效液相色谱法测定了吲哚酚生成硫酸吲哚酚的情况，并测定了磺化率。两种细胞质均磺化吲哚酚，人细胞质的表观K-m值为6.8 +/- 0.9 muM，大鼠细胞质的表观K-m值为3.2 +/- 0.6 muM。为了帮助鉴定硫酸吲哚酚的异构体，将硫酸吲哚酚与人类和大鼠的肝细胞溶胶和PAPS一起在异构体特异性SULT抑制剂的存在下孵育。脱氢表雄酮（一种特定的羟基类固醇硫转移酶抑制剂）和雌酮（一种雌激素硫转移酶抑制剂）均未观察到抑制作用。然而，芳基（苯酚）硫转移酶抑制剂2,6-二氯-4-硝基苯酚（DCNP）抑制硫酸吲哚酚的形成，其IC50值对人为3.2 muM，对大鼠细胞质为1.0 muM，表明人和大鼠芳基（苯酚）硫转移酶负责硫酸吲哚酚的形成。吲哚酚与人重组芳基SULT （SULT1A1*2）孵育后，表观K-m值为5.6 +/- 1.8 muM。对人和大鼠细胞质和人重组SULT1A1*2的动力学研究得出了相似的动力学值，表明人和大鼠芳香基磺基转移酶能有效催化硫酸吲哚酚的形成，这是一种重要的尿毒症毒素代谢物。
The aim of this study was to identify sulfotransferase (SULT) isoform(s) responsible for the formation of indoxyl sulfate from indoxyl (3-hydroxyindole). Indoxyl was incubated together with the co-substrate 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and either human or rat liver cytosol or recombinant sulfotransferase enzymes. Formation of indoxyl sulfate from indoxyl was measured by HPLC and used for determination of sulfonation rates. Both cytosols sulfonated indoxyl with apparent K-m values of 6.8 +/- 0.9 muM for human and 3.2 +/- 0.6 muM for rat cytosol. To help identify the isoform(s) of SULT responsible for indoxyl sulfate formation, indoxyl was incubated with human and rat liver cytosols and PAPS in the presence of isoform-specific SULT inhibitors. No inhibition was observed by DHEA, a specific hydroxysteroid sulfotransferase inhibitor, nor by oestrone, an inhibitor of oestrogen sulfotransferase. However, an aryl (phenol) sulfotransferase inhibitor, 2,6-dichloro-4-nitrophenol (DCNP), inhibited the formation of indoxyl sulfate with a IC50 values of 3.2 muM for human and 1.0 muM for rat cytosol indicating that human and rat aryl (phenol) sulfotransferases are responsible for the formation of indoxyl sulfate. When indoxyl was incubated with SULT1A1*2, a human recombinant aryl SULT, an apparent K-m value of 5.6 +/- 1.8 muM was obtained. Kinetic studies with human and rat cytosols and human recombinant SULT1A1*2 gave similar kinetic values indicating that human and rat aryl sulfotransferases efficiently catalyze the formation of indoxyl sulfate, an important uremic toxin metabolite.