Identification of a novel zebrafish SULTI cytosolic sulfotransferase: Cloning, expression, characterization, and developmental expression study
Identification of a novel zebrafish SULTI cytosolic sulfotransferase: Cloning, expression, characterization, and developmental expression study
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DOI：
10.1016/j.abb.2005.02.029
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发表时间：
2005-05-01
影响因子：
3.9
通讯作者：
Liu, MC
中科院分区：
文献类型：
--
作者：
Liu, MY;Yang, YS;Liu, MC
通过对斑马鱼表达序列标签数据库的检索，我们鉴定了两个编码细胞溶质磺基转移酶（SULT）5 '和3 '区的部分cDNA克隆。利用逆转录-聚合酶链反应（RT-PCR）技术，扩增、克隆和测序了编码该斑马鱼SULT的全长cDNA。序列数据的分析显示，这种新的斑马鱼SULT显示与人SULT 1A 1、小鼠SULT 1D 1和大鼠SULT 1C 1具有49%、46%和45%的氨基酸序列同一性。因此，该斑马鱼SULT似乎属于SULT 1胞质SULT基因家族。重组斑马鱼SULT（命名为SULT 1亚型4），使用pGEX-2 TK原核表达载体表达，并从转化的大肠杆菌细胞纯化，迁移作为一个35 kDa的蛋白十二烷基硫酸钠-聚丙烯酰胺凝胶电泳。在作为底物测试的内源性化合物中，纯化的SULT 1同种型4显示出对甲状腺激素、雌酮的显著硫酸化活性。和脱氢表雄酮。该酶还显示出对一些异生物质的活性，包括一些黄酮类化合物，黄酮类化合物和其他酚类化合物，最适pH为7.0。热稳定性实验表明，该酶在28至37摄氏度的温度范围内相对稳定。在10种二价金属离子中，Fe ~（2+）、Hg ~（2+）、Co ~（2+）、Zn ~（2+）、Cu ~（2+）和Cd ~（2+）对该酶活性有明显的抑制作用。利用RT-PCR对斑马鱼SULT 1亚型4的发育表达进行了研究，结果表明，斑马鱼SULT 1亚型4在胚胎发育过程中的分节期表达水平较低，而在整个仔鱼期逐渐升高，直至成熟。(c)2005年爱思唯尔公司All rights reserved.
By searching the zebrafish expressed sequence tag database, we had identified two partial cDNA clones encoding the 5 '- and 3 ' regions of a putative cytosolic sulfotransferase (SULT). Using the reverse transcription-polymerase chain reaction (RT-PCR) technique, a full-length cDNA encoding this zebrafish SULT was amplified, cloned, and sequenced. Analysis of the sequence data revealed that this novel zebrafish SULT displays 49, 46, and 45% amino acid sequence identity to human SULT1A1, mouse SULT1D1, and rat SULT1C1 This zebrafish SULT therefore appears to belong to the SULT1 cytosolic SULT gene family. Recombinant zebrafish SULT (designated SULT1 isoform 4), expressed using the pGEX-2TK prokaryotic expression vector and purified from transformed Escherichia coli cells, migrated as a 35 kDa protein upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Among the endogenous compounds tested as substrates, the purified SULT1 isoform 4 displayed significant sulfating activities toward thyroid hormones, estrone.. and dehydroepiandrosterone. The enzyme also showed activities toward a number of xenobiotics including some flavonoids, isoflavonoids, and other phenolic compounds, with a pH optimum at 7.0. A thermostability experiment revealed the enzyme to be relatively stable over a temperature range between 28 and 37 degrees C. Among 10 divalent metal cations tested, Fe2+ Hg2+, Co2+ Zn2+ Cu2+, and Cd2+ exhibited dramatic inhibitory effects on the activity of the enzyme. Developmental expression study using RT-PCR revealed that the zebrafish SULT1 isoform 4 showed a low level of expression in the segmentation period during the embryonic development, which gradually increased to a high level of expression throughout the larval stage onto maturity. (c) 2005 Elsevier Inc. All rights reserved.