Yasuda Shin, Liu Ming-Yih, Yang Yuh-Shyong, Snow Rhodora, Takahashi Saki, Liu Ming-Cheh
Biomedical Research Center, The University of Texas Health Center, Tyler, TX 75708, USA.
Arch Biochem Biophys. 2006 Nov 1;455(1):1-9. doi: 10.1016/j.abb.2006.09.004. Epub 2006 Sep 25.
By searching the expressed sequence tag database, two zebrafish cDNAs encoding putative cytosolic sulfotransferases (SULTs) were identified. Sequence analysis indicated that these two zebrafish SULTs belong to the cytosolic SULT2 gene family. The recombinant form of these two novel zebrafish SULTs, designated SULT2 ST2 and SULT2 ST3, were expressed using the pGEX-2TK glutathione S-transferase (GST) gene fusion system and purified from transformed BL21 (DE3) Escherichia coli cells. Purified GST-fusion protein form of SULT2 ST2 and SULT2 ST3 exhibited strong sulfating activities toward dehydroepiandrosterone (DHEA) and corticosterone, respectively, among various endogenous compounds tested as substrates. Both enzymes displayed pH optima at approximately 6.5. Kinetic constants of the two enzymes, as well as the GST-fusion protein form of the previously identified SULT2 ST1, with DHEA and corticosterone as substrates were determined. Developmental stage-dependent expression experiments revealed distinct patterns of expression of SULT2 ST2 and SULT2 ST3, as well as the previously identified SULT2 ST1, during embryonic development and throughout the larval stage onto maturity.
通过搜索表达序列标签数据库，鉴定出两个编码假定胞质磺基转移酶（SULTs）的斑马鱼cDNA。序列分析表明，这两种斑马鱼SULTs属于胞质SULT2基因家族。这两种新型斑马鱼SULTs的重组形式，命名为SULT2 ST2和SULT2 ST3，使用pGEX-2TK谷胱甘肽S-转移酶（GST）基因融合系统进行表达，并从转化的BL21（DE3）大肠杆菌细胞中纯化。在作为底物测试的各种内源性化合物中，纯化的SULT2 ST2和SULT2 ST3的GST融合蛋白形式分别对脱氢表雄酮（DHEA）和皮质酮表现出强烈的硫酸化活性。两种酶的最适pH均约为6.5。测定了这两种酶以及先前鉴定的SULT2 ST1的GST融合蛋白形式以DHEA和皮质酮为底物的动力学常数。发育阶段依赖性表达实验揭示了SULT2 ST2和SULT2 ST3以及先前鉴定的SULT2 ST1在胚胎发育期间以及整个幼体阶段直至成熟阶段的不同表达模式。
