Yasuda Shin, Kumar A Pavan, Liu Ming-Yih, Sakakibara Yoichi, Suiko Masahito, Chen Lanzhuang, Liu Ming-Cheh
Biomedical Research Center, The University of Texas Health Center, Tyler 75708, USA.
FEBS J. 2005 Aug;272(15):3828-37. doi: 10.1111/j.1742-4658.2005.04791.x.
By employing RT-PCR in conjunction with 3'-RACE, a full-length cDNA encoding a novel zebrafish cytosolic sulfotransferase (SULT) was cloned and sequenced. Sequence analysis revealed that this zebrafish SULT (designated SULT1 ST5) is, at the amino acid sequence level, close to 50% identical to human and dog SULT1B1 (thyroid hormone SULT). A recombinant form of zebrafish SULT1 ST5 was expressed using the pGEX-2TK bacterial expression system and purified from transformed BL21 (DE3) cells. Purified zebrafish SULT1 ST5 migrated as a 34 kDa protein and displayed substrate specificity for thyroid hormones and their metabolites among various endogenous compounds tested. The enzyme also exhibited sulfating activities toward some xenobiotic phenolic compounds. Its pH optima were 6.0 and 9.0 with 3,3',5-triiodo-l-thyronine (l-T3) as substrate and 6.0 with beta-naphthol as substrate. Kinetic constants of the enzyme with thyroid hormones and their metabolites as substrates were determined. Quantitative evaluation of the regulatory effects of divalent metal cations on the l-T3-sulfating activity of SULT1 ST5 revealed that Fe2+, Hg2+, Co2+, Zn2+, Cu2+, Cd2+ and Pb2+ exhibited dramatic inhibitory effects, whereas Mn2+ showed a significant stimulation. Developmental stage-dependent expression experiments revealed a significant level of expression of this novel zebrafish thyroid hormone-sulfating SULT at the beginning of the hatching period during embryogenesis, which gradually increased to a high level of expression throughout the larval stage into maturity.
通过结合逆转录聚合酶链反应（RT-PCR）和3'-末端快速扩增技术（3'-RACE），克隆并测序了编码一种新型斑马鱼胞质磺基转移酶（SULT）的全长cDNA。序列分析表明，这种斑马鱼SULT（命名为SULT1 ST5）在氨基酸序列水平上与人和狗的SULT1B1（甲状腺激素SULT）的相似度接近50%。使用pGEX-2TK细菌表达系统表达了重组形式的斑马鱼SULT1 ST5，并从转化的BL21（DE3）细胞中进行了纯化。纯化后的斑马鱼SULT1 ST5迁移为一条34 kDa的蛋白条带，在测试的各种内源性化合物中，对甲状腺激素及其代谢产物表现出底物特异性。该酶对一些外源性酚类化合物也表现出硫酸化活性。以3,3',5-三碘-L-甲状腺原氨酸（L-T3）为底物时，其最适pH值为6.0和9.0，以β-萘酚为底物时最适pH值为6.0。测定了该酶以甲状腺激素及其代谢产物为底物时的动力学常数。对二价金属阳离子对SULT1 ST5的L-T3硫酸化活性的调节作用进行定量评估发现，Fe2+、Hg2+、Co2+、Zn2+、Cu2+、Cd2+和Pb2+表现出显著的抑制作用，而Mn2+则表现出显著的刺激作用。发育阶段依赖性表达实验表明，这种新型斑马鱼甲状腺激素硫酸化SULT在胚胎发育的孵化期开始时表达水平显著，在整个幼体阶段直至成熟逐渐增加到高水平表达。
