Identification of a novel zebrafish SULT1 cytosolic sulfotransferase: cloning, expression, characterization, and developmental expression study.
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 35kDa 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.
Liu
Ming-Yih
MY
Biomedical Research Center, The University of Texas Health Center, Tyler, TX 75708, USA.
Yang
Yuh-Shyong
YS
Sugahara
Takuya
T
Yasuda
Shin
S
Liu
Ming-Cheh
MC
D000595:Amino Acid Sequence; D000818:Animals; D015239:Arylsulfotransferase / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D015870:Gene Expression; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid; D015027:Zebrafish / Q000196:embryology / Q000235:genetics*; D029961:Zebrafish Proteins / Q000096:biosynthesis / Q000235:genetics*
D007527:Isoenzymes; D029961:Zebrafish Proteins; D015239:Arylsulfotransferase; C501250:sult1st4 protein, zebrafish