A novel hydroxysteroid-sulfating cytosolic sulfotransferase, SULT3 ST3, from zebrafish: identification, characterization, and ontogenic study.
To establish the zebrafish as a model for investigating the drug metabolism through sulfation, we had embarked on establishing a complete repertoire of the zebrafish Phase II cytosolic sulfotransferases (SULTs). By searching the expressed sequence tag database, a zebrafish cDNA encoding a putative cytosolic sulfotransferase (SULT) was identified. Based on the sequence analysis, this zebrafish SULT was found to belong to the SULT3 gene family. The recombinant protein of the zebrafish SULT, designated SULT3 ST3, was expressed in and purified from BL21 (DE3) Escherichia coli cells transformed with the pGEX-2TK expression vector harboring SULT3 ST3 cDNA. Upon enzymatic characterization, purified SULT3 ST3 displayed sulfating activity toward hydroxysteroids, particularly pregnenolone and dehydroepiandrosterone (DHEA), as well as several drugs among various endogenous and xenobiotic compounds tested as substrates. The pH-dependence and kinetic constants of this enzyme with DHEA were determined. The regulatory effects of various divalent metal cations on the DHEA-sulfating activity of SULT3 ST3 were quantitatively evaluated. A reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed developmental stage-dependent expression of SULT3 ST3 during embryonic development and throughout the larval stage onto maturity. Collectively, these results suggest a possible involvement of the newly discovered SULT3 ST3 in the metabolism of hydroxysteroids and xenobiotics including drugs in zebrafish.
Yasuda
Shin
S
Department of Pharmacology, College of Pharmacy, The University of Toledo, Toledo, OH 43606, USA.
Burgess
Meredith
M
Yasuda
Tomoko
T
Liu
Ming-Yih
MY
Bhuiyan
Shakhawat
S
Williams
Frederick E
FE
Kurogi
Katsuhisa
K
Sakakibara
Yoichi
Y
Suiko
Masahito
M
Liu
Ming-Cheh
MC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology*; D003687:Dehydroepiandrosterone / Q000378:metabolism; D005260:Female; D018507:Gene Expression Regulation, Developmental; D006863:Hydrogen-Ion Concentration; D006914:Hydroxysteroids / Q000378:metabolism*; D007700:Kinetics; D007814:Larva; D008297:Male; D008969:Molecular Sequence Data; D011284:Pregnenolone / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D015262:Xenobiotics / Q000378:metabolism; D015027:Zebrafish / Q000196:embryology / Q000254:growth & development / Q000378:metabolism*; D029961:Zebrafish Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics
D006914:Hydroxysteroids; D011994:Recombinant Proteins; D015262:Xenobiotics; D029961:Zebrafish Proteins; D003687:Dehydroepiandrosterone; D011284:Pregnenolone; C548300:SULT3 sulfotransferase, zebrafish; D015238:Sulfotransferases