By searching the zebrafish EST (expressed-sequence tag) database, we have identified two partial cDNA clones encoding the 5′ and 3′ regions of a putative zebrafish sulphotransferase (ST). Using the reverse transcription-PCR technique, a full-length cDNA encoding this zebrafish ST was successfully cloned. Sequence analysis revealed that this novel zebrafish ST displays 44%, 43% and 40% amino acid identity with mouse SULT2B1, human SULT2B1b and human SULT2A1 ST respectively. This zebrafish ST therefore appears to belong to the SULT2 cytosolic ST gene family. Recombinant zebrafish ST, expressed using the pGEX-2TK prokaryotic expression system and purified from transformed Escherichia coli cells, migrated as a 34 kDa protein upon SDS/PAGE. Purified zebrafish ST displayed a strong sulphonating activity toward DHEA (dehydroepiandrosterone), with a optimum pH of 9.5. The enzyme also exhibited activities toward several neurosteroids with differential Km and Vmax values. A thermostability experiment revealed the enzyme to be relatively stable over a temperature range between 20 °C and 43 °C. Among ten different divalent metal cations tested, Fe2+ and Cd2+ exhibited small, but significant, stimulatory effects, whereas Hg2+ and Cu2+ displayed considerably stronger inhibitory effects on the DHEA-sulphonating activity of the enzyme. These results constitute the first study on the molecular cloning, expression, and characterization of a zebrafish cytosolic SULT2 ST.
Abbreviations used: Caps, 2-(N-cyclohexylamino)-1-propanesulphonic acid; Ches, 2-(N-cyclohexylamino)ethane sulphonic acid; DHEA, dehydroepiandrosterone; DHEA-S, sulphate-conjugated DHEA; EST, expressed-sequence tag; GST, glutathione S-transferase; HSST, hydroxysteroid ST; IPTG, isopropyl β-d-thiogalactoside; PAPS, 3′-phosphoadenosine 5′-phosphosulphate; PST, phenol ST; RT-PCR, reverse transcription-PCR; ST, sulphotransferase; T3, 3,3′,5-tri-iodo-l-thyronine.
The nucleotide sequence data reported for zebrafish sulphotransferase has been submitted to the DDBJ, EMBL, GenBank® and GSDB Nucleotide Sequence Databases under the accession number AY181063.