Molecular cloning, expression, and functional characterization of a novel zebrafish cytosolic sulfotransferase.
By searching the zebrafish expressed sequence tag (EST) database, we have identified a cDNA clone encoding a putative zebrafish cytosolic sulfotransferase (ST). This cDNA was isolated and subjected to nucleotide sequencing. Analysis of the sequence data revealed that this novel zebrafish ST displays 32-35% amino acid sequence identity to members of all major cytosolic ST gene families. Therefore, this zebrafish ST, while belonging to the cytosolic ST gene superfamily, appears to be independent from all known constituent ST gene families. Recombinant zebrafish ST, expressed using the pET23c prokaryotic expression vector and purified from transformed Escherichia coli cells, migrated as a 34-kDa protein upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified zebrafish ST displayed sulfating activities toward dopamine and thyroid hormones (T(3) and T(4)), with a pH optimum spanning 7-9. The enzyme also exhibited activities toward a number of xenobiotics including some flavonoids, isoflavonoids, and other phenolic compounds. A thermostability experiment revealed the enzyme to be relatively stable over a temperature range between 20 and 48 degrees C. Among 10 divalent metal cations tested, Fe(++), Hg(++), Co(++), Zn(++), Cu(++), and Cd(++) exhibited dramatic inhibitory effects on the activity of the enzyme. These results constitute a first study on the cloning, expression, and characterization of a zebrafish cytosolic ST.
Sugahara
Takuya
T
Biomedical Research Center, The University of Texas Health Center, 11937 US Highway 271, Tyler, TX 75708, USA.
Liu
Chau Ching
CC
Govind Pai
T
T
Liu
Ming Cheh
MC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D013696:Temperature; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*
D002413:Cations, Divalent; D029961:Zebrafish Proteins; D015238:Sulfotransferases