PUBLICATION
Sulphonation of dehydroepiandrosterone and neurosteroids: molecular cloning, expression, and functional characterization of a novel zebrafish SULT2Cytosolic Sulphotransferase
- Authors
- Sugahara, T., Yang, Y.-S., Liu, C.-C., Pai, G.-T., and Liu, M.-C.
- ID
- ZDB-PUB-030730-9
- Date
- 2003
- Source
- The Biochemical journal 375(3): 785-791 (Journal)
- Registered Authors
- Keywords
- dehydroepiandrosterone (DHEA), molecular cloning, neurosteroid, sulphotransferase, SULT2, zebrafish
- MeSH Terms
-
- Amino Acid Sequence
- Base Sequence
- Cations, Divalent/pharmacology
- Cloning, Molecular
- Cytosol/drug effects
- Cytosol/enzymology
- Cytosol/metabolism
- DNA, Complementary/chemistry
- DNA, Complementary/genetics
- Dehydroepiandrosterone/metabolism*
- Enzyme Stability
- Escherichia coli/genetics
- Gene Expression Regulation, Enzymologic
- Hydrogen-Ion Concentration
- Kinetics
- Molecular Sequence Data
- Phylogeny
- Recombinant Proteins/isolation & purification
- Recombinant Proteins/metabolism
- Sequence Analysis, DNA
- Steroids/metabolism*
- Substrate Specificity
- Sulfonic Acids/metabolism
- Sulfotransferases/genetics*
- Sulfotransferases/isolation & purification
- Sulfotransferases/metabolism
- Temperature
- Zebrafish Proteins/genetics*
- Zebrafish Proteins/isolation & purification
- Zebrafish Proteins/metabolism
- PubMed
- 12885295 Full text @ Biochem. J.
Citation
Sugahara, T., Yang, Y.-S., Liu, C.-C., Pai, G.-T., and Liu, M.-C. (2003) Sulphonation of dehydroepiandrosterone and neurosteroids: molecular cloning, expression, and functional characterization of a novel zebrafish SULT2Cytosolic Sulphotransferase. The Biochemical journal. 375(3):785-791.
Abstract
By searching the zebrafish expressed sequence tag (EST) database, we have identified two partial cDNA clones encoding the 5#39- and 3#39-regions of a putative zebrafish sulphotransferase (ST). Using the reverse transcription-polymerase chain reaction 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 to, respectively, mouse SULT2B1, human SULT2B1b, and human SULT2A1 ST. 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 E. coli cells, migrated as a 34 kDa protein upon sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Purified zebrafish ST displayed a strong sulphonating activity toward dehydroepiandrosterone (DHEA), with a pH optimum at 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#176C and 43#176C. Among ten different divalent metal cations tested, Fe++ and Cd++ exhibited small but significant stimulatory effects, whereas Hg++ and Cu++ 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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping