RESUMO
Zebrafish has in recent years emerged as a popular vertebrate model for use in pharmacological and toxicological studies. While there have been sporadic studies on the zebrafish glutathione S-transferases (GSTs), the zebrafish GST gene superfamily still awaits to be fully elucidated. We report here the identification of 15 zebrafish cytosolic GST genes in NCBI GenBank database and the expression, purification, and enzymatic characterization of the zebrafish cytosolic GST Pi-1 (GSTP1). The cDNA encoding the zebrafish GSTP1 was cloned from a 3-month-old female zebrafish, expressed in Eschelichia coli host cells, and purified. Purified GSTP1 displayed glutathione-conjugating activity toward 1-chloro-2,4-dinitrobenzene as a representative substrate. The enzymatic characteristics of the zebrafish GSTP1, including pH-dependency, effects of metal cations, and kinetic parameters, were studied. Moreover, the expression of zebrafish GSTP1 at different developmental stages during embryogenesis, throughout larval development, onto maturity was examined.
Assuntos
Regulação Enzimológica da Expressão Gênica/fisiologia , Glutationa S-Transferase pi , Proteínas de Peixe-Zebra , Peixe-Zebra/metabolismo , Animais , Dinitroclorobenzeno/química , Feminino , Glutationa S-Transferase pi/biossíntese , Glutationa S-Transferase pi/química , Glutationa S-Transferase pi/genética , Especificidade por Substrato , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/biossíntese , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genéticaRESUMO
By searching the GenBank database, we identified sequences encoding three new zebrafish cytosolic sulfotransferases (SULTs). These three new zebrafish SULTs, designated SULT1 ST9, SULT3 ST4, and SULT3 ST5, were cloned, expressed, purified, and characterized. SULT1 ST9 appeared to be mostly involved in the metabolism and detoxification of xenobiotics such as ß-naphthol, ß-naphthylamine, caffeic acid and gallic acid. SULT3 ST4 showed strong activity toward endogenous compounds such as dehydroepiandrosterone (DHEA), pregnenolone, and 17ß-estradiol. SULT3 ST5 showed weaker, but significant, activities toward endogenous compounds such as DHEA and corticosterone, as well as xenobiotics including mestranol, ß-naphthylamine, ß-naphthol, and butylated hydroxyl anisole (BHA). pH-dependency and kinetic constants of these three enzymes were determined with DHEA, ß-naphthol, and 17ß-estradiol as substrates. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to examine the expression of these three new zebrafish SULTs at different developmental stages during embryogenesis, through larval development, and on to maturity.
