To determine the zebrafish as a model for investigating the methylation

To determine the zebrafish as a model for investigating the methylation pathway of drug metabolism we embarked on the molecular cloning of the zebrafish catechol cells transformed with the pGEX-2TK expression vector harboring the zebrafish COMT cDNA. revealed developmental stage-dependent expression of the zebrafish COMT during embryonic development and throughout the larval stage onto maturity. These results provide a foundation for investigating the involvement of COMT-mediated methylation in protection against the adverse effects of catechol drugs and other xenobiotic catechols during the developmental process. the developmental stage-dependent expression of the zebrafish COMT was investigated. Materials and Methods Materials Dopamine epinephrine l-3 4 (l-Dopa) methyl-Dopa S-adenosyl-L-methionine (AdoMet) sodium dodecyl sulfate (SDS) FMK sodium acetate 2 acid (MES) 3 polymerase was a product of Promega Corporation. Takara DNA polymerase was purchased from Fisher Scientific. T4 DNA ligase and HI restriction endonuclease were from New England Biolabs. Oligonucleotide primers were synthesized by MWG Biotech. pSTBlue-1 AccepTor Vector Kit and BL21 (DE3) competent cells were from Novagen. Protein molecular weight standards were from Fermentas Life Sciences. pGEX-2T glutathione DNA polymerase with the first-strand cDNA reverse-transcribed from the total RNA isolated from a 3-month-old adult female zebrafish as the template. Amplification conditions were 2 min at 94°C and 25 cycles of 94°C for 30 s 60 for 35 s and 72°C for 45 s followed by a 5-min incubation at 72°C. The final reaction mixture was applied onto a 1% agarose gel separated by electrophoresis and visualized by ethidium bromide staining. The PCR FMK product band detected was excised from the gel and the DNA therein was isolated by spin filtration. Purified PCR product was cloned into the pSTBlue-1 vector and verified for authenticity by nucleotide sequencing [21]. To amplify a truncated cDNA encoding a “soluble-form” of the zebrafish COMT another set of sense and antisense primers (Table 1) was used in a PCR reaction with pSTBlue-1 harboring the full-length zebrafish COMT cDNA (see above) as the template. Amplification conditions were the same as described above. At Mouse monoclonal to CD53.COC53 monoclonal reacts CD53, a 32-42 kDa molecule, which is expressed on thymocytes, T cells, B cells, NK cells, monocytes and granulocytes, but is not present on red blood cells, platelets and non-hematopoietic cells. CD53 cross-linking promotes activation of human B cells and rat macrophages, as well as signal transduction. the end of the PCR reaction the PCR product was purified subjected to HI limitation and FMK subcloned into HI-restricted pGEX-2TK vector. Expressing the recombinant zebrafish COMT skilled BL21 (DE3) cells changed with pGEX-2TK harboring the COMT cDNA had been expanded in 1 L LB moderate supplemented with 60 μg/ml ampicillin. Following the cell denseness reached 0.6 OD600nm IPTG (0.1 mM FMK last concentration) was put into induce the creation of recombinant zebrafish COMT. After an over night induction at space temperatures the cells had been gathered by centrifugation and homogenized in 25 ml ice-cold lysis buffer (20 mM Tris-HCl pH 8.0 150 mM NaCl and 1 mM EDTA) using an Aminco People from france Press. Twenty μl of 10 mg/ml aprotinin (a protease inhibitor) was put into the crude homogenate. The crude homogenate was put through centrifugation at 10 0 × for 15 min at 4°C. The supernatant gathered was fractionated using 2.5 ml of glutathione-Sepharose as well as the destined GST-fusion protein was eluted by an elution buffer (50 mM Tris-HCl pH 8.0 in addition 10 mM reduced glutathione) at 4°C or treated with 3 ml of the thrombin digestive function buffer (50 mM Tris-HCl pH 8.0 150 mM NaCl and 2.5 mM CaCl2) including 5 unit/ml bovine thrombin at room temperature. Carrying out a 15-min incubation with continuous agitation the planning was put through centrifugation. The recombinant zebrafish COMT was analyzed for purity by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and put through enzymatic characterization. Desk 1 Oligonucleotide primers useful for the cDNA cloning of zebrafish COMT as well as for the quantitative real-time RT-PCR evaluation from the developmental stage-dependent manifestation from the zebrafish COMT Enzymatic assay The methylating activity of purified recombinant zebrafish COMT was assayed using radioactive [14C]-tagged AdoMet as the methyl group donor. The typical assay blend with your final level of 20 μl included 50 mM TrisHCl buffer at pH 7.5 0.1 mM [14C]-labeled S-adenosyl-L-methionine 5 mM DTT 1.5 mM MgCl2 1 mM substrate. Settings with DMSO or drinking water instead of substrate were prepared also. The response was started with the addition of the enzyme permitted to continue FMK for 60 min at 28°C and terminated with the addition of 10 μl of just one 1 N HCl. The precipitates shaped had been cleared by centrifugation as well as the.