Purba ER et al. (2014), Isolation and characterization of Xenopus solub...

XB-ART-48756

Biochim Biophys Acta 2014 Jul 01;18417:954-62. doi: 10.1016/j.bbalip.2014.03.010.

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Isolation and characterization of Xenopus soluble epoxide hydrolase.

Purba ER , Oguro A , Imaoka S .

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Soluble epoxide hydrolase (sEH) contributes to cell growth, but the contribution of sEH to embryonic development is not well understood. In this study, Xenopus sEH cDNA was isolated from embryos of Xenopus laevis. The Xenopus sEH was expressed in Escherichia coli and was purified. The epoxide hydrolase and phosphatase activities of purified sEH were investigated. The Xenopus sEH did not show phosphatase activity toward 4-methylumbelliferyl phosphate or several lysophosphatidic acids although it had EH activity. The amino acid sequence of Xenopus sEH was compared with that reported previously. We found amino acid substitutions of the 29th Thr to Asn and the 146th Arg to His and prepared a sEH mutant (N29T/H146R), designed as mutant 1. Neither wild-type sEH nor mutant 1 had phosphatase activity. Additional substitution of the 11th Gly with Asp was found by comparison with human sEH which has phosphatase activity, but the Xenopus sEH mutant G11D prepared as mutant 2 did not have phosphatase activity. The epoxide hydrolase activity of sEH seemed to be similar to that of human sEH, while Xenopus sEH did not have phosphatase activity toward several substrates that human sEH metabolizes.

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Species referenced: Xenopus laevis
Genes referenced: bag3 ephx2 mdfic tnni3

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	Fig. 1. The epoxide hydrolase and phosphatase activities of Xenopus and human sEH. (A) EH activities of Xenopus wild-type, mutant 1, mutant 2, and chimera sEH were assessed using purified protein (0.5 Î¼g) toward 25 Î¼M PHOME. (B) Purified sEH (5 Î¼g) was incubated with 0.5 mM 4-methylumbelliferyl phosphate. The fluorescence of 4-methylumbelliferone was measured every 1 min for 60 min at 330 nm (excitation) and 465 nm (emission).
	Fig. 2. Sequence alignment between Xenopus sEH and sEH derived from other species. Black boxes indicate the active sites of phosphatase activity. The arrow indicates a different amino acid of the phosphatase active site between human and Xenopus sEH. Thr27 or Arg146 indicated by an open circle was substituted by Asn or His, restrictively, in the isolated Xenopus sEH.
	Fig. 3. EH activities of Xenopus, human and chimera sEH toward 11,12-EET and 14,15-EET and their phosphatase activities toward LPAs. (A and B) Purified Xenopus, human or chimera sEH (6 Î¼g) was incubated with 10 Î¼M 11,12 EET (A) or 14,15 EET (B) at 37 Â°C for 15 min then analyzed by HPLC-ELSD. (C) Purified human sEH, or Xenopus sEH (0.6 Î¼g) was incubated with 10 Î¼M stearoyl-LPA, arachidonoyl-LPA, arachidoyl-LPA, and dipalmitoyl-LPA, and the phosphatase activities were detected by malachite green. Results are expressed as the mean Â± SD of three separate experiments.
	Fig. 4. The effects of ionic strength and pH of buffer, and reaction temperature on catalytic activities of Xenopus and human sEH. (A) Purified Xenopus sEH or human sEH (0.5 Î¼g) was incubated with 25 Î¼M PHOME with 10, 25, 50, or 100 mM Bis Tris buffer pH 7.0 at 30 Â°C. (B) EH activities toward 25 Î¼M PHOME in 25 mM Bis Tris buffer, pH 5.5, 6.0, 6.5, 7.0 or 7.5, at 30 Â°C. (C) EH activities in 25 mM Bis Tris buffer pH 7.0 at 20, 25, 30, or 37 Â°C. (Dâ€“F) Purified sEH (5 Î¼g) were each incubated with 0.5 mM 4-methylumbelliferyl phosphate in the various conditions described above.
	Fig. 5. Inhibition of EH activity by DCU and kinetic analysis of EH activity. (A) Purified Xenopus sEH or human sEH (0.5 Î¼g) was incubated with 25 Î¼M PHOME in the presence of 0.0â€“0.2 Î¼M DCU. (B, C) Lineweaverâ€“Burk plots of the epoxide hydrolase activities toward PHOME and the kinetic parameters were obtained using GraphPad Prism enzyme kinetic software. Results are expressed as the mean Â± SD of triplicate experiments.
	Fig. 6. Detection of 11,12-EET and 14,15-EET in Xenopus liver by LCâ€“MS. Xenopus liver (80 mg) were homogenized and analyzed by UPLC/ESI/MS. (A) The total ion chromatogram of Xenopus liver. (B, C) The selected ion chromatogram with m/z 319 (EETs). (D) Selected ion chromatogram with m/z 319 of Xenopus liver by the addition of authentic 11,12-EET and 14,15-EET (0.4 pmol). TIC, total ion chromatogram; XIC, extracted ion chromatogram.
	Fig. 7. The mRNA and protein levels of sEH in Xenopus embryos. (A) Xenopus sEH mRNA levels were detected at the stages 0, 10.5, 18, 23, 26, 30, and 38 of Xenopus embryo. Total RNA was isolated from embryos and RT-PCR was performed. Amplified DNA fragments were analyzed by electrophoresis with 2% agarose gel. The values on the graphs are the mean Â± SD of three separate samples. Band intensity was measured by NIH Image and normalized to that of histone. (B) sEH protein levels in Xenopus embryos were detected by Western blotting. Ten embryos were homogenized and separated into three distinct layers: lipid, cytoplasmic, and pigment granule/yolk. The cytoplasmic layer (30 Î¼g protein) was subjected to SDS-polyacrylamide gel electrophoresis and analyzed by Western blotting with the antibody against human sEH or Î²-actin. Xenopus sEH protein levels were normalized by the expression level of Î²-actin protein. The values on the graphs are the means Â± SD of three separate samples.
	Fig. 8. sEH mRNA distribution in several tissues of Xenopus laevis. Total RNA was isolated from the brain, heart, liver, lung, kidney, eye, pancreas, and spleen and converted to cDNA. The amplified DNA fragments were run on an agarose gel (1%) and visualized with ethidium bromide staining. The band intensity of sEH was normalized by histone and quantified using NIH Image. The values on the graphs are each the means Â± SD of three separate samples.
	Fig. 9. Localization of Xenopus sEH by WISH. (A and B) Embryos were hybridized by a sense probe (A) and an antisense probe (B). The localization of Xenopus sEH was detected in the head region in olfactory placode (op), cement gland (cg), pharyngeal cavity (pc), eye vesicle (ev), otic vesicle (ov), and notochord (n).
	ephx2 (epoxide hydrolase 2, cytoplasmic) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 30, lateral view, anterior left, dorsal up.

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