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Human intestinal acyl-coa synthetase 5 is sensitive to the inhibitor triacsin c

World Journal of Gastroenterology 2011年第44期17卷 4883-4889页

作者： Elke Kaemmerer Anne Peuscher Andrea Reinartz christian Liedtke Ralf Weiskirchen Jürgen Kopitz Nikolaus Gassler Department of Pediatrics RWTH Aachen UniversityAachenGermany Institute of Pathology RWTH Aachen University Department of Plant Biotechnology Fraunhofer Institute for Molecular Biology and Applied Ecology (IME) Institute of Pathology RWTH Aachen University Department of Medicine Ⅲ RWTH Aachen University Institute of clinical chemistry and Pathobiochemistry RWTH Aachen University Institute of Pathology University of Heidelberg

AIM:To investigate whether human acyl-coa synthetase 5(AcSL5) is sensitive to the AcSL inhibitor triacsin ***:The AcSL isoforms AcSL1 and AcSL5 from rat as well as human AcSL5 were cloned and recombinantly expressed as 6xHis-tagged *** 2+-affinity purified recombinant enzymes were assayed at pH 7.5 or pH 9.5 in the presence or absence of triacsin *** addition,AcSL5 transfected caco2 cells and intestinal human mucosa were ***5 expression in cellular systems was verified using Western blot and *** AcSL assay mix included TrisHcl(pH 7.4),ATP,coa,EDTA,DTT,Mgcl 2,[9,103 H] palmitic acid,and triton *** 200 μL reaction was initiated with the addition of solubilized,purified recombinant proteins or cellular *** were terminated after 10,30 or 60 min of incubation with Doles ***:Expression of soluble recombinant AcSL proteins was found after incubation with isopropyl betaD-1-thiogalactopyranoside and after ultracentrifugation these were further purified to near homogeneity with Ni 2+-affinity *** c selectively and strongly inhibited recombinant human AcSL5 protein at pH 7.5 and pH 9.5,as well as recombinant rat AcSL1(sensitive control),but not recombinant rat AcSL5(insensitive control).The Ic50 for human AcSL5 was about 10 μmol/*** inhibitory triacsin c effect was similar for different incubation times(10,30 and 60 min) and was not modified by the N-or c-terminal location of the *** order to evaluate AcSL5 sensitivity to triacsin c in a cellular environment,stable human AcSL5 caco2 transfectants and mechanically dissected normal human intestinal mucosa with high physiological expression of AcSL5 were *** both models,AcSL5 peak activity was found at pH 7.5 and pH 9.5,corresponding to the properties of recombinant human AcSL5 *** the presence of triacsin c(25 μmol/L),total AcSL activity was dramatically diminished in human AcSL5 transfectants as well as in Ac

关键词： acyl-coa synthetase 5 fatty acid metabolism mitochondria triacsin c

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