Human intestinal acyl-CoA synthetase 5 is sensitive to the inhibitor triacsin C.

ABSTRACT:

Aim

To investigate whether human acyl-CoA synthetase 5 (ACSL5) is sensitive to the ACSL inhibitor triacsin C.

Methods

The ACSL isoforms ACSL1 and ACSL5 from rat as well as human ACSL5 were cloned and recombinantly expressed as 6xHis-tagged enzymes. Ni(2+)-affinity purified recombinant enzymes were assayed at pH 7.5 or pH 9.5 in the presence or absence of triacsin C. In addition, ACSL5 transfected CaCo2 cells and intestinal human mucosa were monitored. ACSL5 expression in cellular systems was verified using Western blot and immunofluorescence. The ACSL assay mix included TrisHCl (pH 7.4), ATP, CoA, EDTA, DTT, MgCl(2), [9,10-(3)H] palmitic acid, and triton X-100. The 200 μL reaction was initiated with the addition of solubilized, purified recombinant proteins or cellular lysat

SUBMITTER: Kaemmerer E 

PROVIDER: S-EPMC3235631 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature