Expression of rat liver long-chain acyl-CoA synthetase and characterization of its role in the metabolism of R-ibuprofen and other fatty acid-like xenobiotics

Roland Bruggera, Christine Reichel, Begonia Garcia Alia, Kay Brune, Tokuo Yamamoto, Irmgard Tegeder, Gerd Geissinger

Research output: Contribution to journalArticlepeer-review

26 Citations (Scopus)

Abstract

Our investigations of fatty acid metabolism and epimerization of the 2-arylpropionic acid derivative, R-ibuprofen, resulted in the successful purification of an acyl-CoA synthetase from rat liver microsomes that catalyzes the formation of both palmitoyl-CoA and R-ibuprofenoyl-CoA. To investigate whether R-ibuprofenoyl-CoA synthetase and long-chain acyl-CoA synthetase (LACS) are identical enzymes, we cloned the cDNA from LACS into the pQE30 expression vector and transformed the construct into Escherichia coli M15[pREP4]. Induction of the bacterial protein synthesis with 0.2 mM isopropyl-beta-D-galactoside resulted in a strong, time-dependent increase in LACS protein as determined by Western blot analysis using a polyclonal rabbit anti-LACS antibody. Incubations of the recombinantly expressed protein with palmitic acid as physiological LACS substrate or R-ibuprofen in the presence of Mg2+, ATP, and CoA resulted in a 5-fold increase in the thioesterification of both substrates. Western blot analysis using tissue homogenates of rat liver, heart, kidney, lung, brain, and ileum showed that LACS was found in every tissue investigated, with the greatest expression in the liver. Similar results were obtained with activity measurements using R-ibuprofen and palmitic acid as substrates. Northern blot analysis revealed a hybridization with a 3.8-kb mRNA transcript in rat liver, heart, and kidney, but no signal was observed in lung, brain and ileum, suggesting the expression of different LACS isoform(s) in these organs. In summary, our results further show that R-ibuprofenoyl-CoA synthetase and long-chain acyl-CoA synthetase are identical enzymes that are involved in the metabolism of various xenobiotics.

Original languageEnglish
Pages (from-to)651-656
Number of pages6
JournalBiochemical Pharmacology
Volume61
Issue number6
DOIs
Publication statusPublished - 2001 Mar 15

Keywords

  • Chiral inversion
  • Ibuprofen
  • Long-chain acyl-CoA synthetase (EC 6.1.2.3.)
  • NSAIDs
  • Palmitoyl-CoA

ASJC Scopus subject areas

  • Biochemistry
  • Pharmacology

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