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

Citation
R. Brugger et al., 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, BIOCH PHARM, 61(6), 2001, pp. 651-656
Citations number
34
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Pharmacology & Toxicology
Journal title
BIOCHEMICAL PHARMACOLOGY
ISSN journal
0006-2952 → ACNP
Volume
61
Issue
6
Year of publication
2001
Pages
651 - 656
Database
ISI
SICI code
0006-2952(20010315)61:6<651:EORLLA>2.0.ZU;2-7
Abstract
Our investigations of fatty acid metabolism and epimerization of the 2-aryl propionic acid derivative, R-ibuprofen, resulted in the successful purifica tion of an acyl-CoA synthetase from rat liver microsomes that catalyzes the formation of both palmitoyl-CoA and R-ibuprofenoyl-CoA. To investigate whe ther R-ibuprofenoyl-CoA synthetase and long-chain acyl-CoA synthetase (LACS ) are identical enzymes, we cloned the cDNA from LACS into the pQE30 expres sion 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 a s determined by Western blot analysis using a polyclonal rabbit anti-LACS a ntibody. Incubations of the recombinantly expressed protein with palmitic a cid 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 bo th 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 res ults were obtained with activity measurements using R-ibuprofen and palmiti c 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 identi cal enzymes that are involved in the metabolism of various xenobiotics. (C) 2001 Elsevier Science Inc. All rights reserved.