Document Detail


Fluorescently labelled bovine acyl-CoA-binding protein acting as an acyl-CoA sensor: interaction with CoA and acyl-CoA esters and its use in measuring free acyl-CoA esters and non-esterified fatty acids.
MedLine Citation:
PMID:  12071849     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Long-chain acyl-CoA esters are key metabolites in lipid synthesis and beta-oxidation but, at the same time, are important regulators of intermediate metabolism, insulin secretion, vesicular trafficking and gene expression. Key tools in studying the regulatory functions of acyl-CoA esters are reliable methods for the determination of free acyl-CoA concentrations. No such method is presently available. In the present study, we describe the synthesis of two acyl-CoA sensors for measuring free acyl-CoA concentrations using acyl-CoA-binding protein as a scaffold. Met24 and Ala53 of bovine acyl-CoA-binding protein were replaced by cysteine residues, which were covalently modified with 6-bromoacetyl-2-dimethylaminonaphthalene to make the two fluorescent acyl-CoA indicators (FACIs) FACI-24 and FACI-53. FACI-24 and FACI-53 showed fluorescence emission maximum at 510 and 525 nm respectively, in the absence of ligand (excitation 387 nm). Titration of FACI-24 and FACI-53 with hexadecanoyl-CoA and dodecanoyl-CoA increased the fluorescence yield 5.5-and 4.7-fold at 460 and 495 nm respectively. FACI-24 exhibited a high, and similar increase in, fluorescence yield at 460 nm upon binding of C14-C20 saturated and unsaturated acyl-CoA esters. Both indicators bind long-chain (>C14) acyl-CoA esters with high specificity and affinity (K(d)=0.6-1.7 nM). FACI-53 showed a high fluorescence yield for C8-C12 acyl chains. It is shown that FACI-24 acts as a sensitive acyl-CoA sensor for measuring the concentration of free acyl-CoA, acyl-CoA synthetase activity and the concentrations of free fatty acids after conversion of the fatty acid into their respective acyl-CoA esters.
Authors:
Majken C T Wadum; Jens K Villadsen; Søren Feddersen; Rikke S Møller; Thomas B F Neergaard; Birthe B Kragelund; Peter Højrup; Nils J Faergeman; Jens Knudsen
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Publication Detail:
Type:  In Vitro; Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  The Biochemical journal     Volume:  365     ISSN:  0264-6021     ISO Abbreviation:  Biochem. J.     Publication Date:  2002 Jul 
Date Detail:
Created Date:  2002-06-19     Completed Date:  2002-07-26     Revised Date:  2009-11-18    
Medline Journal Info:
Nlm Unique ID:  2984726R     Medline TA:  Biochem J     Country:  England    
Other Details:
Languages:  eng     Pagination:  165-72     Citation Subset:  IM    
Affiliation:
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.
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MeSH Terms
Descriptor/Qualifier:
Acyl Coenzyme A / analysis*,  chemistry,  metabolism
Animals
Base Sequence
Cattle
Coenzyme A Ligases / analysis
DNA / genetics
Diazepam Binding Inhibitor / chemistry*,  metabolism*
Escherichia coli Proteins / analysis
Esterification
Fatty Acids, Nonesterified / analysis*,  chemistry,  metabolism
Fluorescent Dyes / chemistry
Kinetics
Ligands
Models, Molecular
Mutagenesis, Site-Directed
Protein Structure, Tertiary
Recombinant Proteins / chemistry,  genetics,  metabolism
Chemical
Reg. No./Substance:
0/Acyl Coenzyme A; 0/Diazepam Binding Inhibitor; 0/Escherichia coli Proteins; 0/Fatty Acids, Nonesterified; 0/Fluorescent Dyes; 0/Ligands; 0/Recombinant Proteins; 9007-49-2/DNA; EC 6.2.1.-/Coenzyme A Ligases; EC 6.2.1.-/acyl-coenzyme A synthetase, E coli
Comments/Corrections

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