Characterization of the Acyl-CoA synthetase activity of purified murine fatty acid transport protein 1.
Fatty acid transport protein 1 (FATP1) is an approximately 63-kDa plasma membrane protein that facilitates the influx of fatty acids into adipocytes as well as skeletal and cardiac myocytes. Previous studies with FATP1 expressed in COS1 cell extracts suggested that FATP1 exhibits very long chain acyl-CoA synthetase (ACS) activity and that such activity may be linked to fatty acid transport. To address the enzymatic activity of the isolated protein, murine FATP1 and ACS1 were engineered to contain a C-terminal Myc-His tag expressed in COS1 cells via adenoviral-mediated infection and purified to homogeneity using nickel affinity chromatography. Kinetic analysis of the purified enzymes was carried out for long chain palmitic acid (C16:0) and very long chain lignoceric acid (C24:0) as well as for ATP and CoA. FATP1 exhibited similar substrate specificity for fatty acids 16-24 carbons in length, whereas ACS1 was 10-fold more active on long chain fatty acids relative to very long chain fatty acids. The very long chain acyl-CoA synthetase activity of the two enzymes was comparable as were the Km values for both ATP and coenzyme A. Interestingly, FATP1 was insensitive to inhibition by triacsin C, whereas ACS1 was inhibited by micromolar concentrations of the compound. These data represent the first characterization of purified FATP1 and indicate that the enzyme is a broad substrate specificity acyl-CoA synthetase. These findings are consistent with the hypothesis that that fatty acid uptake into cells is linked to their esterification with coenzyme A.
Hall
Angela M
AM
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Smith
Anne J
AJ
Bernlohr
David A
DA
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001692:Biological Transport; D019556:COS Cells; D002352:Carrier Proteins / Q000378:metabolism*; D003066:Coenzyme A Ligases / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D050612:Fatty Acid Transport Proteins; D005227:Fatty Acids / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D026901:Membrane Transport Proteins; D051379:Mice; D017830:Octoxynol / Q000494:pharmacology; D019308:Palmitic Acid / Q000378:metabolism; D011485:Protein Binding; D013379:Substrate Specificity; D013997:Time Factors; D014226:Triazenes / Q000494:pharmacology
D002352:Carrier Proteins; D003902:Detergents; D004791:Enzyme Inhibitors; D050612:Fatty Acid Transport Proteins; D005227:Fatty Acids; D026901:Membrane Transport Proteins; C493569:Slc27a1 protein, mouse; C493572:Slc27a4 protein, mouse; D014226:Triazenes; D019308:Palmitic Acid; C034613:triacsin C; D017830:Octoxynol; D003066:Coenzyme A Ligases; C010210:lignoceric acid