Interaction of fatty acids with recombinant rat intestinal and liver fatty acid-binding proteins.
Intestinal enterocytes contain two homologous fatty acid-binding proteins, intestinal fatty acid-binding protein (I-FABP)2 and liver fatty acid-binding protein (L-FABP). Since the functional basis for this multiplicity is not known, the fatty acid-binding specificity of recombinant forms of both rat I-FABP and rat L-FABP was examined. A systematic comparative analysis of the 18 carbon chain length fatty acid binding parameters, using both radiolabeled (stearic, oleic, and linoleic) and fluorescent (trans-parinaric and cis-parinaric) fatty acids, was undertaken. Results obtained with a classical Lipidex-1000 binding assay, which requires separation of bound from free fatty acid, were confirmed with a fluorescent fatty acid-binding assay not requiring separation of bound and unbound ligand. Depending on the nature of the fatty acid ligand, I-FABP bound fatty acid had dissociation constants between 0.2 and 3.1 microM and a consistent 1:1 molar ratio. The dissociation constants for L-FABP bound fatty acids ranged between 0.9 and 2.6 microM and the protein bound up to 2 mol fatty acid per mole of protein. Both fatty acid-binding proteins exhibited relatively higher affinity for unsaturated fatty acids as compared to saturated fatty acids of the same chain length. cis-Parinaric acid or trans-parinaric acid (each containing four double bonds) bound to L-FABP and I-FABP were displaced in a competitive manner by non-fluorescent fatty acid. Hill plots of the binding of cis- and trans- parinaric acid to L-FABP showed that the binding affinities of the two sites were very similar and did not exhibit cooperativity. The lack of fluorescence self-quenching upon binding 2 mol of either trans- or cis-parinaric acid/mol L-FABP is consistent with the presence of two binding sites with dissimilar orientation in the L-FABP. Thus, the difference in binding capacity between I-FABP and L-FABP predicts a structurally different binding site or sites.
Nemecz
G
G
Department of Pharmacology and Cell Biophysics, University of Cincinnati Medical Center, Ohio 45267-0004.
Hubbell
T
T
Jefferson
J R
JR
Lowe
J B
JB
Schroeder
F
F
D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D007413:Intestinal Mucosa / Q000378:metabolism*; D007700:Kinetics; D019787:Linoleic Acid; D008041:Linoleic Acids / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D008433:Mathematics; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D019301:Oleic Acid; D009829:Oleic Acids / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D013229:Stearic Acids / Q000378:metabolism
D002352:Carrier Proteins; C493320:Fabp1 protein, mouse; C493321:Fabp1 protein, rat; C490174:Fabp7 protein, rat; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D005231:Fatty Acids, Unsaturated; D008041:Linoleic Acids; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D009829:Oleic Acids; D011994:Recombinant Proteins; D013229:Stearic Acids; D019301:Oleic Acid; C031183:stearic acid; D019787:Linoleic Acid; C013641:parinaric acid