Interaction of fatty acids with recombinant rat intestinal and liver fatty acid-binding proteins

Abstract

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.

The Crystal Structure of the Liver Fatty Acid-binding Protein

“…The second molecule of fatty acid (oleate 128) is bound with its hydrocarbon tail in the space occurring between the limbs of the "U" and has its carboxylate head group on the surface of the LFABP molecule in what appears to be a fully solvent-accessible location. The semiperpendicular orientation of the two oleic acids is consistent with a lack of fluorescence self-quenching observed when cis-or trans-parinaric acids were bound to LFABP (52,65). The conformation of the ligands will be described in a separate section below.…”

Structure and Function of the Sterol Carrier Protein-2 N-Terminal Presequence

“…Quenching of tryptophan fluorescence by the small molecule acrylamide is an effective method of further analyzing the surface and aqueous exposure of Trp in Trp-containing proteins (37,42). To this end, 50 Figure 4A).…”

Ligand Specificity and Conformational Dependence of the Hepatic Nuclear Factor-4α (HNF-4α)

“…Fluorescent Ligands-Direct binding assays not requiring separation of bound from free ligand were performed using cis-parinaroyl-CoA as described (25)(26)(27)(28)(29)(30). To establish specificity for LCFA-CoA, this assay was repeated using fluorescent fatty acids: cis-parinaric acid and NBDstearic acid.…”

“…Fluorescence emission spectra were obtained using a PC1 photon counting fluorimeter (ISS Inc., Urbana, IL) and maximal intensities measured. The K d and number of binding sites (n) were calculated as previously described (26,27,30). Where it was possible (e.g.…”