Interactions of fatty acids with neutral fatty‐acid‐binding protein from bovine liver

Abstract

Hepatic‐type fatty‐acid‐binding protein (hFABP) from the cytosol of bovine liver is a 14.4‐kDa neutral protein with a blocked N‐terminus and a disulfide system located on the surface of the protein. It binds two molecules of fatty acid in one binding site, apparent dissociation constants of the oleic acid/hFABP complex are 0.24 μM and 2.15 μM. Computer analysis of circular dichroic spectra predicts that hFABP contains about 12%α‐helix, 45%β‐structure, 15%β‐turn and 27% unordered structure. Ellipticities indicative of secondary structure are not affected by fatty acid binding. Cationic amino acid residues of hFABP (1 His, 15 Lys, 2 Arg) were screened for ionic fatty acid/protein interactions. His was excluded, as 1H‐NMR analysis of His‐C2 and His‐C4 protons indicated that binding of oleic acid shifts the pK of His from 6.9 to 7.1 only in hFABP with the disulfide system in the oxidized state; acylation of His with diethylpyrocarbonate does not affect the binding of the fatty acid. Acetylation of Lys reduces binding marginally, whereas modification of Arg with phenylglyoxal lowers the binding activity by 65%. From 1H‐NMR investigations, conformational changes within the protein, due to a sort of disaggregation of hFABP upon fatty acid binding, were derived. Most of the proton resonances sharpen up with ligand binding, and some of the methyl resonances shift positions, possibly because they are directly involved in the fatty acid/protein interaction.

Ligand Specificity of Brain Lipid-binding Protein

“…As shown in Fig. 2A and Table I, recombinant BLBP bound oleic acid with K d of ϳ0.4 M and B max of ϳ100 pmol/g, as expected from similar studies of other fatty acidbinding proteins (8,9,15,16). Also as expected, binding of arachidonic acid to BLBP was determined to have K d (0.25 M) and B max (47 pmol/g) similar to previously studied FABPs ( Fig.…”

“…As shown in Fig. 2D, a nonsaturable binding curve of retinoic acid to BLBP was observed, demonstrating that BLBP did not specifically bind retinoic acid, similarly to other previously studied FABPs (15,16).…”

“…First, we have established that BLBP is a fatty acid-binding protein with ligand binding properties that are similar to but different from those of previously characterized members of this family. BLBP binds oleic acid and arachidonic acid with similar affinities as the heart or liver fatty acid-binding proteins (15,16), but unlike these family members it does not bind saturated fatty acids such as palmitic or arachidinic acid. The strong preference of BLBP for long chain, polyunsaturated fatty acids may be a distinguishing feature of BLBP.…”

Pro-sterol Carrier Protein-2

“…Parinaric acid binding was done as described (32)(33)(34). Fluorescent sterol exchange assays were done at 37°C as described previously (35,36).…”

Sequence‐specific 1H‐NMR assignment and determination of the secondary structure of bovine heart fatty‐acid‐binding protein

“…The sequence similarity of FABP types from the same organ but related species was found to be much higher than between different FABP types expressed in a different or even the same organ of the same species (Spener et al, 1989). Fatty acids are the foremost ligands of these binding proteins, from which they can be rapidly mobilized in the cytosol (Schulenberg-Schell et al, 1988;Bass, 1988;Spener et al, 1989). The fatty-acid-binding proteins belong to a large family of non-enzymic hydrophobic ligand-binding proteins that is characterized by structural similarities indicating a common ancestral gene (Gordon and Lowe, 1985;Sacchettini et al, 1988).…”