SELECTIVE BINDING OF CHOLESTEROL BY RECOMBINANT FATTY-ACID BINDING-PROTEINS

The sterol binding specificity of rat recombinant liver fatty acid binding protein (L-FABP) and intestinal fatty acid binding protein (I-FABP) was characterized with [H-3]cholesterol and a fluorescent sterol analog dehydroergosterol. Ligand binding analysis, fluorescence spectroscopy, and activation of microsomal acyl-CoA:cholesterol acyltransferase activity showed that L-FABP-bound sterols. 1) Lipidex-1000 assay showed a dissociation constant K(d) = 0.78 +/- 0.18-mu-M and stoichiometry of 0.47 +/- 0.16 mol/mol for [H-3] cholesterol binding to L-PABP. 2) With [H-3]cholesterol/phosphatidylcholine liposomes, the cholesterol binding parameters for L-FABP were K(d) = 1.53 +/- 0.28-mu-M and stoichiometry 0.83 +/- 0.07 mol/mol. 3) L-FABP interaction with dehydroergosterol altered the fluorescence intensity and polarization of dehydroergosterol. Dehydroergosterol bound to L-FABP with K(d) = 0.37-mu-M and a stoichiometry of 0.83 mol/mol. 4) Cholesterol and dehydroergosterol decreased L-FABP tyrosine lifetime. Dehydroergosterol binding produced sensitized emission of bound dehydroergosterol with longer lifetime. 5) L-FABP bound two cis-parinaric acid molecules/molecule of protein. Cholesterol displaced one of these bound cis-parinaric acids. 6) L-FABP enhanced acyl-CoA:cholesterol acyltransferase in a concentration-dependent manner. In contrast, these assays indicated that I-FABP did not bind sterols. Thus, L-FABP appears able to bind 1 mol of cholesterol/mol of L-FABP, the L-FABP sterol binding site is equivalent to one of the two fatty acid binding sites, and L-FABP stimulates acyl-CoA:cholesterol acyltransferase by transfer of cholesterol.