Fatty-Acid-Binding Proteins. Occurrence of Two Fatty-Acid-Binding Proteins in Bovine Liver Cytosol and Their Binding of Fatty Acids, Cholesterol, and Other Lipophilic Ligands

Abstract

Fatty-acid-binding proteins (FABP) are known as cytosolic binding sites for fatty acids and their CoA esters. Radioactively labeled and fluorescent fatty acids were used to locate and identify these proteins in bovine liver cytosol. The occurrence of 2 species of FABP was demonstrated and these were designated pI [isoelectric point] 6.0-FABP and pI 7.0-FABP according to their isoelectric points in the delipidated state. Oleic acid/FABP binding ratios were 1 with pI 6.0-FABP and 2 with pI 7.0-FABP. Upon binding of oleic acid the pI values of ligand FABP shifted to pH 5.0-5.1 in each case. Both proteins were purified by removing non-binding proteins by acid and heat denaturation and subsequent gel filtration. By making use of the pI shifts observed upon lipidation and delipidation of the binding proteins with ligand fatty acids, final purification was achieved in 2 fractionations by isoelectric focusing. The binding proteins (MW 11,800 .+-. 1000) had similar amino-acid compositions (no Trp) and were not covalently modified by carbohydrate and fatty acid. Fatty acids and their CoA esters were complexed by either FABP, cholesterol only by pI-7.0-FABP, though non-stoichiometrically. 16-(9-Anthroyloxy)palmitic acid was bound by pI-7.0-FABP in a 1:1 ratio and precluded the additional binding of a straight-chain fatty acid. Electrophoretic titration curves indicated dissociation of the oleic acid/pI 7.0-FABP complex below pH 5.0. It appears that fatty acids and their CoA esters are the foremost binding partners of FABP in vivo. The results are discussed in terms of a single binding site for fatty acids per molecule FABP.