Reversible binding of long-chain fatty acids to purified FAT, the adipose CD36 homolog.
Transport of long-chain fatty acids into rat adipocytes was previously shown to be inhibited by the reactive derivative sulfosuccinimidyl oleate consequent to its binding to a membrane protein FAT, which is homologous to CD36. In this report, the ability of the purified protein to bind native fatty acids was investigated. CD36 was isolated from rat adipocytes by phase partitioning into Triton X-114 followed by chromatography on DEAE and then on wheat germ agglutinin. Fatty acid binding was determined by incubating CD36, solubilized in buffer containing 0.1 Triton X-100, with fatty acids at 37 degrees C, and then by adsorbing the unbound ligand with Lipidex 1,000 at 0 degrees C. Bovine serum albumin was used as a positive control and gelatin, a protein that does not bind fatty acids, as a negative control. Measurements with albumin yielded reproducible binding values which were not altered by the presence of 0.1% Triton X-100. Under the same conditions, gelatin yielded reproducibly negative measurements that did not differ significantly from zero. CD36 bound various long-chain fatty acids at low ligand to protein ratios. Warming the protein-FA-Lipidex mixture to 37 degrees C removed the FA off the protein. Thus, binding was reversible and distinct from the palmitoylation of the protein known to occur on an extracellular domain. Comparison of the predicted secondary sequence of CD36 with that of human muscle fatty acid binding protein suggested that a potential binding site for the fatty acid on CD36 may exist in its extracellular segment between residues 127 and 279.
Baillie
A G
AG
Department of Physiology and Biophysics, State University of New York at Stony Brook, Stony Brook, NY 11794-8661, USA.
Coburn
C T
CT
Abumrad
N A
NA
D017667:Adipocytes / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D018955:CD36 Antigens / Q000378:metabolism; D002478:Cells, Cultured; D005227:Fatty Acids / Q000378:metabolism*; D006801:Humans; D008297:Male; D008562:Membrane Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D027361:Organic Anion Transporters; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D017386:Sequence Homology, Amino Acid
D018955:CD36 Antigens; D005227:Fatty Acids; D008562:Membrane Glycoproteins; D027361:Organic Anion Transporters