The adipocyte fatty acid-binding protein binds to membranes by electrostatic interactions

The adipocyte fatty acid-binding protein (AFABP) is believed to transfer un esterified fatty acids (FA) to phospholipid membranes via a collisional mec hanism that involves ionic interactions between lysine residues on the prot ein surface and phospholipid headgroups, This hypothesis is derived largely from kinetic analysis of FA transfer from AFABP to membranes. In this stud y, we examined directly the binding of AFABP to large unilamellar vesicles (LUV) of differing phospholipid compositions, AFABP bound LW containing eit her cardiolipin or phosphatidic acid, and the amount of protein bound depen ded upon the mol % anionic phospholipid. The K-a for CL or PA in LUV contai ning 25 mol % of these anionic phospholipids was approximately 2 x 10(3) M- 1 No detectable binding occurred when AFABP was mixed with zwitterionic mem branes, nor when acetylated AFABP in which surface lysines had been chemica lly neutralized was mixed with anionic membranes. The binding of AFABP to a cidic membranes depended upon the ionic strength of the incubation buffer: greater than or equal to 200 mM NaCl reduced protein-lipid complex formatio n in parallel with a decrease in the rate of FA transfer from AFABP to nega tively charged membranes. It was further found that AFABP, but not acetylat ed AFABP, prevented cytochrome c, a well characterized peripheral membrane protein, from binding to membranes. These results directly demonstrate that AFABP binds to anionic phospholipid membranes and suggest that, although g enerally described as a cytosolic protein, AFABP may behave as a peripheral membrane protein to help target fatty acids to and/or from intracellular s ites of utilization.