The Adipocyte Fatty Acid-binding Protein Binds to Membranes by Electrostatic Interactions

Elizabeth R Smith,Judith Storch

doi:10.1074/jbc.274.50.35325

Abstract

The adipocyte fatty acid-binding protein (AFABP) is believed to transfer unesterified fatty acids (FA) to phospholipid membranes via a collisional mechanism that involves ionic interactions between lysine residues on the protein surface and phospholipid headgroups. This hypothesis is derived largely from kinetic analysis of FA transfer from AFABP to membranes. In this study, we examined directly the binding of AFABP to large unilamellar vesicles (LUV) of differing phospholipid compositions. AFABP bound LUV containing either cardiolipin or phosphatidic acid, and the amount of protein bound depended upon the mol % anionic phospholipid. The K(a) for CL or PA in LUV containing 25 mol % of these anionic phospholipids was approximately 2 x 10(3) M(-1). No detectable binding occurred when AFABP was mixed with zwitterionic membranes, nor when acetylated AFABP in which surface lysines had been chemically neutralized was mixed with anionic membranes. The binding of AFABP to acidic membranes depended upon the ionic strength of the incubation buffer: >/=200 mM NaCl reduced protein-lipid complex formation in parallel with a decrease in the rate of FA transfer from AFABP to negatively charged membranes. It was further found that AFABP, but not acetylated 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 generally described as a cytosolic protein, AFABP may behave as a peripheral membrane protein to help target fatty acids to and/or from intracellular sites of utilization.

Highlights

§ To whom correspondence should be addressed: Dept. of Nutritional Sciences, Rutgers University, 96 Lipman Drive, New Brunswick, NJ 08901-8525
Anionic phospholipids incorporated into egg phosphatidylcholine (EPC) small unilamellar vesicles (SUV) increased the anthroyloxy-labeled FA (AOFA) transfer rate relative to zwitterionic SUV, whereas transfer was slowed to positively charged SUV. 2) High salt concentrations diminished the rate of fatty acids (FA) transfer to anionic membranes but had no effect on transfer to neutral vesicles [7]. 3) Selective chemical neutralization of lysine residues located on the protein surface resulted in a greater than 35-fold decrease in the rate of fluorescent FA transfer and shifted the mechanism of fatty acid transfer from collisional to diffusional [8]. 4) Fourier transform infrared spectroscopic studies demonstrated large decreases in lipid phase transition temperatures of anionic but not zwitterionic vesicles in the presence of adipocyte fatty acid-binding protein (AFABP), indicative of proteinmembrane interactions [9]
This study demonstrates that the major fatty acid-binding protein of the adipocyte behaves like a peripheral membrane protein

Summary

EXPERIMENTAL PROCEDURES

Materials—The fluorescent probe 2-(9-anthroyloxy)palmitic acid (2AP) was obtained from Molecular Probes (Eugene, OR). Protein concentrations were determined using the molar extinction coefficient for AFABP at 280 nm, 1.55 ϫ 104 MϪ1 cmϪ1 [16]. The concentration of acetylated AFABP was determined by the Lowry assay and by using the molar extinction coefficient at 280 nm, 1.71 ϫ 104 MϪ1 cmϪ1 [8]. The phospholipid concentrations were determined by quantitation of inorganic phosphate [20] and for vesicles containing CL have been corrected for two phosphate groups/CL molecule. Fluorescent FA Transfer Assay—The rate of 2-AP transfer from recombinant murine AFABP to acceptor membrane vesicles containing 10% NBD-PE was determined using a resonance energy transfer assay, as described previously [3, 8]. The apparent binding affinity of AFABP for phospholipids in LUV was determined using the sucrose-loaded vesicle binding assay and calculated using Equation 1 as described under “Experimental Procedures.”.

65 Ϯ 17 111 Ϯ 91

RESULTS

DISCUSSION