title: Thermodynamic and kinetic properties of fatty acid interactions with rat liver fatty acid-binding protein.
abstract: Fatty acid-binding protein from rat liver (L-FABP) binds 2 fatty acids (FA) per protein, in contrast to FABPs from adipocyte, heart, and intestine, for which binding and structural studies are consistent with a single FA binding site. To understand better the unique characteristics of L-FABP, we have carried out equilibrium binding and kinetic measurements of long chain FA using the fluorescent probes of free fatty acids (FFA), ADIFAB and ADIFAB2, to monitor the concentration of FFA in the reaction of FA with L-FABP. We found that the dissociation constants (Kd) ranged from about 1 nM to 4 microM, being largest for myristate at 45 degrees C and smallest for oleate at 10 degrees C, and that 2 FA were bound per L-FABP for all temperatures and FA. The binding measurements also revealed that at temperatures below 37 degrees C, affinities for the two binding sites differ by between 5- and 20-fold but as the temperature was increased, the affinities converge toward equal values. Off-rate constants (koff) were similar for all FA and for temperatures between 10 and 45 degrees C, ranged from about 0.1 s-1 to 50 s-1. Moreover, for all FA, koff values for dissociation from both the high and low affinity sites were similar, indicating that binding affinity differences at the lower temperatures reflect lower on-rates for binding to the low affinity site. The temperature at which the affinities of the two sites become equivalent depends upon the FA; higher temperatures (45-50 degrees C) are required for the unsaturated FA and myristate than for the longer chain saturated FA (<37 degrees C). This transition from different to equivalent affinity binding sites at specific temperatures reflects a nonlinear van't Hoff behavior of the high affinity site, which in turn is a reflection of large heat capacity changes (between -0.6 and -1.2 kcal K-1 mol-1) that accompany FA binding to the high affinity site. These heat capacity changes, which are unique to L-FABP, do not appear to be correlated with a significant conformational change upon ligand binding. The differences between long chain saturated and unsaturated FA suggest that the conformation of FA bound to L-FABP may differ with both FA type and temperature, and that, in comparison to other FABPs, L-FABP may have distinctly different effects on saturated and unsaturated FA metabolism.
authors.affiliation: Medical Biology Institute, La Jolla, California 92037, USA.
authors.forename: G V
authors.initials: GV
authors.lastname: Richieri
authors.forename: R T
authors.initials: RT
authors.lastname: Ogata
authors.forename: A M
authors.initials: AM
authors.lastname: Kleinfeld
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D018996:Myelin P2 Protein / Q000378:metabolism*; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D051381:Rats; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
chemical_list: D002352:Carrier Proteins; C493320:Fabp1 protein, mouse; C493321:Fabp1 protein, rat; C490174:Fabp7 protein, rat; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D005456:Fluorescent Dyes; D018996:Myelin P2 Protein; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; C079339:acrylodated intestinal fatty acid binding protein, recombinant; C102579:acrylodated intestinal fatty acid-binding protein 2