Probing the ligand binding sites of fatty acid and sterol carrier proteins: effects of ethanol.
Direct effects of ethanol on the interaction of cytosolic lipid transfer proteins with ligands are not known. In this study, recombinant liver fatty acid binding protein (L-FABP) and sterol carrier protein-2 (SCP-2) were used in conjunction with a series of fluorescent fatty acid probe molecules to compare the relative dielectric properties of the ligand binding sites and to examine the effects of ethanol in vitro on ligand interaction with these proteins. L-FABP and SCP-2 exhibited broad but distinct ligand specificities. Although NBD-stearic acid bound with high affinity to both proteins, emission spectra showed that the relative dielectric constant of the ligand binding site in SCP-2 was significantly lower than in L-FABP, 2 vs 24. Furthermore, affinities of L-FABP for NBD-fatty acid probes were NBD-stearic acid > NBD-lauric acid >>> NBD-hexanoic acid, NBD-acetic acid. In contrast, SCP-2 bound only NBD-stearic acid with a Kd of 0.23 microM and Bmax of 0.98 mol/mol. This observation of SCP-2 specifically binding the fluorescent NBD-stearic acid was confirmed with RdB-stearic acid and the naturally fluorescent cis-parinaric acid, both of which had similar affinities and stoichiometries. Ethanol in vitro had no effect on L-FABP-NBD-stearic acid binding. However, ethanol at physiological concentrations (25 mM) dramatically inhibited NBD-stearic acid binding to SCP-2. In conclusion, the data show that both L-FABP and SCP-2 specifically bind fluorescent fatty acids. However, the ligand binding sites of L-FABP and SCP-2 differed dramatically in their dielectric properties and their sensitivity to ethanol.
Schroeder
F
F
Department of Physiology and Pharmacology, Texas A&M University, TVMC, College Station 77843-4466, USA.
Myers-Payne
S C
SC
Billheimer
J T
JT
Wood
W G
WG
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D004563:Electrochemistry; D000431:Ethanol / Q000494:pharmacology*; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids / Q000378:metabolism*; D005456:Fluorescent Dyes; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008024:Ligands; D015335:Molecular Probes; D018996:Myelin P2 Protein / Q000737:chemistry / Q000378:metabolism*; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D010940:Plant Proteins; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013228:Stearates / Q000378:metabolism; D013261:Sterols / Q000378:metabolism*; D025521:Tumor Suppressor Proteins
D002352:Carrier Proteins; C493319:FABP1 protein, human; C107608:FABP7 protein, human; 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; D008024:Ligands; D015335:Molecular Probes; D018996:Myelin P2 Protein; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D010940:Plant Proteins; D011994:Recombinant Proteins; D013228:Stearates; D013261:Sterols; D025521:Tumor Suppressor Proteins; C072087:sterol carrier proteins; C060027:12-N-methyl-7-nitrobenzo-2-oxa-1,3-diazolamino stearate; D000431:Ethanol; D009327:4-Chloro-7-nitrobenzofurazan