PROBING THE LIGAND-BINDING SITES OF FATTY-ACID AND STEROL CARRIER PROTEINS - EFFECTS OF ETHANOL

被引:102
作者
SCHROEDER, F
MYERSPAYNE, SC
BILLHEIMER, JT
WOOD, WG
机构
[1] DUPONT CO INC,DEPT CARDIOVASC,WILMINGTON,DE 19898
[2] VET ADM MED CTR,CTR GERIATR RES EDUC & CLIN,MINNEAPOLIS,MN 55417
[3] UNIV MINNESOTA,SCH MED,DEPT PHARMACOL,MINNEAPOLIS,MN 55417
关键词
D O I
10.1021/bi00037a033
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 K-d Of 0.23 mu M and B-max 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.
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页码:11919 / 11927
页数:9
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