Both lipids and proteins are sites of ethanol action. There have been an extensive number of studies on effects of ethanol on lipid structure. However, while quite a few studies examined effects of ethanol on protein function, there is little information known on how ethanol actually alters protein activity. It has been previously proposed that ethanol directly binds to proteins, but that conclusion has been largely based on effects of ethanol o protein function. Another issue is if ethanol directly binds to a protein what are the consequences of that binding on protein function. Preliminary data indicate that ethanol indeed directly binds to certain lipid transfer proteins, i.e., sterol carrier binding protein-2 (SCP-2), liver-fatty acid binding protein (L-FABP) and bovine serum albumin (BSA). In addition, we observed that ethanol displaced cis-parinaric acid from some, but not all fatty acid-binding sites on BSA. Thus, not only does ethanol directly bind to some proteins but it selectively binds to certain hydrophobic sites. It is the overall hypothesis of this new application that ethanol directly binds to hydrophobic areas of SCP-2 and L-FABP, two proteins that are affected by chronic ethanol consumption. We further proposed that ethanol inhibits the binding of endogenous ligands to SCP-2 and L-FABP and modifies sterol transport between membranes. These hypotheses will be examined using the powerful combination of fluorescence spectroscopy and 13C NMR relaxation technique.
The specific aims of this application are: 1) characteristics of cholesterol phospholipids and fatty acids binding to SCP-2 and L-FABP will be examined in the absence and presence of ethanol in vitro using fluorescence techniques; 2) the binding geometry of ethanol and fatty acids to SCP-2 and L-FABP will be studied using 13C- ethanol NMR relaxation data; and 3) effects of SCP-2 and L-FABP enhanced sterol exchange between liver membranes will be evaluated. Chronic alcohol abuse is the most important cause of morbidity and mortality from liver disease in the United States. Approximately 90 percent of heavy alcohol drinkers develop fatty liver, resulting mainly from marked abnormalities in hepatic lipid metabolism, specifically, from accumulation of triacylglycerols and other lipids. In addition to lipids accumulating in liver with chronic ethanol consumption, proteins accumulate as well. Cytosolic proteins are largely responsible for this increase and L-FABP which binds fatty acids and cholesterol accounted for 22 percent of the total increase in liver cytosolic proteins in chronic ethanol treated rats. Therefore, it is important to understand how ethanol may directly act on L- FABP and SCP-2 with respect to binding, displacement of endogenous ligands, and lipid transport.
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