LIVER STEROL CARRIER PROTEINS--EFFECTS OF ETHANOL

描述(摘自申请人摘要)：脂类和蛋白质都是 乙醇作用的部位。已经有大量的研究 乙醇对脂质结构的影响。然而，虽然相当多的人 研究了乙醇对蛋白质功能的影响，但几乎没有 关于乙醇实际上如何改变蛋白质活性的已知信息。它 之前已经提出乙醇直接与蛋白质结合， 但这一结论在很大程度上是基于乙醇对人体的影响。 蛋白质的功能。另一个问题是，如果乙醇直接与 这种结合对蛋白质功能的影响是什么。 初步数据表明，乙醇确实直接与某些 脂转移蛋白，即固醇载体结合蛋白-2(SCP-2)， 肝脂肪酸结合蛋白L与牛血清白蛋白 (BSA)。此外，我们还观察到乙醇取代了顺式-侧柏酸 酸来自牛血清白蛋白上的一些，但不是所有的脂肪酸结合部位。因此，不是 乙醇只与一些蛋白质直接结合，但它选择性地 结合到某些疏水部位。这是一个总的假设 乙醇直接结合到疏水区域的这一新应用 慢性乙醇对SCP-2和L-FABP的影响 消费。我们进一步提出，乙醇抑制细胞外基质的结合 SCP-2和L-FABP的内源性配体与调节甾醇转运 在膜之间。这些假设将用强大的 荧光光谱与~(13)C核磁共振弛豫相结合 技术。本申请的具体目的是：1) 与胆固醇结合的磷脂和脂肪酸的特性 SCP-2和L-FABP将在无乙醇和有乙醇存在的情况下进行检测 在体外使用荧光技术；2)结合构型 乙醇和脂肪酸对SCP-2和L-FABP的作用将用~(13)C- 乙醇核磁共振弛豫数据；3)SCP-2和L-FABP的增强效应 将评估肝膜之间的甾醇交换。慢性 酗酒是导致发病和死亡的最重要原因。 在美国死于肝病。约90%的 大量饮酒者会患上脂肪肝，主要原因是 肝脂代谢异常，尤其是 三酰甘油和其他类脂的堆积。除了……之外 慢性酒精摄入导致肝脏脂肪堆积，蛋白质 积累也是如此。胞质蛋白在很大程度上对此负责。 增加和结合脂肪酸和胆固醇的L-脂肪酸结合蛋白 占肝脏胞浆蛋白总增加量的22% 慢性乙醇处理的大鼠。因此，重要的是要了解 乙醇如何直接作用于L-FABP和SCP-2 内源性配体的结合、置换和脂类运输。

DESCRIPTION (from Applicant's Abstract): 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.