MECHANISMS OF PROTECTION BY GLYCINE AGAINST CELL DEATH

甘氨酸防止细胞死亡的机制

• 批准号: 2458846
• 负责人: MANJERI A VENKATACHALAM
• 金额: $ 18.45万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2458846
• 关键词: affinity chromatography; cell death; cell membrane; chloride channels; confocal scanning microscopy; fluorescent dye /probe; glycine; membrane proteins; membrane structure; method development; pore forming protein; protein purification; protein structure function; tissue /cell culture

We propose research to investigate the mechanisms of how glycine and related amino acids prevent plasma membrane damage in energy compromised cells. The effects of amino acids are not related to metabolism. Moreover, they occur in spite of severe ATP depletion and break down of ion homeostasis. Our preliminary studies show that the cellular defect in ATP depleted cells that is specifically corrected by glycine is a unique plasma membrane abnormality. Analysis of the dysfunction revealed that the membrane behaves as if it is perforated by pores of molecular dimensions with definable size exclusion limits, although it remains structurally intact in other respects. This type of pathology is either suppressed or aggravated by a number of other agents which have activities at inhibitory glycine receptors in the central nervous system (CNS). The alkaloid Strychnine and the anthelmintic Avermectin protect cells, and their effects are additive to those of glycine. Other agents, (RU5135, pitrazepin and thebaine) antagonize the protective effects of glycine in a specific manner. The pharmacology of these effects suggests strongly that the beneficial effects of glycine may be explained by its actions on a target protein in the plasma membrane that is analogous to the CNS glycine receptor chloride channel. According to the hypothesis, ATP depletion leads to an unknown abnormality of the glycine receptor like protein which is responsible for the porous membrane defect. Dose responses to some of the pharmacological agents (Avermectin, RU5135, pitrazepin) suggest high affinity interactions in the ATP depletion model and point towards strategies that may be used to identify and isolate the protein at which their effects are directed. Our objectives are threefold. Firstly, we will characterize the glycine specific membrane defect by functional as well as morphological techniques. Secondly, we will develop methodology to obtain ultra pure preparations of plasma membranes. Thirdly, we will use derivatives of Avermectin B1a to affinity label and affinity purify plasma membrane proteins to which they may bind. We believe that this approach will succeed in the identification and purification of the putative target protein for glycine. The studies will be done in a cultured kidney epithelial cell line, but will be generally relevant to other cell types.

我们提出研究来调查甘氨酸和 相关氨基酸可防止能量受损时质膜损伤 细胞。氨基酸的作用与新陈代谢无关。而且， 尽管 ATP 严重耗尽且离子分解，它们仍会发生 体内平衡。我们的初步研究表明细胞中 ATP 缺陷 经甘氨酸专门校正的耗尽细胞是一种独特的 质膜异常。对功能障碍的分析表明， 膜的行为就好像它被分子尺寸的孔穿孔一样 具有可定义的尺寸排除限制，尽管它在结构上仍然存在 其他方面完好无损。这种类型的病理要么被抑制，要么 许多其他具有抑制活性的药物会加剧这种情况 中枢神经系统（CNS）中的甘氨酸受体。生物碱 士的宁和驱虫阿维菌素保护细胞及其 效果与甘氨酸的效果相加。其他代理，（RU5135， 吡嗪平和蒂巴因）拮抗甘氨酸的保护作用 一种特定的方式。这些作用的药理学强烈表明 甘氨酸的有益作用可以通过其对 质膜中的靶蛋白，类似于中枢神经系统 甘氨酸受体氯离子通道。根据假设，ATP 耗尽会导致甘氨酸受体的未知异常，例如 造成多孔膜缺陷的蛋白质。剂量 对某些药物的反应（阿维菌素、RU5135、 吡嗪平）表明 ATP 耗竭模型中存在高亲和力相互作用 并指出可用于识别和隔离的策略 它们的作用所针对的蛋白质。我们的目标有三个。 首先，我们将通过以下方式表征甘氨酸特异性膜缺陷： 功能技术和形态技术。其次，我们将发展 获得质膜超纯制剂的方法。 第三，我们将使用阿维菌素B1a的衍生物进行亲和标记和 亲和纯化它们可能结合的质膜蛋白。我们 相信这种方法将成功地识别和 甘氨酸的假定目标蛋白的纯化。这些研究将 在培养的肾上皮细胞系中完成，但通常会 与其他细胞类型相关。