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.

我们提出的研究来研究甘氨酸和如何 相关氨基酸防止能量损害的质膜损伤 细胞。氨基酸的作用与代谢无关。而且， 尽管发生了严重的ATP耗竭并分解离子，它们仍发生 稳态。我们的初步研究表明，ATP的细胞缺陷 通过甘氨酸特异性校正的耗尽的细胞是独特的 质膜异常。功能障碍的分析表明 膜表现得好像是被分子尺寸的孔穿孔的 具有可定义的排除限制，尽管它在结构上保持 在其他方面完好无损。这种类型的病理要么被抑制或 许多其他有抑制作用活动的代理人加重 中枢神经系统（CNS）中的甘氨酸受体。生物碱 士宁宁和驱虫敏敏素保护细胞，它们的细胞及其 影响是甘氨酸的添加剂。其他特工（Ru5135， pitrazepin和thebaine）拮抗甘氨酸的保护作用 特定的方式。这些作用的药理学表明 甘氨酸的有益作用可以通过其对 类似于CNS的质膜中的靶蛋白 甘氨酸受体氯化物通道。根据假设，ATP 耗竭导致甘氨酸受体的未知异常 负责多孔膜缺损的蛋白质。剂量 对某些药理学剂的反应（Avermectin，Ru5135， Pitrazepin）建议在ATP耗竭模型中高亲和力相互作用 并指出可能用于识别和隔离的策略 其作用的蛋白质。我们的目标是三倍。 首先，我们将表征甘氨酸特异性膜缺陷 功能和形态技术。其次，我们将发展 获得质膜的超纯制剂的方法。 第三，我们将使用平均B1a的衍生物来亲和力标签 亲和力纯化它们可能结合的质膜蛋白。我们 相信这种方法将在识别中取得成功，并且 甘氨酸推定靶蛋白的纯化。研究会 可以在培养的肾脏上皮细胞系中完成，但通常将是 与其他细胞类型有关。