VACUOLE FUNCTION IN NEUROSPORA

神经孢子虫中的液泡功能

• 批准号: 3290764
• 负责人: RICHARD Louis WEISS
• 金额: $ 16.72万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-04-01 至 1994-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3290764
• 关键词: Neurospora; aminoacid metabolism; aminoacid transport; antibody formation; arginase; arginine; bioenergetics; cofactor; complementary DNA; cytoplasm; enzyme mechanism; fungal genetics; gel electrophoresis; genetic library; intracellular membranes; membrane permeability; metalloenzyme; molecular cloning; mutant; nitrogen metabolism; nuclear magnetic resonance spectroscopy; ornithine; structural genes; vesicle /vacuole

The goal of this project is to characterize the function of the vascular compartmentation of amino acids in Neurospora crassa. Arginine, ornithine and a number of other amino acids are sequestered in the vacuoles in this organism. A specific arginine carrier has been identified in the tonoplast. The protein will be purified sufficiently to obtain a partial amino acid sequence. The amino acid sequence will be used to synthesize gene-specific oligonucleotide probes and polyclonal antibodies to be used to isolate the structural gene. Once isolated, the cloned gene will be used to construct mutants defective in the ability to accumulate arginine in the vacuoles. Attempts will also be made to isolate mutants by standard genetic techniques. Such mutants will be characterized for physiological defects. The identity and specificity of other tonoplast carriers will be determined. Genetic, biochemical and biophysical techniques will be used to investigate the mechanisms responsible for regulating the uptake, retention and release of amino acids from the vacuoles. The effect of environmental alterations on vacuolar pH and the association of vacuolar arginine with polyphosphates will be investigated using in situ 15N-nuclear magnetic resonance spectroscopy. The relationship between nitrogen metabolite regulation and mobilization of vacuolar arginine will be investigated using mutants affecting nitrogen metabolite regulation. The physiological metal ion cofactor for the catabolic enzyme arginase will be identified and the kinetic properties of the enzyme will be measured under physiological conditions to test the hypothesis that the availability of arginine to the enzyme is the primary determinant in controlling arginine catabolism. The structural gene will be cloned and used to determine the origin of the multiple immunologically related species detected with antibodies to the purified enzyme. The results may provide insight into the function of amino acid compartment in eukaryotic cells and organisms.

该项目的目标是表征 粗糙脉孢菌中氨基酸的维管区室。 精氨酸、鸟氨酸和许多其他氨基酸 隔离在该生物体的液泡中。 特定的精氨酸 已在液泡膜中鉴定出载体。 蛋白质将是 充分纯化以获得部分氨基酸序列。 这 氨基酸序列将用于合成基因特异性 寡核苷酸探针和多克隆抗体用于 分离结构基因。 一旦分离，克隆的基因将 用于构建积累能力有缺陷的突变体 液泡中的精氨酸。 也将尝试隔离 通过标准遗传技术产生突变体。 这样的突变体将是 以生理缺陷为特征。 身份和 其他液泡膜载体的特异性将被确定。 遗传、生物化学和生物物理技术将被用于 研究负责调节吸收的机制， 氨基酸从液泡中的保留和释放。 效果 环境变化对液泡 pH 值的影响及其关联 将使用多磷酸盐研究液泡精氨酸 原位15N-核磁共振波谱。 这 氮代谢调节与氮代谢的关系 将使用以下方法研究液泡精氨酸的动员 影响氮代谢调节的突变体。 这 分解代谢酶精氨酸酶的生理金属离子辅助因子 将被识别并且酶的动力学特性将 在生理条件下进行测量以检验假设 精氨酸对酶的可用性是主要的 控制精氨酸分解代谢的决定因素。 结构性 基因将被克隆并用于确定基因的起源 用抗体检测到多个免疫相关物种 到纯化的酶。 结果可以提供深入了解 真核细胞中氨基酸区室的功能和 有机体。