ISOLATION AND CHARACTERZATION OF KIDNEY ENDOSOMES

肾内体的分离和表征

• 批准号: 2292180
• 负责人: Dennis Brown
• 金额: $ 2.17万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-04-01 至 1998-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2292180
• 关键词: G protein; adenosinetriphosphatase; antibody; apical membrane; basolateral membrane; calcium; cell population study; cell type; cellular polarity; chlorine; enzyme activity; epithelium; guanine nucleotide binding protein; hydrogen; immunocytochemistry; in situ hybridization; ion transport; kidney cell; laboratory rabbit; membrane transport proteins; microtubules; protein purification; protein structure function; sodium; vesicle /vacuole

The specific aims of this FIRCA proposal are: 1) To isolate pure endocytic vesicles from different kidney regions (cortex, inner stripe of the outer medulla, papilla). Vesicles purified by traditional methods are contaminated with other membranes, and we plan to develop and use affinity- purification techniques with specific antibodies to improve vesicle purity while maintaining a high yield; 2) To characterize enzyme and transport activities of the resulting endosomal preparations, including the H+-ATPase (H-pump), Na+/H+-exchange, Ca2+ /H+-exchange, Ca2+=ATPase (Ca-pump), Cl- channel, and different types of water channels; 3) To generate immune sera by immunizing rabbits with preparations of highly purified endocytic vesicles; 4) To purify particular antibodies by immunoadsorption techniques from immune sera. A battery of antibodies, thus obtained, will be used in immunocytochemical studies on renal tissue sections in order to specify one or more antibodies that specifically label endocytic vesicles in defined populations of renal cells. These antibodies will be used as tools for the further affinity purification of vesicle populations as well as for studies on vesicle transport pathways that are part of the parent grant. The results will allow us to describe in more detail the molecular and functional heterogeneity of endosomal vesicles from various tubules in the mammalian kidney and to study their behaviour in a variety of conditions with disturbances in salt, water, and protein reabsorption.

该FIRCA提案的具体目的是：1）隔离纯内吞 来自不同肾脏区域的囊泡（皮层，外部的内部条纹 髓质，乳头）。 传统方法纯化的囊泡是 被其他膜污染，我们计划开发和使用亲和力 - 具有特定抗体的纯化技术可改善囊泡纯度 同时保持高收益； 2）表征酶和运输 所得的内体制剂的活动，包括H+-ATPase （H-PUMP），Na+ /H+-Exchange，Ca2+ /H+-exchange，Ca2+= ATPase（Ca-Pump），Cl- 通道和不同类型的水道； 3）产生免疫血清 通过用高度纯化的内吞作用的兔子免疫兔子 囊泡； 4）通过免疫吸附纯化特定抗体 免疫血清的技术。 因此获得的一系列抗体将 用于肾脏组织切片的免疫细胞化学研究 指定一种或多种专门标记内吞囊泡的抗体 在确定的肾细胞种群中。 这些抗体将用作 还可以进一步纯化囊泡群体的工具 至于对囊泡传输途径的研究，这是父母的一部分 授予。 结果将使我们能够更详细地描述分子 来自各个小管的内体囊泡的功能异质性 哺乳动物肾脏，并研究他们的行为 盐，水和蛋白质重吸收的情况。