ATP-LINKED EFFECTORS OF NA+/K+ ATPASE AND CATARACT

NA /K ATP酶和白内障的ATP连锁效应子

• 批准号: 2161161
• 负责人: MARGARET H GARNER
• 金额: $ 20.86万
• 依托单位: UNIVERSITY OF NORTH TEXAS HLTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 1996-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2161161
• 关键词: active sites; adenosine triphosphate; antiserum; cataract; diabetic cataract; dogs; enzyme substrate analog; gene expression; glycation; high performance liquid chromatography; human tissue; insulin; insulinlike growth factor; ion transport; isozymes; laboratory rat; membrane fusion; membrane reconstitution /synthesis; organ culture; oxidation; posttranslational modifications; potassium; protein sequence; sodium; sodium potassium exchanging ATPase

Increased lens Na+ concentrations correlate both with increasing lens color and with increasing opacity in the outer fiber cell region of the lens, the lens cortex. Lens Na+ concentrations are maintained by 3 lenticular Na+ -pumps, Na,K-ATPases, which use the energy from the hydrolysis of ATP to transport K+ into lens cells and Na+ out of lens cells, presumably at a Na:K:ATP ratio of 3:2:1. In cataract lenses, ATP hydrolysis and/or K+ transport do not always change concurrently with lens Na+ concentration increases. This would suggest the Na,K-ATPase dependent Na:K transport efficiency can be altered so that ATP hydrolysis and/or K+ transport can be normal while Na+ transport is reduced. Transport efficiency may be related to gene expression (isoform function differences), to metabolite or hormone regulation, or to post- translational modification. The major long term goal of this project is to determine factors that lead to Na:K transport efficiency changes in human cataract. Na,K-ATPase function changes in other eye tissues with aging and with the same systemic diseases that correlate with human cataract. The second longterm goal is to determine if the agents that alter lenticular Na,KATPase function also cause Na,K-ATPase dysfunction in other eye tissues. The lens is unique, in that the 3 isoforms of the catalytic subunit of Na,K-ATPase, that are expressed in different regions of the lens, can be easily separated from one another by dissection. In other eye tissues mixtures of isoforms are often expressed in the same cell. Studies are proposed to characterize, by specific antibodies, catalytic subunit isoform expression in noncataractous and cataractous human lenses. The mechanism of action and Na:K transport efficiency will be determined for each lens isoform (normal and cataractous lenses) using a combination of techniques including: lens organ culture, membrane vesicle reconstitution, fusion of lens membranes into Na,K-ATPase depleted erythrocytes, Fourier Transform Infared Spectroscopy, and fluorescent substrate analog binding. Factors that affect isoform expression or that affect 150-form transport efficiency will be identified. The sites of post-translational modification of lens Na,K-ATPase isoforms by glucose, H202 and ascorbate oxidation products will be identified by sequence analysis of modified peptides isolated from proteolytic digests. Antisera to these modified peptides will be used to assess post-translational modification by these agents in human cataract.

增加的透镜Na+浓度与增加的透镜 颜色和增加不透明度的外部纤维细胞区域的 透镜，透镜皮质。透镜Na+浓度维持在3 晶状体Na+泵，Na，K-ATP酶，其使用来自晶状体的能量。 水解ATP将K+转运到透镜细胞中并将Na+转运到透镜外 细胞，推测Na：K：ATP比例为3：2：1。在白内障晶状体中，ATP 水解和/或K+转运并不总是与透镜同时改变 Na+浓度增加。这表明Na，K-ATP酶依赖于 Na：K转运效率可以改变，使得ATP水解和/或K+ 运输可以正常，而Na+运输减少。运输 效率可能与基因表达（亚型功能）有关 差异），代谢物或激素调节，或后 翻译修饰。该项目的主要长期目标是 确定导致人体Na：K转运效率变化的因素 白内障 眼其他组织Na，K-ATP酶功能的增龄变化 以及与人类白内障相关的相同系统性疾病。 第二个长期目标是确定是否改变的代理人 晶状体Na，K-ATP酶功能也可引起其他组织Na，K-ATP酶功能障碍 眼组织该透镜是独特的，因为催化剂的3种异构体 Na，K-ATP酶的亚基，其表达于细胞的不同区域。 透镜可以通过解剖容易地彼此分离。另一只眼睛 同种型的组织混合物通常在同一细胞中表达。研究 提出通过特异性抗体表征催化亚基 非白内障和白内障人晶状体中同种型表达。的 作用机制和Na：K转运效率将被确定为 每种透镜同种型（正常晶状体和白内障晶状体）使用以下组合 技术包括：透镜器官培养，膜囊泡重建， 透镜膜融合成Na，K-ATP酶耗竭的红细胞，傅立叶 变换红外光谱和荧光底物类似物结合。 影响同种型表达或影响150型转运的因素 效率将得到确定。翻译后位点 葡萄糖、H2 O2和抗坏血酸对透镜Na，K-ATP酶同工型的修饰 氧化产物将通过修饰的DNA的序列分析来鉴定。 从蛋白水解酶中分离的肽。这些修饰的抗血清 肽将被用来评估翻译后修饰， 人类白内障中的药物