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

晶状体钠离子浓度升高与晶状体增大相关 颜色，并且随着外层纤维细胞区域中不透明度的增加 晶状体，晶状体皮质。晶状体Na+浓度维持3% 晶状体Na+泵，Na，K-ATPase，它使用来自 ATP水解液将K+输送到晶状体细胞内，将Na+转运出晶状体 细胞，推测Na：K：ATP的比例为3：2：1。在白内障晶状体中，ATP 水解性和/或K+转运并不总是与晶状体同时发生变化 Na+浓度增加。这表明Na，K-ATPase依赖于 可以改变Na：K的运输效率，从而使ATP水解和/或K+ 当Na+转运减少时，转运可以正常。运输 效率可能与基因表达有关(异构体功能 差异)，代谢或激素调节，或后 翻译修饰。这个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。眼部其他组织Na，K-ATPase功能随增龄的变化 以及与人类白内障相关的相同的系统性疾病。 第二个长期目标是确定是否会改变 晶状体Na，K-ATPase功能也可导致其他 眼部纸巾。晶状体是独一无二的，因为催化的3种亚型 Na，K-ATPase亚基，在不同区域表达 透镜，可以很容易地通过解剖彼此分离。在另一只眼睛里 不同亚型的组织混合物通常在同一细胞中表达。研究 被提议通过特定的抗体来表征催化亚单位 亚型在非白内障和白内障人晶状体中的表达。这个 将确定其作用机理和Na：K转运效率 每种晶状体(正常晶状体和白内障晶状体)使用以下组合 技术包括：晶状体器官培养、膜泡重建、 晶状体膜与Na，K-ATPase耗尽的红细胞的融合 变换红外光谱，和荧光底物模拟结合。 影响异构体表达或影响150-亚型转运的因素 效率将被确定。翻译后的网站 葡萄糖、H202和抗坏血酸对晶状体Na，K-ATPase异构体的修饰 氧化产物将通过对修饰的序列进行分析来鉴定 从蛋白水解物中分离出的多肽。这些修饰的抗血清 这些多肽将被用来评估翻译后修饰 人类白内障中的毒剂。

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.