Micromethodology for measuring ATPase activity in renal tubules: mineralocorticoid influence.
Micromethodology for measuring ATPase activity in renal tubules: mineralocorticoid influence.
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测量肾小管 ATP 酶活性的微方法：盐皮质激素的影响。
DOI：
10.1152/ajpcell.1984.247.5.c314
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发表时间：
1984
期刊：
影响因子：
--
通讯作者：
Dubinsky,WP
中科院分区：
文献类型：
--
作者：
O'Neil,RG;Dubinsky,WP
使用微技术测量兔离体肾小管片段的 ATP 酶活性，其中 ATP 的水解与碱转化的、高荧光形式的烟酰胺腺嘌呤二核苷酸的出现相耦合。该方法简单、重现性好、灵敏度高，可以轻松测量皮摩尔量的水解 ATP。评估了几种透化细胞膜以测量 Na+-K+-ATP 酶活性的方法，包括渗透（蒸馏水或 300 mM 咪唑）和温度（冷冻）冲击以及添加非离子洗涤剂辛基葡萄糖苷。发现 0.5% 的辛基葡萄糖苷浓度可引起 Na+-K+-ATP 酶的最大激活，并且与通过暴露于蒸馏水和冷冻使小管透化时观察到的结果相当。在 300 mM 咪唑中孵育小管对细胞膜的透化效果较差。在所有后续研究中，细胞都像其他人一样通过暴露于蒸馏水并冷冻来透化。该方法用于测定浅表近曲小管、浅表近曲小管和皮质集合管中 Na+-K+-ATP 酶的基础水平，结果平均分别为 44.9 +/- 6.3、26.4 +/- 2.4 和 11.8 +/- 2.2 pmol ADP X mm-1 X min-1。此外，每日注射醋酸脱氧皮质酮（2 mg X kg-1 X day-1）4-15天可提高血浆盐皮质激素水平，导致皮质集合管的Na+-K+-ATP酶活性加倍，这证实了其他人的结果。所提出的方法可以很容易地适用于其他 ATP 酶的微量分析。
The ATPase activity of rabbit isolated renal tubule segments was measured using a microtechnique in which the hydrolysis of ATP was enzymatically coupled to the appearance of an alkali-converted, highly fluorescent form of nicotinamide adenine dinucleotide. The methods are simple, reproducible, and have a high sensitivity in which picomole quantities of hydrolyzed ATP can readily be measured. Several methods for permeabilizing the cell membranes for measurement of Na+-K+-ATPase activity were evaluated, including osmotic (distilled water or 300 mM imidazole) and temperature (freezing) shock and addition of the nonionic detergent octylglucoside. An octylglucoside concentration of 0.5% was found to cause a maximum activation of the Na+-K+-ATPase and was comparable with that observed when tubules were permeabilized by exposure to distilled water and freezing. Incubation of tubules in 300 mM imidazole was less effective in permeabilizing the cell membranes. In all subsequent studies, the cells were permeabilized by exposure to distilled water and freezing as done by others. The methods were used to assay for the basal levels of Na+-K+-ATPase in the superficial proximal convoluted tubule, the superficial proximal straight tubule, and the cortical collecting tubule and were found to average 44.9 +/- 6.3, 26.4 +/- 2.4, and 11.8 +/- 2.2 pmol ADP X mm-1 X min-1, respectively. Furthermore, elevation of plasma mineralocorticoids by daily injections of deoxycorticosterone acetate (2 mg X kg-1 X day-1) for 4-15 days caused a doubling in the Na+-K+-ATPase activity of the cortical collecting duct, confirming the results of others. The methods presented can easily be adapted for microanalysis of other ATPases.