Purification of active Na+-K+-ATPase using a new ouabain-affinity column.
Purification of active Na+-K+-ATPase using a new ouabain-affinity column.
复制标题
使用新型哇巴因亲和柱纯化活性 Na -K -ATP 酶。
DOI：
10.1152/ajpcell.1998.275.4.c1167
复制
发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Schiebinger,R
中科院分区：
文献类型：
--
作者：
Yingst,DR;Yang,SY;Schiebinger,R
将Na ~+-K ~+-ATP酶的特异性抑制剂哇巴因（Ouabain）通过13个原子的间隔基与环氧琼脂糖偶联，制备了与Na ~+-K ~+-ATP酶特异性结合的亲和柱。从大鼠和狗肾脏的Na+-K+-ATP酶结合到柱上，并作为酶构象的函数洗脱，通过添加特定的配体组合来改变。在Na + ATP + Mg存在下和在含有30 mM K的溶液中，来自两种来源的Na+-K+-ATP酶与柱结合。在存在Na或Na + ATP的情况下未观察到结合。这些实验表明，Na+-K+-ATP酶在与未束缚的哇巴因结合的相同条件下与柱结合。Na+-K+-ATP酶与过量的游离Na +哇巴因或Na + ATP竞争洗脱。后者洗脱活性酶。对于相当量的结合Na+-K+-ATP酶，Na +哇巴因和Na + ATP洗脱大鼠Na+-K+-ATP酶多于犬Na+-K+-ATP酶，这与大鼠Na+-K+-ATP酶对哇巴因的较低亲和力一致。用哇巴因亲和层析柱从大鼠肾微粒体和大鼠肾上腺肾小球细胞中纯化活性Na ~+-K ~+-ATP酶。肾脏酶的比活性从0.02增加到15 μmol Pi·mg−1·min−1。从肾小球细胞纯化的Na+-K+-ATP酶用[32 P]正磷酸盐预标记，在α亚基上磷酸化，表明这些细胞含有磷酸化Na+-K+-ATP酶的激酶。
Ouabain, a specific inhibitor of Na+-K+-ATPase, was coupled to epoxy agarose via a 13-atom spacer to make an affinity column that specifically binds Na+-K+-ATPase. Na+-K+-ATPase from rat and dog kidney was bound to the column and was eluted as a function of enzyme conformation, altered by adding specific combinations of ligands. Na+-K+-ATPase from both sources bound to the column in the presence of Na + ATP + Mg and in solutions containing 30 mM K. No binding was observed in the presence of Na or Na + ATP. These experiments suggest that Na+-K+-ATPase binds to the column under the same conditions that it binds to untethered ouabain. Na+-K+-ATPase already bound to the column was competitively eluted with excess free Na + ouabain or with Na + ATP. The latter eluted active enzyme. For comparable amounts of bound Na+-K+-ATPase, Na + ouabain and Na + ATP eluted more rat than dog Na+-K+-ATPase, consistent with the lower affinity of the rat Na+-K+-ATPase for ouabain. The ouabain-affinity column was used to purify active Na+-K+-ATPase from rat kidney microsomes and rat adrenal glomerulosa cells. The specific activity of the kidney enzyme was increased from ∼2 to 15 μmol Pi⋅ mg−1⋅ min−1. Na+-K+-ATPase purified from glomerulosa cells that were prelabeled with [32P]orthophosphate was phosphorylated on the α-subunit, suggesting that these cells contain a kinase that phosphorylates Na+-K+-ATPase.