Eosin, a fluorescent probe of ATP binding to the (Na+ + K+)-ATPase.
Eosin, a fluorescent probe of ATP binding to the (Na+ + K+)-ATPase.
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曙红，一种与 (Na K )-ATP 酶结合的 ATP 荧光探针。
DOI：
10.1016/0005-2736(81)90251-0
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发表时间：
1981
期刊：
影响因子：
--
通讯作者：
M. Esmann
中科院分区：
文献类型：
--
作者：
Jens Christian Skou;M. Esmann
在K+存在下，与（Na++ K+）-ATP酶结合的曙红实际上具有与不含酶的曙红相同的荧光，而在Na+存在下，荧光更高，激发最大值从518 nm移动到524 nm，发射最大值从538 nm移动到542 nm，并且在激发曲线上在约490 nm处出现肩部。(2)曙红结合量随K+浓度增加而增加，但亲和力较低。当Na+和K+浓度相等时，在存在Na+的情况下结合更多，150 mM Na+和150 mM K+之间的差异显示每个32 P标记位点有一个高亲和力曙红结合位点（KD 0.45 μM）。与较低浓度的阳离子有一个和两个之间的Na+依赖性高亲和力曙红结合位点每32 P-标记网站。(3)ATP（和ADP）阻止高亲和力Na+依赖的曙红结合，在Na+（+ Mg 2+）存在下，曙红和ATP之间存在水解竞争。(4)曙红与ATP一样，增加Na+相对于K+的亲和力（Na++ K+= 150 mM），以激活Na+水解和保护Na+免受N-乙基马来酰亚胺灭活。(5)结果表明，曙红高亲和力结合位点是ATP结合位点，它位于低极性环境中的酶上，即Na+引起的构象变化打开了一个ATP高亲和力位点，而K+关闭了该位点（或使亲和力降低到低水平）。实验还表明，增加内部位点Na+相对于K+亲和力的ATP不是水解的ATP，即在Na++ K+存在下的周转循环中，系统与两种不同的ATP分子反应。
Eosin bound to the (Na++ K+)-ATPase in the presence of K+ has practically the same fluorescence as eosin without enzyme while in the presence of Na+ the fluorescence is higher, the excitation maximum is shifted from 518 to 524 nm, the emission maximum from 538 to 542 nm, and a shoulder appears at about 490 nm on the excitation curve.(2) The amount of eosin bound increases with the K+ concentration but with a low affinity. With equal concentrations of Na+ and K+ more is bound in the presence of Na+, and the difference between 150 mM Na+ and 150 mM K+ shows one high-affinity eosin binding site per 32 P-labelling site (K D 0.45 μM). With lower concentrations of the cations there are between one and two Na+-dependent high-affinity eosin binding sites per 32 P-labelling site.(3) ATP (and ADP) prevents the hig-affinity Na+-dependent eosin binding and there is competition between eosin and ATP for the hydrolysis in the presence of Na+(+ Mg 2+).(4) Eosin, like ATP, increases the Na+ relative to K+ affinity (Na++ K+= 150 mM) for Na+ activation of hydrolysis and for Na+ protection against inactivation by N-ethylmaleimide.(5) The results suggest that the high affinity eosin binding site is an ATP binding site and that it is located on the enzyme in an environment with a low polarity, ie, the conformational change induced by Na+ opens a high-affinity site for ATP while K+ closes the site (or decreases the affinity to a low level). The experiments suggest, furthermore, that the ATP which increases the Na+ relative to K+ affinity of the internal sites is not the ATP which is hydrolyzed, ie, in a turnover cycle in the presence of Na++ K+ the system reacts with two different ATP molecules.