Effect of magnesium ions on the high-affinity binding of eosin to the (Na+ + K+)-ATPase.
Effect of magnesium ions on the high-affinity binding of eosin to the (Na+ + K+)-ATPase.
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镁离子对伊红与 (Na K )-ATP 酶高亲和力结合的影响。
DOI：
10.1016/0005-2736(83)90373-5
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发表时间：
1983
期刊：
影响因子：
--
通讯作者：
M. Esmann
中科院分区：
文献类型：
--
作者：
J. Skou;M. Esmann
(1)在(Na++ K+)- atp酶存在下，Mg2+增强了伊红Y的荧光。Mg2+的增强作用大于Na+（Skou， J.C. and Esmann， M. (1981) Biochim）。Biophys。学报，647,232-240)。Mg2+将激发最大值从518 nm移至524 nm，发射最大值从538 nm移至542 nm。在约490 nm的激发曲线上也出现了一个肩形，这也是在Na+中观察到的。(2) Mg2+依赖性的荧光增强可以被K+和ATP逆转。在Mg2++ Pi(即。在磷酸化条件下，荧光增强可以被瓦巴因逆转。与Mg2+和低浓度的K+(即。在钒酸盐结合的条件下，荧光增强可以被钒酸盐逆转。(3)随着Mg2+浓度的增加，曙红具有低亲和力结合。当激发波长大于520 nm时，荧光略有增加。低亲和结合是K+-， ATP-，瓦巴因-和钒酸盐不敏感。(4)结合实验的Scatchard分析表明，在5mm Mg2+存在的情况下，每个32p标记位点存在两个高亲和力的伊红结合位点，这两个位点均对瓦巴因、钒酸盐和atp敏感。在5m Mg2++ 0.25 Pi条件下，kd值分别为0.14 μM和1.3 μM。Mg2+为5 mM， Na+为150 mM时，kd值分别为0.45 μM和3.2 μM。对于5mm Mg2+， K+的加入使亲和性明显降低，但不减少结合位点的数量（每个32p标记位点保持两个）。当5mm Mg2++ 150mm K+时，两个结合位点在kd17 μM处的亲和力完全相同。(5)采用止流法测定构象转变速率。从Mg2+形态到K+形态的转变速率很高。低霉素对速率的影响很小（如果有的话）。在Mg2+存在的情况下加入Na+不会明显改变K+形式的转化速率，其速率常数约为110 s−。然而，在Mg2++ Na+存在的情况下添加寡霉素会产生深远的影响：转化为K+形式的速率降低了2000倍，约为0.063 s−1。这表明单独使用Mg2+的构象与单独使用Na+的构象不同。(6) K+、瓦巴因、钒酸盐和ATP对伊红高亲和力结合的影响表明，每个32p标记位点结合的两个伊红分子与ATP位点结合。
(1) The fluorescence of eosin Y in the presence of (Na++ K+)-ATPase is enhanced by Mg2+. The enhancement by Mg2+is larger than that obtained with Na+(Skou, J.C. and Esmann, M. (1981) Biochim. Biophys. Acta 647, 232–240). Mg2+shifts the excitation maximum from 518 to 524 nm, the emission maximum from 538 to 542 nm. Also a shoulder appears at about 490 nm on the excitation curve, as was also observed with Na+. (2) The Mg2+-dependent enhancement of fluorescence can be reversed by K+as well as by ATP. In the presence of Mg2++ Pi(i.e. under conditions of phosphorylation), the fluorescence enhancement can be reversed by ouabain. With Mg2+and a low concentation of K+(i.e. conditions for vanadate binding), the enhancement of fluorescence can be reversed by vanadate. (3) There is a low-affinity binding of eosin which increases with the Mg2+concentration. This is observed as a slight increase in the fluorescence when the excitation wavelength is above 520 nm. The low-affinity binding is K+-, ATP-, ouabain- and vanadate-insensitive. (4) Scatchard analysis of the binding experiments suggests that there are two high-affinity eosin-binding sites per32P-labelling site in the presence of 5 mM Mg2+both of which are ouabain-, vanadate- and ATP-sensitive. With 5 M Mg2++ 0.25 Pi, the Kdvalues are 0.14 μM and 1.3 μM, respectively. With 5 mM Mg2+, 150 mM Na+, the Kdvalues are 0.45 μM and 3.2 μM, respectively. With 5 mM Mg2+, the addition of K+gives a pronounced decrease in affinity but does not decrease the number of binding sites (which remains at two per32P-labelling site). With 5 mM Mg2++ 150 mM K+, the affinities of the two binding sites become identical, at a Kdof 17 μM. (5) The rate of conformational transitions was measured using the stopped-flow method. The rate of the transition from the Mg2+-form to the K+-form is high. Oligomycin has only a small (if any) effect on the rate. Addition of Na+in the presence of Mg2+does not appreciably change the rate of conversion to the K+-form, giving a rate constant of about 110 s−. However, the addition of oligomycin in the presence of Mg2++ Na+had a profound effect: the rate of conversion to the K+-form was decreased by a factor of 2000 to about 0.063 s−1. This suggests that the conformation with Mg2+alone is different from the conformation with Na+alone. (6) The effects of K+, ouabain, vanadate and ATP on the high-affinity binding of eosin suggest that the two eosin molecules bound per32P-labelling site are bound to ATP sites.