Electrogenic Partial Reactions of the SR-Ca-ATPase Investigated by a Fluorescence Method
Electrogenic Partial Reactions of the SR-Ca-ATPase Investigated by a Fluorescence Method
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荧光法研究 SR-Ca-ATP 酶的生电部分反应
DOI：
--
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发表时间：
1999
影响因子：
2.4
通讯作者：
H. Apell
中科院分区：
文献类型：
--
作者：
C. Butscher;M. Roudná;H. Apell
抽象的。采用荧光法研究了SR-Ca-ATP酶膜制品中的主动离子转运。苯乙烯基染料RH 421以前用于研究Na，K-ATP酶被取代的类似物，2BITC，以获得优化的荧光变化后，底物诱导的部分反应。假设局部电场的变化是由摄取/释放或蛋白质内离子移动产生的荧光变化的来源，2BITC允许确定泵循环中的生电部分反应。结果发现，钙离子结合的细胞质和内腔侧的泵是产电的，而磷酸化和构象转换显示只有轻微的产电性。Ca 2+平衡滴定实验在pH 7.2的两个主要的构象的蛋白质表示合作的两个Ca 2+离子的结合状态E1的表观半饱和浓度，KM为600 nm。在状态P-E2中，测定了两个KM值（5 μm和2.2 mM），与已发表的数据基本一致。从具有不同pH的缓冲液中的Ca 2+滴定和从P-E2中的pH滴定，可以证明H+结合是产电的，并且Ca 2+和H+竞争相同的结合位点。Tharpsiglide诱导的抑制的Ca-ATP酶导致一个状态与一个特定的荧光水平相媲美的状态E1与未占用的离子位点，独立的缓冲液组成。
Abstract. A fluorescence method was adapted to investigate active ion transport in membrane preparations of the SR-Ca-ATPase. The styryl dye RH421 previously used to investigate the Na,K-ATPase was replaced by an analogue, 2BITC, to obtain optimized fluorescence changes upon substrate-induced partial reactions. Assuming changes of the local electric field to be the source of fluorescence changes that are produced by uptake/release or by movement of ions inside the protein, 2BITC allowed the determination of electrogenic partial reactions in the pump cycle. It was found that Ca2+ binding on the cytoplasmic and on the lumenal side of the pump is electrogenic while phosphorylation and conformational transition showed only minor electrogenicity. Ca2+ equilibrium titration experiments at pH 7.2 in the two major conformations of the protein indicated cooperative binding of two Ca2+ ions in state E1 with an apparent half-saturation concentration, KM of 600 nm. In state P-E2 two KM values, 5 μm and 2.2 mM, were determined and are in fair agreement with published data. From Ca2+ titrations in buffers with various pH and from pH titrations in P-E2, it could be demonstrated that H+ binding is electrogenic and that Ca2+ and H+ compete for the same binding site(s). Tharpsigargin-induced inhibition of the Ca-ATPase led to a state with a specific fluorescence level comparable to that of state E1 with unoccupied ion sites, independent of the buffer composition.
DOI：
10.1016/s0021-9258(18)83639-0
发表时间：
1989-04
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
G. Inesi;L. Meis
通讯作者：
G. Inesi;L. Meis
影响因子：
3.9
作者：
INESI, G;SAGARA, Y
通讯作者：
SAGARA, Y
与钾与 Na /K( )-ATP 酶结合和释放相关的构象变化动力学。
DOI：
10.1016/s0005-2736(96)00162-9
发表时间：
1996
期刊：
Biochimica et biophysica acta
影响因子：
--
作者：
Pratap,PR;Palit,A;Grassi-Nemeth,E;Robinson,JD
通讯作者：
Robinson,JD