Champeil P, Henao F, de Foresta B
Département de Biologie Cellulaire et Moléculaire, Commissariat à l'Energie Atomique, Gif-sur-Yvette, France.
Biochemistry. 1997 Oct 7;36(40):12383-93. doi: 10.1021/bi9709699.
Sequential dissociation of the two Ca2+ ions bound to non-phosphorylated sarcoplasmic reticulum Ca2+-ATPase was triggered by addition, in a stopped-flow experiment, of quin2, which acted both as a high-affinity chelator and as a Ca2+-sensitive fluorescent probe. The kinetics of Ca2+ dissociation were deduced from the observed changes in quin2 fluorescence in the visible region (with lambdaex = 313 nm), while fluorescence detection in the UV region (with lambdaex = 290 nm) made it possible to monitor the tryptophan fluorescence changes accompanying this dissociation under the same ionic conditions. In the absence of KCl or NaCl, at pH 6 or 7, the observed changes in quin2 fluorescence were monoexponential, with rate constants very close to those of the changes in ATPase tryptophan fluorescence, which also appeared monophasic. In the presence of 100 mM KCl, quin2 fluorescence changes, although still monoexponential, were faster than in the absence of the monovalent ions but distinctly slower than the changes in tryptophan fluorescence, which were accelerated to a larger extent. In addition, the apparent kinetics of the Trp fluorescence changes depended on the excitation wavelength. Using an excitation wavelength of 296 nm, the Trp fluorescence drop was still faster than with an excitation wavelength of 290 nm, and in the presence of NaCl it even displayed a clear undershoot. We conclude that in the presence of KCl or NaCl and with an excitation wavelength of 290 nm, the rapid drop in tryptophan fluorescence mainly monitors the dissociation of the first of the two Ca2+ ions to be released from Ca2+-ATPase, while excitation at 296 nm optically selects a subpopulation of Trp residues whose fluorescence level is lower in the ATPase species with one Ca2+ ion bound than in the Ca2+-deprived ATPase species. The latter conditions result in an initial drop in Trp fluorescence whose apparent rate constant (in single-exponential analysis) is faster than the true rate of dissociation of the first Ca2+ ion and in a subsequent slower rise related to dissociation of the second Ca2+ ion. The difference between results obtained in the absence and in the presence of K+ or Na+ is due to an antagonizing effect of these cations on proton-induced conformational rearrangement of Ca2+-free ATPase, a conformational rearrangement which changes the ATPase Trp fluorescence level and significantly affects the cooperativity of Ca2+ binding at equilibrium.
在停流实验中加入喹啉-2（quin2）可引发与非磷酸化肌浆网Ca²⁺-ATP酶结合的两个Ca²⁺离子的顺序解离，喹啉-2既是高亲和力螯合剂，又是Ca²⁺敏感荧光探针。Ca²⁺解离动力学由可见光区域（激发波长λex = 313 nm）观察到的喹啉-2荧光变化推导得出，而紫外区域（激发波长λex = 290 nm）的荧光检测则能在相同离子条件下监测伴随这种解离的色氨酸荧光变化。在不存在KCl或NaCl、pH为6或7的情况下，观察到的喹啉-2荧光变化呈单指数形式，速率常数与ATP酶色氨酸荧光变化的速率常数非常接近，色氨酸荧光变化也呈单相。在存在100 mM KCl的情况下，喹啉-2荧光变化虽然仍呈单指数形式，但比不存在单价离子时更快，但明显慢于色氨酸荧光变化，色氨酸荧光变化加速程度更大。此外，色氨酸荧光变化的表观动力学取决于激发波长。使用296 nm的激发波长时，色氨酸荧光下降仍比290 nm激发波长时更快，并且在存在NaCl的情况下甚至出现明显的下冲。我们得出结论，在存在KCl或NaCl且激发波长为290 nm时，色氨酸荧光的快速下降主要监测从Ca²⁺-ATP酶释放的两个Ca²⁺离子中第一个的解离，而在296 nm激发时，光学选择了一部分色氨酸残基，其在结合一个Ca²⁺离子的ATP酶物种中的荧光水平低于无Ca²⁺的ATP酶物种。后一种情况导致色氨酸荧光最初下降，其表观速率常数（在单指数分析中）快于第一个Ca²⁺离子的真实解离速率，随后与第二个Ca²⁺离子解离相关的上升较慢。在不存在和存在K⁺或Na⁺时获得的结果之间的差异是由于这些阳离子对质子诱导的无Ca²⁺ATP酶构象重排的拮抗作用，这种构象重排会改变ATP酶色氨酸荧光水平并显著影响平衡时Ca²⁺结合的协同性。
