Binding kinetics of calbindin-D28k determined by flash photolysis of caged Ca2+
Binding kinetics of calbindin-D28k determined by flash photolysis of caged Ca2+
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DOI：
10.1016/s0006-3495(00)76537-4
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发表时间：
2000-12-01
影响因子：
3.4
通讯作者：
Vergara, JL
中科院分区：
文献类型：
--
作者：
Nägerl, UV;Novo, D;Vergara, JL
我们使用 DM-硝基酚的紫外闪光光解结合基于模型的 Oregon Green 488 BAPTA-5N 荧光瞬态分析来研究 Ca2+ 与 calbindin-D-28K 结合的动力学。实验使用 [Ca2+] 为 1.5 μM 的饱和 DM-硝基酚。在这些条件下，在不存在 calbindin-D-28K 的情况下，紫外激光闪光会产生 [Ca2+] 的快速阶梯式增加，而在其存在下，闪光诱导的荧光衰减仅归因于蛋白质的 Ca2+ 缓冲。我们开发了一种新的动力学参数推导方法，并使用合成的 Ca2+ 缓冲液 EGTA 来证实其有效性。我们提供的证据表明 calbindin-D-28K 至少以两种不同的动力学模式结合 Ca2+，一种是由高亲和力位点结合 Ca2+，其 k(on) 与 EGTA 相当（即类似于 1 x 10(7) M-1 s(-1)），另一种具有较低的亲和力和大约八倍的 k(on) 速度。鉴于传统方法无法充分解析 Ca2+ 缓冲液的快速 Ca2+ 结合动力学，该方法对于研究其他生物学上重要的 Ca2+ 结合蛋白的 Ca2+ 结合特性非常有价值。
We have used UV flash photolysis of DM-nitrophen in combination with model-based analysis of Oregon Green 488 BAPTA-5N fluorescence transients to study the kinetics of Ca2+ binding to calbindin-D-28K. The experiments used saturated DM-nitrophen at a [Ca2+] of 1.5 muM. Under these conditions, UV laser flashes produced rapid steplike increases in [Ca2+] in the absence of calbindin-D-28K, and in its presence the decay of the flash-induced fluorescence was due solely to the Ca2+ buffering by the protein. We developed a novel method for kinetic parameter derivation and used the synthetic Ca2+ buffer EGTA to confirm its validity. We provide evidence that calbindin-D-28K binds Ca2+ in at least two distinct kinetic patterns, one arising from high-affinity sites that bind Ca2+ with a k(on) comparable to that of EGTA (i.e., similar to1 x 10(7) M-1 s(-1)) and another with lower affinity and an approximately eightfold faster k(on). In view of the inability of conventional approaches to adequately resolve rapid Ca2+ binding kinetics of Ca2+ buffers, this method promises to be highly valuable for studying the Ca2+ binding properties of other biologically important Ca2+ binding proteins.