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首页> 外文期刊>Biochimica et biophysica acta: international journal of biochemistry and biophysics >3-O-methylfluorescein phosphate as a fluorescent substrate for plasma membrane Ca2+-ATPase.
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3-O-methylfluorescein phosphate as a fluorescent substrate for plasma membrane Ca2+-ATPase.

机译：3-O-甲基荧光素磷酸酯作为质膜Ca2 + -ATPase的荧光底物。

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摘要

3-O-methylfluorescein phosphate hydrolysis, catalyzed by purified erythrocyte Ca2+-ATPase in the absence of Ca2+, was slow in the basal state, activated by phosphatidylserine and controlled proteolysis, but not by calmodulin. p-Nitrophenyl phosphate competitively inhibits hydrolysis in the absence of Ca2+, while ATP inhibits it with a complex kinetics showing a high and a low affinity site for ATP. Labeling with fluorescein isothiocyanate impairs the high affinity binding of ATP, but does not appreciably modify the binding of any of the pseudosubstrates. In the presence of calmodulin, an increase in the Ca2+ concentration produces a bell-shaped curve with a maximum at 50 microM Ca2+. At optimal Ca2+ concentration, hydrolysis of 3-O-methylfluorescein phosphate proceeds in the presence of fluorescein isothiocyanate, is competitively inhibited by p-nitrophenyl phosphate and, in contrast to the result observed in the absence of Ca2+, it is activated by calmodulin. In marked contrast with other pseudosubstrates, hydrolysis of 3-O-methylfluorescein phosphate supports Ca2+ transport. This highly specific activity can be used as a continuous fluorescent marker or as a tool to evaluate partial steps from the reaction cycle of plasma membrane Ca2+-ATPases.
机译：在不存在Ca2 +的情况下，纯化的红细胞Ca2 + -ATPase催化的3-O-甲基荧光素磷酸水解在基础状态下缓慢，并由磷脂酰丝氨酸激活并控制蛋白水解，但不受钙调蛋白的影响。在不存在Ca2 +的情况下，对硝基苯基磷酸酯竞争性地抑制水解，而ATP以复杂的动力学抑制水解，显示出对ATP的高和低亲和力。用异硫氰酸荧光素标记会损害ATP的高亲和力结合，但不会明显改变任何假底物的结合。在钙调蛋白的存在下，Ca2 +浓度的增加会产生一个钟形曲线，最大值为50 microM Ca2 +。在最佳的Ca2 +浓度下，在异硫氰酸荧光素的存在下，3-O-甲基荧光素磷酸酯的水解会受到对硝基苯基磷酸酯的竞争性抑制，并且与在不存在Ca2 +的情况下观察到的结果相反，它会被钙调蛋白活化。与其他伪底物形成鲜明对比的是，磷酸3-O-甲基荧光素支持Ca2 +转运。这种高度特异性的活性可以用作连续荧光标记，也可以用作评估质膜Ca2 + -ATPases反应周期中部分步骤的工具。

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