Approximately 40% of prescribed drugs exert their activity via GTP-binding protein-coupled receptors (GPCRs). Once activated, these receptors cause transient changes in the concentration of second messengers, e.g., cyclic adenosine 3′,5′-monophosphate (cAMP). Specific and efficacious genetically encoded biosensors have been developed to monitor cAMP fluctuations with high spatial and temporal resolution in living cells or tissue. A well characterized biosensor for cAMP is the Förster resonance energy transfer (FRET)-based Epac1-camps protein. Pharmacological characterization of newly developed ligands acting at GPCRs often includes numerical quantification of the second messenger amount that was produced. To quantify cellular cAMP concentrations, we bacterially over-expressed and purified Epac1-camps and applied the purified protein in a cell-free detection assay for cAMP in a multi-well format. We found that the biosensor can detect as little as 0.15 pmol of cAMP, and that the sensitivity is not impaired by non-physiological salt concentrations or pH values. Notably, the assay tolerated desiccation and storage of the protein without affecting Epac1-camps cyclic nucleotide sensitivity. We found that determination cAMP in lysates obtained from cell assays or tissue samples by purified Epac1-camps is a robust, fast, and sensitive assay suitable for routine and high throughput analyses.

中文翻译：

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建立敏感的基于荧光的环状核苷酸定量方法。

大约40％的处方药通过GTP结合蛋白偶联受体（GPCR）发挥其活性。一旦激活，这些受体会引起第二信使（例如环腺苷3'，5'-单磷酸酯（cAMP））的浓度发生瞬时变化。已经开发出了特定且有效的遗传编码生物传感器来监测活细胞或组织中具有高时空分辨率的cAMP波动。针对cAMP的特征鲜明的生物传感器是基于Förster共振能量转移（FRET）的Epac1-camps蛋白。对GPCR起作用的新开发配体的药理学表征通常包括所产生的第二信使量的数值定量。为了量化细胞cAMP浓度，我们以细菌的形式过度表达和纯化了Epac1-camps，并将纯化的蛋白质应用于多孔形式的cAMP的无细胞检测测定中。我们发现该生物传感器只能检测到0.15 pmol的cAMP，并且其灵敏度不受非生理盐浓度或pH值的影响。值得注意的是，该测定法可耐受蛋白质的干燥和存储，而不会影响Epac1-camps的环状核苷酸敏感性。我们发现，通过纯化的Epac1-camps从细胞测定或组织样品中获得的裂解物中测定cAMP是一种健壮，快速且灵敏的测定，适用于常规和高通量分析。并且非生理盐浓度或pH值不会影响灵敏度。值得注意的是，该测定法可耐受蛋白质的干燥和存储，而不会影响Epac1-camps的环状核苷酸敏感性。我们发现，通过纯化的Epac1-camps从细胞测定或组织样品中获得的裂解物中测定cAMP是一种健壮，快速且灵敏的测定，适用于常规和高通量分析。并且非生理盐浓度或pH值不会影响灵敏度。值得注意的是，该测定法可耐受蛋白质的干燥和存储，而不会影响Epac1-camps的环状核苷酸敏感性。我们发现，通过纯化的Epac1-camps从细胞测定或组织样品中获得的裂解物中测定cAMP是一种健壮，快速且灵敏的测定，适用于常规和高通量分析。