Biochemistry of Macromolecular Interactions Unit, Department of Structural Biology and Chemistry, Institut Pasteur, Université Paris Cité, CNRS UMR 3528, 75015 Paris, France.
Université Paris Cité, 75014 Paris, France.
Toxins (Basel). 2022 Oct 8;14(10):691. doi: 10.3390/toxins14100691.
Various bacterial pathogens are producing toxins that target the cyclic Nucleotide Monophosphate (cNMPs) signaling pathways in order to facilitate host colonization. Among them, several are exhibiting potent nucleotidyl cyclase activities that are activated by eukaryotic factors, such as the adenylate cyclase (AC) toxin, CyaA, from or the edema factor, EF, from . The characterization of these toxins frequently requires accurate measurements of their enzymatic activity in vitro, in particular for deciphering their structure-to-function relationships by protein engineering and site-directed mutagenesis. Here we describe a simple and robust in vitro assay for AC activity based on the spectrophotometric detection of cyclic AMP (cAMP) after chromatographic separation on aluminum oxide. This assay can accurately detect down to fmol amounts of CyaA and can even be used in complex media, such as cell extracts. The relative advantages and disadvantages of this assay in comparison with other currently available methods are briefly discussed.
多种细菌病原体产生毒素，靶向环核苷酸单磷酸 (cNMPs) 信号通路，以促进宿主定植。其中，一些毒素具有很强的核苷酸环化酶活性，被真核因子激活，如来自 的腺苷酸环化酶 (AC) 毒素 CyaA，或来自 的水肿因子 EF。这些毒素的特性通常需要在体外准确测量其酶活性，特别是通过蛋白质工程和定点突变来破译它们的结构-功能关系。在这里，我们描述了一种基于氧化铝层析后分光光度法检测环 AMP (cAMP) 的简单而稳健的 AC 活性体外测定法。该测定法可以准确检测低至 fmol 数量的 CyaA，甚至可以在复杂的介质中使用，如细胞提取物。简要讨论了与其他当前可用方法相比，该测定法的相对优缺点。
