Bruzzone Santina, Guse Andreas H
Department of Experimental Medicine (DIMES), Section of Biochemistry, University of Genova, 16132 Genova, Italy.
Cold Spring Harb Protoc. 2013 Jun 1;2013(6):564-8. doi: 10.1101/pdb.prot072991.
Cyclic ADP-ribose (cADPR) is a Ca(2+)-mobilizing second messenger involved in the regulation of various physiological processes. The ability to detect changes in endogenous cADPR is a fundamental step in the identification of its role in signal transduction triggered by hormones and other stimuli. Because the intracellular concentration of cADPR can be very low, depending on the expression level of the ADP-ribosyl cyclase activity (forming cADPR and nicotinamide from NAD) in the cell type of interest, very sensitive and selective methods are required. The method presented here exploits the ability of the ADP-ribosyl cyclase to catalyze the reverse reaction (i.e., to synthesize NAD stoichiometrically starting from cADPR) in the presence of an excess of nicotinamide. The generation of NAD can be coupled to a cycling assay using the enzymes alcohol dehydrogenase and diaphorase. The former reduces NAD to NADH in the presence of ethanol and the latter oxidizes NADH to NAD in the presence of resazurin and flavin mononucleotide. The formation of the fluorescent reduced resazurin (resofurin) can be detected with a plate reader. Thus, this cycling assay for cADPR determination can be considered a high-throughput method, potentially screening cADPR concentration simultaneously in many samples.
环磷酸腺苷核糖（cADPR）是一种参与多种生理过程调节的钙离子动员第二信使。检测内源性cADPR变化的能力是确定其在激素和其他刺激引发的信号转导中作用的基本步骤。由于cADPR的细胞内浓度可能非常低，这取决于目标细胞类型中ADP - 核糖基环化酶活性（从NAD形成cADPR和烟酰胺）的表达水平，因此需要非常灵敏和特异的方法。本文介绍的方法利用了ADP - 核糖基环化酶在过量烟酰胺存在下催化逆反应（即从cADPR化学计量合成NAD）的能力。NAD的生成可以与使用醇脱氢酶和黄递酶的循环测定法偶联。前者在乙醇存在下将NAD还原为NADH，后者在刃天青和黄素单核苷酸存在下将NADH氧化为NAD。荧光还原型刃天青（试卤灵）的形成可以用酶标仪检测。因此，这种用于测定cADPR的循环测定法可被视为一种高通量方法，有可能同时在许多样品中筛选cADPR浓度。
