Cycling assay for determining intracellular cyclic adp-ribose levels

TY - JOUR

T1 - Cycling assay for determining intracellular cyclic adp-ribose levels

AU - Bruzzone, Santina

AU - Guse, Andreas H

PY - 2013/6/1

Y1 - 2013/6/1

N2 - 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.

AB - 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.

KW - ADP-ribosyl Cyclase

KW - Alcohol Dehydrogenase

KW - Cyclic ADP-Ribose

KW - Cytoplasm

KW - Ethanol

KW - NAD

KW - NADH Dehydrogenase

KW - Niacinamide

KW - Oxazines

KW - Xanthenes

U2 - 10.1101/pdb.prot072991

DO - 10.1101/pdb.prot072991

M3 - SCORING: Journal article

C2 - 23734016

VL - 2013

SP - 564

EP - 568

JO - Cold Spring Harbor protocols

JF - Cold Spring Harbor protocols

SN - 1559-6095

IS - 6

ER -