Radioimmunoassay of cyclic AMP can provide a highly sensitive assay for adenylate cyclase, even at very high ATP concentrations.
Modifications of the cyclic AMP radioimmunoassay of Cailla et al. [in Hormones and Cell Regulation (J. Dumont and J. Nunez, eds.), Vol. 4, pp. 1-24, Elsevier/North-Holland, Amsterdam/New York (1980)] allowed its use in the determination of adenylate cyclase activity, which was otherwise precluded by high blank values. These high values originate mainly from chemically formed cyclic AMP and from ATP cross-reactivity. The simultaneous presence of ATP and magnesium ions generates cyclic AMP under the alkaline conditions used to succinylate the sample; this interference can be dealt with either by chelation of Mg2+ ions with EDTA during succinylation or by periodic acid oxidation of samples prior to succinylation. In addition, ATP itself contributes to blank values by its cross-reactivity, especially when working with high concentrations of this substrate. This interference can be decreased by a batch adsorption of ATP or oxidized ATP on alumina. Detailed procedures were discussed, with the choice of the additional steps to the standard method of Cailla et al. having to be made on the basis of the sensitivity requirements. When preventing ATP cyclization, the radioimmunoassay was as sensitive as methods using [alpha-32P]ATP as substrate. Elimination of ATP can improve the sensitivity by one order of magnitude. This method is especially interesting with high ATP concentrations and/or with low cyclic AMP production.
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D015056:1-Methyl-3-isobutylxanthine / Q000032:analysis; D000255:Adenosine Triphosphate / Q000032:analysis*; D000262:Adenylyl Cyclases / Q000032:analysis*; D000327:Adsorption; D000537:Aluminum Oxide; D002384:Catalysis; D055598:Chemical Phenomena; D002621:Chemistry; D000242:Cyclic AMP / Q000032:analysis*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D009711:Nucleotides / Q000032:analysis; D010084:Oxidation-Reduction; D010504:Periodic Acid; D011863:Radioimmunoassay
D009711:Nucleotides; D010504:Periodic Acid; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D008274:Magnesium; D000537:Aluminum Oxide; D015056:1-Methyl-3-isobutylxanthine