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A new method for separating cyclic AMP from 5′-AMP with application to the assay for cyclic AMP phosphodiesterase

✍ Scribed by Asru K. Sinha; Robert W. Colman

Publisher
Elsevier Science
Year
1981
Tongue
English
Weight
567 KB
Volume
113
Category
Article
ISSN
0003-2697

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✦ Synopsis

A new method for assay of cyclic AMP phosphodiesterase (EC 3.1.4.17) has been developed based on the observation that a mixture of cyclic AMP and AMP can be resolved on a column of florisil (activated magnesium silicate) at pH 7.0. The cyclic nucleotide is retained by the silicate and the AMP which is not adsorbed is virtually quantitatively recovered. The adsorption of cyclic AMP by florisil is greatly influenced by the pH of the buffer but independent of its ionic strength. In the actual assay cyclic['H]AMP is incubated with the enzyme source in the presence of Mg*+ and the reaction is stopped by the addition of CCI,COOH (0.3 M). The mixture is then neutralized by dilution with 10 vol of 0.5 M sodium phosphate buffer, pH 7.0, and applied on a small (0.4 X 4.0~cm) florisil column equilibrated with the same buffer. The column is eluted with 3 vol of the buffer and the radioactivity of the eluate which contains only [jH]AMP is measured. The use of cyclic[3H]AMP of high specific activity in the assay allows a high degree of sensitivity while the addition of CCI$OOH instantaneously terminates the reaction allowing for increased precision. The assay compares favorably in simplicity and speed with those currently employed for cyclic AMP phosphodiesterase.

Methods

Chemicals. Florisil (activated magnesium silicate), 100-200 mesh, was brought from Sigma Chemical Company,

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