Assay of adenosine 3', 5' cyclic monophosphate by stimulation of protein kinase: a method not involving radioactivity
Handa, A.K.; Bressan, R.A.
Analytical Biochemistry 102(2): 332-339
1980
ISSN/ISBN: 0003-2697 PMID: 6252798 Document Number: 157406
To meet a need for a c cyclic AMP assay which is not subject to interference by compounds in plant extracts, and which is suitable for use on occasions separated by many 32P half-lives, an assay based on cAMP-dependent protein kinase was developed which does not require the use of .gamma.-32P ATP. Instead of measuring the cAMP-stimulated increase in the rate of transfer of .gamma.-32P phosphate from .gamma.-32P ATP to protein, the rate of loss of ATP from the reaction mixture is determined. The ATP remaining after the protein kinase reaction is assayed by ATP-dependent chemiluminescence of the firefly luciferin-luciferase system. Under conditions of the protein kinase reaction in which a readily measurable decrease in ATP concentration occurs, the logarithm of the concentration of ATP decreases in proportion to the cAMP concentration, i.e., the reaction can be described by the equation: ATP = ATP O e- cAMP kt. The assay based on this relationship can detect less than 1 pmol of cAMP. The levels of cAMP found with this assay after partial purification of the cAMP from rat tissue, algal cells and the media in which the cells were grown agreed with measurements made by the cAMP binding-competition assay of Gilman, and the protein kinase stimulation assay based on transfer of 32P phosphate from .gamma.-32P ATP to protein. All of the enzymes and chemicals required for the assay of cAMP by protein kinase catalyzed loss of ATP can be stored frozen for months, making the assay suitable for occasional use.