Assay of adenosine 3',5' cyclic monophosphate by stimulation of protein kinase: a method not involving radioactivity
In order to meet a need for a cAMP assay which is not subject to interference by compounds in plant extracts, and which is suitable for use on occasions separated by many /sup 32/P half-lives, an assay based on cAMP-dependent protein kinase has been developed which does not require the use of (..gamma..-/sup 32/P)ATP. Instead of measuring the cAMP-stimulated increase in the rate of transfer of (..gamma..-/sup 32/P) phosphate from (..gamma..-/sup 32/P)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)/sub 0/ e/sup -(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 potein kinase stimulation assay based on transfer of (/sup 32/P) phosphate from (..gamma..-/sup 32/P)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.
- Research Organization:
- Michigan State Univ., East Lansing
- DOE Contract Number:
- EY-76-C-02-1338
- OSTI ID:
- 5368144
- Journal Information:
- Anal. Biochem.; (United States), Journal Name: Anal. Biochem.; (United States) Vol. 102:2; ISSN ANBCA
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
AMP
ATP
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOASSAY
BIOCHEMICAL REACTION KINETICS
CHEMILUMINESCENCE
COMPARATIVE EVALUATIONS
DAYS LIVING RADIOISOTOPES
ENZYMES
EQUATIONS
ISOTOPES
KINETIC EQUATIONS
KINETICS
LIGHT NUCLEI
LUCIFERASE
LUMINESCENCE
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
OXIDASES
OXIDOREDUCTASES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOSPHOTRANSFERASES
QUANTITY RATIO
RADIOASSAY
RADIOISOTOPES
REACTION KINETICS
TRANSFERASES