Cyclic AMP assay

Cyclic AMP assay. Cyclic AMP levels were measured on 6% cold trichloracetic acid extracts of sonicated cell extracts as described previously (14). The succinylated tyrosyl derivative of cyclic AMP... 

For the cyclic AMP assay [y- P]ATP (10 uM) is incubated with 10 mM magnesium acetate, cyclic AMP standard (0-10 pmol) or water, histone mixture, cyclic AMP-activated protein kinase, and 0.1 M sodium acetate buffer, pH 6.0, for 5 min at 30°C. The reaction is terminated by the addition of trichloroacetic-acid-tungstate-sulfuric acid, the precipitate is dissolved in N NaOH, and the radioactivity counted in scintillation fluid. 

One other cannabinoid receptor agonist of note is 3-(5 -cyano-l, l -dimclhyl-pentyl)-l-(4-N-morpholinobutyryloxy)-A -THC hydrochloride (0-1057). Thus, unlike all established cannabinoid receptor agonists, this is readily soluble in water and yet, compared to CP55940, its potency in the cyclic AMP assay is just... 

Group II assays consist of those monitoring cellular second messengers. Thus, activation of receptors to cause Gs-protein activation of adenylate cyclase will lead to elevation of cytosolic or extracellularly secreted cyclic AMP. This second messenger phosphorylates numerous cyclic AMP-dependent protein kinases, which go on to phosphorylate metabolic enzymes and transport and regulatory proteins (see Chapter 2). Cyclic AMP can be detected either radiometrically or with fluorescent probe technology. 

Enzyme Assay. Na , K -ATPase, and sarcoplasmic reticulum Ca - ATPase were prepared from rat hearts (22) and dog hearts (23), respectively. Bovine heart cyclic AMP phosphodiesterase was purchased from Sigma. The enzyme reaction was carried out after 5-min pretreatment with the drug, and the amount of inorganic phosphate liberated during the reaction period was determined. 

Gabriel, D., Vernier, M., Pfeifer, M.J., Dasen, B., Tenaillon, L., and Bouhelal, R., High throughput screening technologies for direct cyclic AMP measurement, Assay Drug Dev. Technol., 1, 291, 2003. 

Dopaminergic neurons synapse upon the parenchymal cells of the intermediate lobe (IL) of the rat pituitary gland. Dopamine decreases the capacity of the IL cells to synthesize cyclic AMP and inhibits the release of otMSH and other peptides from this tissue. The presence of a D-2 receptor accounts for both of these phenomena. This D-2 dopamine receptor can be studied in a binding assay using [3H]-spiroperidol, a dopamine... 

Cyclic AMP phosphodiesterase was partially purified from rat brain and from rat heart. The assay was applicable at different levels of enzyme purification. 

Bovine serum albumin (BSA) and cyclic AMP (cAMP) are determined by a competitive binding enzyme immunoassay (315). With urease as label, an ammonia gas-sensing electrode is used to measure the amount of urease-labeled antigen bound to a double-antibody solid phase by continuously measuring the rate of ammonia produced from urea as substrate. The method yields accurate and sensitive assays for proteins (BSA less than 10 ng/mL) and antigens (cAMP less than 10 nM), with fairly good selectivity over cGMP, AMP, and GMP. 

For the assay of adenylate cyclase activity of washed dog heart particles, a medium has been employed which contained 40 mM Tris or 20 mM potassium phosphate buffer (pH 7.5) with 4 mM ATP, 6.6 mM magnesium sulphate, 13 mM caffeine, and 2-5 ixglml of bovine serum albumin in a volume of 1.8 ml [95]. The reaction was stopped by heating the vessels for 3 min in a boiling water bath. After centrifugation, the supernatant fluid was assayed for cyclic AMP by the dog liver phosphorylase-activation method. 

The most serious problem encountered with the assay system is a quantitative variability that is, a particular set of extracts assayed under identical conditions in separate assays may give figures which are quantitatively different. The reason for this is unknown. The relative cyclic AMP activity in the extracts is the same that is, when sample B contains twice as much cyclic AMP as sample A on day 1, it will also contain twice as much on day 2. However, the absolute values of cyclic AMP may vary. 

Skeletal muscle phosphorylase has also been used for the assay of cyclic AMP [135]. The muscle system is even more tedious than that with liver phosphorylase because of the high concentrations of phosphorylase b which must be used. This is necessitated by the stimulation of muscle phosphorylase b activity by 5 -AMP, the major catabolite of cyclic AMP. 

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