Systems and methods for determining cyclic nucleotide concentration and related pathway activation
The present invention relates in general to cellular analysis tools and more particularly to methods and systems for detecting or determining cyclic nucleotide concentrations and related pathway activation in samples. Samples containing cyclic nucleotides may be contacted with a cyclic nucleotide-dependent protein kinase (e.g., cAMP- and cGMP-dependent protein kinases) and a detection system that includes a substrate capable of being phosphorylated by the cyclic nucleotide-dependent protein kinase. The activity of cyclic nucleotide related cell signaling pathways may be measured based on detecting the activity of the cyclic nucleotide-dependent protein kinase.
1. A method for determining the cyclic nucleotide phosphodiesterase activity in a sample using a detection system, comprising:
(a) contacting the sample which may contain cyclic nucleotide phosphodiesterase activity with (I) an inactive enzyme capable of being activated by a cyclic nucleotide, wherein said inactive enzyme is Protein Kinase A, (II) ATP, (III) a cyclic nucleotide, and (IV) a detection system capable of detecting the activity of activated Protein Kinase A and generating a detectable signal, wherein said detection system comprises a substrate capable of being phosphorylated by Protein Kinase A; and
(b) determining the phosphodiesterase activity present in the sample based on said signal generated by the detection system.
2. The method of claim 1 , wherein said sample comprises a purified cyclic nucleotide phosphodiesterase (PDE) enzyme.
3. The method of claim 1 , wherein said substrate comprises SEQ ID NO: 1.
4. The method of claim 1 , wherein said detection system further comprises an enzyme capable of utilizing ATP to generate a luminescent signal.
5. The method of claim 4 , wherein the enzyme is a luciferase.
6. The method of claim 1 , wherein said substrate comprises a fluorescently -labelled substrate.
7. The method of claim 1 , further comprising the addition of an inhibitor of phosphodiesterase activity.
8. The method of claim 1 , wherein the cyclic nucleotide is cAMP.
9. The method of claim 1 , wherein the sample comprises a lysate.