Methods for cyclic nucleotide determination

Abstract

The present invention relates in general to cellular analysis tools and more particularly to methods for detecting or determining cyclic nucleotide concentrations in samples.

1 . A method for determining the amount of cyclic nucleotides in a sample comprising: 
 a) providing a sample which may contain a cyclic nucleotide,    b) adding to said sample an inactive enzyme capable of being activated by said cyclic nucleotide,    c) adding a detection system capable of detecting the activity of said activated enzyme and generating a detectable signal, and    d) determining the amount of cyclic nucleotide present in said sample based on said signal.    
     
     
         2 . The method of  claim 1 , wherein the sample comprises a lysate.  
     
     
         3 . The method of  claim 2 , wherein said lysate is derived from eukaryotic cells.  
     
     
         4 . The method of  claim 1 , wherein said sample comprises plasma membranes.  
     
     
         5 . The method of  claim 1 , wherein said cyclic nucleotide is cAMP or cGMP.  
     
     
         6 . The method of  claim 1 , wherein said inactive enzyme is a cAMP dependent protein kinase or a cGMP dependent protein kinase.  
     
     
         7 . The method of  claim 1 , wherein said detection system comprises a substrate capable of being phosphorylated by cAMP dependent protein kinase or cGMP dependent protein kinase.  
     
     
         8 . The method of  claim 7 , wherein said substrate comprises SEQ ID NO: 1.  
     
     
         9 . The method of  claim 7 , wherein said detection system further comprises an enzyme capable of utilizing ATP to generate a luminescent signal.  
     
     
         10 . The method of  claim 9 , wherein said enzyme is a luciferase.  
     
     
         11 . The method of  claim 7 , wherein said substrate comprises a radioactively labeled biotinylated substrate.  
     
     
         12 . The method of  claim 11 , wherein said substrate further comprises SEQ ID NO: 1.  
     
     
         13 . The method of  claim 11 , wherein said detection system further comprises a streptavidin binding surface.  
     
     
         14 . The method of  claim 7 , wherein said substrate comprises a fluorescently labeled substrate.  
     
     
         15 . The method of  claim 14  wherein said substrate further comprises SEQ ID NO: 1.  
     
     
         16 . The method of  claim 14 , wherein said fluorescent label is a rhodamine moiety.  
     
     
         17 . The method of  claim 1 , further comprising the addition of one or more inhibitors of phosphodiesterases.  
     
     
         18 . The method of  claim 1 , further comprising the addition of an agonist or antagonist capable of affecting cyclic nucleotide amounts in said sample.  
     
     
         19 . The method of  claim 18 , wherein said agonist or antagonist modulates adenylyl cyclase activity.  
     
     
         20 . The method of  claim 18 , wherein said agonist or antagonist modulates G- protein coupled receptor activity.  
     
     
         21 . The method of  claim 18 , wherein said agonist or antagonist modulates phosphodiesterase activity.  
     
     
         22 . A method for determining adenylyl cyclase activity in a sample comprising: 
 a) providing a sample which may contain adenylyl cyclase,    b) adding to said sample an inactive enzyme capable of being activated by cAMP    c) adding a detection system capable of detecting the activity of said activated enzyme and generating a detectable signal, and    d) determining adenylyl cyclase activity present in said sample based on said signal.    
     
     
         23 . The method of  claim 22 , wherein said sample comprises a lysate.  
     
     
         24 . The method of  claim 23 , wherein said lysate is derived from eukaryotic cells.  
     
     
         25 . The method of  claim 22 , wherein said sample comprises plasma membranes.  
     
     
         26 . The method of  claim 22 , wherein said inactive enzyme is a cAMP dependent protein kinase.  
     
     
         27 . The method of  claim 22 , wherein said detection system comprises a substrate capable of being phosphorylated by cAMP dependent protein kinase.  
     
     
         28 . The method of  claim 27 , wherein said substrate comprises SEQ ID NO: 1.  
     
     
         29 . The method of  claim 27 , wherein said detection system further comprises an enzyme capable of utilizing ATP to generate a luminescent signal.  
     
     
         30 . The method of  claim 29 , wherein said enzyme is luciferase.  
     
     
         31 . The method of  claim 27 , wherein said substrate comprises a radioactively labeled biotinylated substrate.  
     
     
         32 . The method of  claim 31 , wherein said substrate further comprises SEQ ID NO:1.  
     
     
         33 . The method of  claim 31 , wherein said detection system further comprises a streptavidin binding surface.  
     
     
         34 . The method of  claim 27 , wherein said substrate comprises a fluorescently labeled substrate.  
     
     
         35 . The method of  claim 34 , wherein said substrate further comprises SEQ ID NO:1.  
     
     
         36 . The method of  claim 34 , wherein said fluorescent label is a rhodamine moity.  
     
     
         37 . The method of  claim 22 , further comprising the addition of one or more inhibitors of phosphodiesterases.  
     
     
         38 . The method of  claim 22 , further comprising the addition of an agonist or antagonist of adenylyl cyclase activity.  
     
     
         39 . A method for determining phosphodiesterase activity in a sample comprising: 
 a) providing a sample which may contain a phosphodiesterase,    b) adding to said sample an inactive enzyme capable of being activated by cAMP,    c) adding a detection system capable of detecting the activity of said activated enzyme and generating a detectable signal, and    d) determining phosphodiesterase activity present in said sample based on said signal.    
     
     
         40 . The method of  claim 39 , wherein said phosphodiesterase is a cyclic nucleotide phosphodiesterase.  
     
     
         41 . The method of  claim 40 , wherein said cyclic nucleotide is cAMP or cGMP.  
     
     
         42 . The method of  claim 39 , wherein said sample comprises a lysate.  
     
     
         43 . The method of  claim 39 , wherein said lysate is derived from eukaryotic cells.  
     
     
         44 . The method of  claim 39 , wherein said sample comprises plasma membranes.  
     
     
         45 . The method of  claim 39 , wherein said inactive enzyme is a cAMP dependent protein kinase or a cGMP dependent protein kinase.  
     
     
         46 . The method of  claim 39 , wherein said detection system comprises a substrate capable of being phosphorylated by cAMP dependent protein kinase or cGMP dependent protein kinase.  
     
     
         47 . The method of  claim 46 , wherein said substrate comprises SEQ ID NO: 1.  
     
     
         48 . The method of  claim 46 , wherein said detection system further comprises an enzyme capable of utilizing ATP to generate a luminescent signal.  
     
     
         49 . The method of  claim 48 , wherein said enzyme is a luciferase.  
     
     
         50 . The method of  claim 46 , wherein said substrate comprises a radioactively labeled biotinylated substrate.  
     
     
         51 . The method of  claim 50 , wherein said substrate further comprises SEQ ID NO:1.  
     
     
         52 . The method of  claim 46 , wherein said detection system further comprises a streptavidin binding surface.  
     
     
         53 . The method of  claim 46 , wherein said substrate comprises a fluorescently labeled substrate.  
     
     
         54 . The method of  claim 53 , wherein said substrate further comprises SEQ ID NO:1.  
     
     
         55 . The method of  claim 53 , wherein said fluorescent label is a rhodamine moity.  
     
     
         56 . The method of  claim 39 , further comprises the addition of one or more inhibitors of phosphodiesterase activity.  
     
     
         57 . A method for determining G-protein coupled receptor activity in a sample comprising: 
 a) providing a sample which may contain a G-protein coupled receptor,    b) adding to said sample an inactive enzyme capable of being activated by cAMP,    c) adding a detection system capable of detecting the activity of said activated enzyme and generating a detectable signal, and    d) determining G-protein coupled receptor activity present in said sample based on said signal.    
     
     
         58 . The method of  claim 57 , wherein said sample comprises a lysate.  
     
     
         59 . The method of  claim 58 , wherein said lysate is derived from eukaryotic cells.  
     
     
         60 . The method of  claim 57 , wherein said sample comprises plasma membranes.  
     
     
         61 . The method of  claim 57 , wherein said inactive enzyme is a cAMP dependent protein kinase.  
     
     
         62 . The method of  claim 57 , wherein said detection system comprises a substrate capable of being phosphorylated by cAMP dependent protein kinase.  
     
     
         63 . The method of  claim 62 , wherein said substrate comprises SEQ ID NO:1.  
     
     
         64 . The method of  claim 62 , wherein said detection system further comprises an enzyme capable of utilizing ATP to generate a luminescent signal.  
     
     
         65 . The method of  claim 64 , wherein said enzyme is luciferase.  
     
     
         66 . The method of  claim 62 , wherein said substrate comprises a radioactively labeled biotinylated substrate.  
     
     
         67 . The method of  claim 66 , wherein said substrate further comprises SEQ ID NO:1.  
     
     
         68 . The method of  claim 66 , wherein said detection system further comprises a streptavidin binding surface.  
     
     
         69 . The method of  claim 62 , wherein said substrate comprises a fluorescently labeled substrate.  
     
     
         70 . The method of  claim 69 , wherein said substrate further comprises SEQ ID NO:1.  
     
     
         71 . The method of  claim 69 , wherein said fluorescent label is a rhodamine moity.  
     
     
         72 . The method of  claim 57 , further comprising the addition of one or more inhibitors of phosphodiesterases.  
     
     
         73 . The method of  claim 57 , further comprising the addition of an agonist or antagonist of G-protein coupled receptor activity.  
     
     
         74 . A kit for determining the concentration of a cyclic nucleotide in a sample comprising: 
 a) a cyclic nucleotide,    b) a protein kinase,    c) a protein kinase substrate,    d) ATP, and    e) instructions for using said kit to determine said concentration of said cyclic nucleotide in said sample.    
     
     
         75 . A kit for determining the cyclic nucleotide phosphodiesterase activity in a sample comprising: 
 a) substrates for cAMP and cGMP,    b) a protein kinase,    c) a protein kinase substrate,    d) ATP, and    e) instructions for using said kit to determine said activity of said cyclic nucleotide phosphodiesterase in said sample.