Method for measuring nucleotides

A method of identifying a compound which increases the amount of a nucleotide generated by a testing medium, the method comprising:

• i) performing a trial comprising the steps of:
• (a) contacting a test compound with the testing medium in a testing vessel;
• (b) maintaining the test compound in contact with the testing medium in the testing vessel for a time sufficient to allow nucleotides to be generated in the testing medium;
• (c) releasing nucleotides generated in the testing medium into the testing vessel;
• (d) adding a radiolabeled nucleotide ligand and a fixed amount of a nucleotide binding protein to the testing vessel, wherein the radiolabeled nucleotide ligand competes with nucleotides generated in the testing medium to bind to the nucleotide binding protein;
• (e) maintaining the testing vessel at a temperature for a period of time sufficient to allow nucleotides generated in the testing medium and the radiolabeled nucleotide ligand to bind to the nucleotide binding protein to form a nucleotide binding protein complex;
• (f) separating the nucleotide binding protein complex from uncomplexed radiolabeled nucleotide ligand;
• and
• (g) measuring the level of radioactivity of the nucleotide binding protein complex, wherein the level of radioactivity is inversely proportional to the amount of the nucleotide generated in said testing medium;
• and
• ii) comparing the results from the trial to results produced from either:
• (a) a negative control in which no compound is contacted with the testing medium;
• (b) a positive control using a positive control compound as the test compound, wherein the positive control compound is a compound that increases the amount of the nucleotide generated in the testing medium;
• or
• (c) both a positive control and a negative control;
• wherein an amount of nucleotide generated in the testing medium is greater than the nucleotide that appears in the testing medium in the negative control and an amount of nucleotide generated in the testing medium is greater than or equal to the amount of nucleotide generated in the testing medium in the positive control is indicative of a test compound which can increase the amount of nucleotide generated in the a testing medium.

A method of identifying a compound with biological activity, the method comprising:

• i) performing a trial comprising the steps of:
• (a) contacting a test compound with a cell in a microtiter plate;
• (b) maintaining the test compound in contact with the cell in the microtiter plate for a time sufficient to allow cyclic adenosine 3′,5′-monophosphate to be generated in the cell;
• (c) releasing cyclic, adenosine 3′,5′-monophosphate generated in the cell into the microtiter plate;
• (d) adding radiolabeled cyclic adenosine 3′,5′-monophosphate and a fixed amount of a protein derived from a bovine adrenal gland to the microtiter plate, wherein the radiolabeled cyclic adenosine 3′,5′-monophosphate competes with cyclic adenosine 3′,5′-monophosphate generated in the cell to bind to the protein derived from a bovine adrenal gland;
• (e) maintaining the microtiter plate at a temperature for a period of time sufficient to allow cyclic adenosine 3′,5′-monophosphate generated in the cell and the radiolabeled cyclic adenosine 3′,5′-monophosphate to bind to the protein derived from a bovine adrenal gland to form a cyclic adenosine 3′,5′-monophosphate binding protein complex;
• (f) separating the cyclic adenosine 3′,5′-monophosphate binding protein complex from uncomplexed radiolabeled cyclic adenosine 3′,5′-monophosphate;
• and
• (g) measuring the level of radioactivity of the cyclic adenosine 3′,5′-monophosphate binding protein complex, wherein the level of radioactivity is inversely proportional to the amount of the cyclic adenosine 3′,5′-monophosphate generated in said cell;
• and
• ii) comparing the results from the trial to results produced from either:
• (a) a negative control in which no compound is contacted with the cell;
• (b) a positive control using a positive control compound as the test compound, wherein the positive control compound is a compound that increases the amount of the cyclic adenosine 3′,5′-monophosphate generated in a cell;
• or
• (c) both a positive and a negative control;
• wherein an amount of cyclic adenosine 3′,5′-monophosphate generated in the cell is greater than the cyclic adenosine 3′,5′-monophosphate that appears in the cell in the negative control and an amount of cyclic adenosine 3′,5′-monophosphate generated in the cell is greater than or equal to the amount of cyclic adenosine 3′,5′-monophosphate generated in the cell in the positive control is indicative of a test compound which can increase the amount of cyclic adenosine 3′,5′-monophosphate generated in a cell.