Enzymatic fluorometric assay for adenylate cyclase
AssigneeRegents of the University of Minnesota
InventorsKeith G. Lurie, Phi Wiegn
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Abstract
A method of measuring adenylate cyclase (AC) in a sample of physiological material which does not employ radioactive reagents is provided, comprising: (a) providing a physiological sample of physiological material comprising cAMP produced by endogenous AC, and other endogenous adenine nucleotides selected from the group consisting of ATP, AMP, ADP and mixtures thereof; (b) combining the sample with effective amounts of apyrase, 5'-nucleotidase and adenosine deaminase, so as to enzymatically eliminate the other endogenous adenine nucleotides in the sample; (c) enzymatically converting the cAMP into AMP; and (d) measuring the amount of AMP, the amount providing a measure of the amount of cAMP and AC in the sample.
Claims15
A method measuring adenylate cyclase (AC) activity in a sample of physiological material comprising:
- (a) combining a sample of physiological material comprising cAMP produced by endogenous AC and other endogenous adenine nucleotides selected from the group consisting of ATP, AMP, ADP and mixtures thereof with effective amounts of apyrase, 5'-nucleotidase and adenosine deaminase, to enzymatically eliminate said other endogenous adenine nucleotides in said sample;
- (b) enzymatically converting the cAMP into AMP;
- and
- (c) measuring the amount of AMP without the use of radioactive reagents, said amount providing a measure of the amount of cAMP and AC in said sample.
A fluorometric method to measure adenylate cyclase (AC) comprising:
- (a) combining a sample of a physiological material comprising cAMP produced by endogenous AC, and at least one other noncyclic adenine nucleotide with a mixture of apyrase, 5'-nucleotidase and adenosine deaminase in aqueous buffer to destroy at least one other adenine nucleotide, while retaining said cAMP in the resulting reaction mixture;
- (b) converting said cAMP to AMP by combining said reaction mixture with phosphodiesterase, glycogen, glycogen phosphorylase a, inorganic phosphate, phosphoglucomutase, glucose-6-phosphate dehydrogenase, glucose-1,6-diphosphate, Mg.sup.2+ and NADP.sup.+ so that said glycogen is converted into NADPH and 6-phosphonogluconolactone;
- and
- (c) measuring the concentration of NADP fluorometrically, said concentration of NADPH providing a measure of the concentration of cAMP and AC in said sample.
Dependencies are inferred from the claim text.