Enzymatic fluorometric assay for adenylate cyclase
AssigneeRegents of the University of Minnesota
InventorsKeith G. Lurie, Phi Wiegn, Atsushi Sugiyama
Life of this patent
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Abstract
A method for measuring the amount of adenylate cyclase without the use of radioactive reagents is provided. The method comprises combining a sample of physiological material containing an amount of cAMP with (a) a mixture of enzymes effective to eliminate any other endogenous adenine nucleotides which may be present in the sample; and (b) an amount of alkaline phosphatase effective to eliminate any glucose-6-phosphate present in the sample. The cAMP present in said sample is then converted to AMP and the amount of AMP measured, which may then be correlated to the amount of cAMP and AC present in the sample.
Claims16
A method of measuring an amount of adenylate cyclase (AC) in a sample of physiological material comprising:
- (a) combining a sample of physiological material comprising (i) cAMP produced by endogenous AC, (ii) other endogenous adenine nucleotides selected from the group consisting of ATP, AMP, ADP and mixtures thereof, and (iii) glucose-6-phosphate, with effective amounts of apyrase, 5'-nucleotidase and adenosine deaminase, to enzymatically eliminate said other endogenous adenine nucleotides in said sample and with an amount of alkaline phosphatase effective to enzymatically eliminate said glucose-6-phosphate in said sample;
- (b) enzymatically converting the cAMP to AMP;
- and
- (c) measuring the AMP without the use of radioactive reagents, said measurement indicating 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 (i) cAMP produced by endogenous AC, (ii) at least on other noncyclic adenine nucleotide;
- and (iii) glucose-6-phosphate;
- with a mixture of apyrase, 5'-nucleotidase, adenosine deaminase and alkaline phosphatase in aqueous buffer to from a reaction mixture so as to destroy at least on other adenine nucleotide and the glucose-6-phosphate, while retaining said cAMP in a 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-phosphogluconolactone;
- and
- (c) measuring the concentration of NADPH fluorometrically, said concentration of NADPH providing a measure of the concentration of cAMP and AC in said sample.
The method of claims or 8 or 9 wherein excess glucose-6-phosphate, excess alpha-ketoglutarate, glutamate dehydrogenase, and glucose-6-phosphate dehydrogenase are combined with the amount of NADPH prior to fluorometry so that the alpha-ketoglutarate is converted to glutamate and NADP.sup.+, and the glucose-6-phosphate and the NADP.sup.+ are converted to 6-phosphogluconolactone and NADPH.
Dependencies are inferred from the claim text.