ADP detection based luminescent phosphotransferase or ATP hydrolase assay
View Patent ↗The invention provides compositions and methods to determine or detect the activity of enzymes, including phosphotransferases such as kinases (e.g., protein, lipid, and sugar kinases) and ATP hydrolases such as ATPases, e.g., HSP90, that employ ATP as a substrate and form ADP as a product by monitoring changes in ADP. Methods, kits and compositions for monitoring changes in metabolites such as ADP are disclosed.
1. A kit for detecting ADP in a solution comprising:
(i) an adenylate cyclase;
(ii) an enzyme that is capable of catalyzing the conversion of ADP to ATP;
(iii) a luciferase enzyme; and
(iv) a substrate for the luciferase enzyme.
2. The kit of claim 1 further comprising one or more of:
(v) a phosphate group donor;
(vi) a pyrophosphatase enzyme;
(vii) one or more detergents;
(viii) one or more buffer solutions; and/or
(ix) one or more salts.
3. The kit of claim 1 further comprising a phosphate group donor, wherein the enzyme that is capable of catalyzing the conversion of the ADP to ATP is a pyruvate kinase.
4. The kit of claim 3 wherein the phosphate donor group is phosphoenolpyruvate.
5. The kit of claim 1 , wherein the adenylate cyclase is a bacterial adenylate cyclase, or a fragment of a full length bacterial adenylate cyclase with adenylate cyclase activity.
6. The kit of claim 5 , wherein the bacterial adenylate cyclase or the fragment is from Bordetella pertussis or Bacillus anthracis.
7. The kit of claim 1 , wherein the adenylate cyclase is a eukaryotic adenylate cyclase, an algal adenylate cyclase, a porcine, non-human primate, canine, bovine, rodent, or human adenylate cyclase, or a fragment thereof with adenylate cyclase activity.
8. The kit of claim 7 , wherein the adenylate cyclase is selected from the group consisting of a calmodulin activated eukaryotic adenylate cyclase, a Spirulina platenis adenylate cyclase, ADCY1, ADCY2, ADCY3, ADCY4, ADCY5, ADCY6, ADCY7, ADCY8, ADCY9, ADCY10, adenylate cyclase isoform I, adenylate cyclase isoform III, adenylate cyclase isoform VIII, adenylate cyclase isoform V, adenylate cyclase isoform VI, and a fragment thereof with adenylate cyclase activity.
9. The kit of claim 8 , wherein the adenylate cyclase is adenylate cyclase isoform I, adenylate cyclase isoform III or adenylate cyclase isoform VIII, and a fragment thereof with adenylate cyclase activity, wherein the adenylate cyclase is stimulated by Ca 2+ /calmodulin.
10. The kit of claim 8 , wherein the adenylate cyclase is isoform V, adenylate cyclase isoform VI, and a fragment thereof with adenylate cyclase activity, wherein the adenylate cyclase is inhibited by Ca 2+ in a calmodulin-independent manner.
11. The kit of claim 1 further comprising phosphoenolpyruvate.