ADP detection based methods using adenylate cyclase and bioluminescence
View Patent ↗The disclosure provides compositions and methods to determine or detect ADP or 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.
1. A method for detecting the presence or absence of or determining the amount of adenosine diphosphate (ADP) in a solution, comprising:
(a) converting substantially all ATP in the solution to cAMP and pyrophosphate using an adenylate cyclase:
(b) after step (a) converting ADP in the solution to ATP using an enzyme that catalyzes the conversion of ADP to ATP; and
(c) detecting the presence or absence of or determining the amount of the ATP produced in (b) using a bioluminescent reaction.
2. The method of claim 1 further comprising
removing substantially all pyrophosphate produced in (a) prior to (b) or (c).
3. The method of claim 1 further comprising
inhibiting the adenylate cyclase prior to (b) or (c) with a composition comprising an inhibitor.
4. The method of claim 1 wherein ADP is formed in the solution prior to (a) by catalysis with an ADP-generating enzyme, and wherein the bioluminescent reaction provides a measure of the ADP-generating enzyme.
5. The method of claim 4 wherein the ADP-generating enzyme is an enzyme that catalyzes the conversion of ATP to ADP.
6. The method of claim 5 wherein the ADP-generating enzyme is a kinase or an ATP hydrolase.
7. The method of claim 6 further comprising
performing a kinase reaction or an ATP hydrolase reaction in the presence or absence of a known or putative inhibitor of the kinase reaction or the ATP hydrolase reaction prior to step (a).
8. The method of claim 1 wherein the adenylate cyclase is an active fragment of a full length bacterial adenylate cyclase with adenylate cyclase activity.
9. The method of claim 1 wherein the enzyme that is capable of catalyzing the conversion of the ADP to ATP is a pyruvate kinase.
10. The method of claim 1 wherein the bioluminescent reaction comprises a luciferase enzyme and a substrate for the luciferase enzyme.
11. The method of claim 2 wherein (d) is performed with a pyrophosphatase enzyme.
12. The method of claim 1 , wherein the wherein the adenylate cyclase is a bacterial adenylate cyclase, or a fragment of a full length bacterial adenylate cyclase with adenylate cyclase activity.
13. The method of claim 1 , wherein the bacterial adenylate cyclase or the fragment is from Bortedella pertussis or Bacillus anthracis.
14. The method of claim 1 , wherein the adenylate cyclase is a eukaryotic adenylate cyclase, an algal adenylate cyclase or an isoform of adenylate cyclase selected from I, III and VIII, which are stimulated by Ca 2+ /calmodulin, and V and VI, which are inhibited by Ca 2+ in a calmodulin-independent manner, or a fragment thereof with adenylate cyclase activity.
15. The method of claim 1 , wherein the bioluminescent reaction of (c) occurs simultaneously with the conversion of ADP to ATP of (b).
16. The method of claim 1 , wherein the adenylate cyclase is an active recombinant fragment of a bacterial adenylate cyclase.
17. The method of claim 16 , wherein the bacterial adenylate cyclase is from Bortedella pertussis or Bacillus anthracis.
18. The method of claim 1 , wherein steps (a) (b) and (c) are carried out as a homogeneous assay.
19. The method of claim 1 , wherein steps (a) (b) and (c) are performed in a single reaction vessel.
20. The method of claim 10 , wherein the luciferase enzyme is a recombinant luciferase enzyme.