Claims
- 1. A method for determining ATPase activity, comprising the steps of:
a. mixing radiolabeled [γ-33P]ATP with an ATP hydrolyzing enzyme; b. incubating reaction mixture a sufficient time to afford orthophosphate to be released from hydrolysis; c. adding a solution of molybdate to said reaction mixture to form a phosphomolybdate complex; d. contacting said phosphomolybdate complex with a scintillant hydrophobic surface; and e. measuring scintillation of said scintillant as a means to calculate the amount of said orthophosphate.
- 2. The method according to claim 1, wherein said ATP hydrolyzing enzyme is the E1 helicase protein from human papillomavirus.
- 3. The method according to claim 1, wherein said molybdate solution and hydrophobic surface are added simultaneously to said reaction mixture.
- 4. The method according to claim 1, further comprising the step of:
f. adding a solution of CsCl to said reaction mixture prior to counting.
- 5. The method according to claim 4, further comprising the step of:
g. adding a solution of citric acid to said CsCl-containing mixture prior to counting.
- 6. The method according to claim 5, wherein said CsCl and citric acid are added simultaneously to the reaction mixture.
- 7. The method according to claim 1, wherein said molybdate is at a final concentration of from 0.05% to 0.3% w/v.
- 8. The method according to claim 7, wherein said ammonium molybdate is at a final concentration of from 0.1% to 0.2% w/v.
- 9. The method according to claim 8, wherein said ammonium molybdate is at a final concentration of about 0.17% w/v.
- 10. The method according to claim 1, wherein said hydrophobic surface is selected from the group consisting of: polyvinyltoluene (PVT), Sephadex, latex, polystyrene, polyacrylamide, acrylamide, agarose, polypropylene, polycarbonate, and Sepharose.
- 11. The method according to claim 10, wherein said surface is polyvinyl toluene beads.
- 12. The method according to claim 1, wherein said CsCl is at a final concentration higher than 1M.
- 13. The method according to claim 12, wherein said CsCl is at a final concentration ranging from 2M and 4M.
- 14. The method according to claim 13, wherein said CsCl is at a final concentration of about 3.5M.
- 15. The method according to claim 1, wherein said citric acid is at a final concentration ranging from 0.05 and 0.2M.
- 16. The method according to claim 15, wherein said citric acid is at a final concentration of about 0.1M.
- 17. A method for screening inhibitors of a phosphate-hydrolyzing enzyme activity comprising the steps of: carrying out steps a to e according to the method of claim 1, in the presence and absence of a candidate inhibitor; and comparing the amount of phosphate in each case to calculate the levels of inhibition.
- 18. A method for screening inhibitors of a phosphate-hydrolyzing enzyme activity comprising the steps of: carrying out steps a to g according to the method of claim 5, in the presence and absence of a candidate inhibitor; and comparing the amount of phosphate in each case to calculate the levels of inhibition.
- 19. The method according to claim 18, wherein said phosphate-hydrolyzing enzyme is selected from the group consisting of: helicase, ATPase, and phosphatase.
- 20. The method according to claim 18, wherein said enzyme is an ATPase.
- 21. The method according to claim 18, wherein said ATPase is the E1 helicase from human papillomavirus.
Parent Case Info
[0001] This application is a Divisional of U.S. patent application Ser. No. 09/595,833 filed Jun. 16, 2000 and claims the priority benefits of U.S. Provisional Application No. 60/139,629 filed Jun. 17, 1999, the disclosures of which are incorporated by reference in their entireties.
Provisional Applications (1)
|
Number |
Date |
Country |
|
60139629 |
Jun 1999 |
US |
Divisions (1)
|
Number |
Date |
Country |
Parent |
09595833 |
Jun 2000 |
US |
Child |
10041522 |
Oct 2001 |
US |