Method for assaying enzyme activity
View Patent ↗The present invention relates to methods for assessing enzyme activity utilizing a phase partition system. In addition, the present invention also relates to methods for screening and identifying compounds that may be used, for example, for the treatment of diabetes, diabetes-related disorders, obesity, cardiovascular disease, cancer, and other diseases or disorders, using this phase partition system.
1. A method for measuring enzyme activity of a reaction comprising
combining an enzyme and radiolabeled substrate(s);
adding a phase-partition scintillation fluid;
incubating the reaction for at least two hours; and
detecting enzyme activity,
wherein said reaction does not require mixing following addition of the phase-partition scintillation fluid and wherein said reaction is conducted in a single reaction vessel.
2. The method of claim 1 , further comprising
terminating the reaction by acidification.
3. The method of claim 1 , wherein the reaction is conducted in organic resistant multiwell plates.
4. The method of claim 3 , wherein the multiwell plate comprises up to 96 wells.
5. The method of claim 3 , wherein the multiwell plate comprises greater than 96 wells.
6. The method of claim 3 , wherein the multiwell plate comprises up to 384 wells.
7. The method of claim 3 , wherein the multiwell plate comprises greater than 384 wells.
8. The method of claim 1 , wherein said method is used for high throughput screening.
9. The method of claim 1 , wherein said enzyme is selected from fatty acid synthase; acetyl CoA carboxylase; diacyiglycerol acetyltransferase; farnesyl diphosphate synthase; glycerol-3-phosphate O-acyltransferase; carnitine O-palmitoyltransferase 1; serine C-palmitoyltransferase; or phospholipase C gamma.
10. The method of claim 1 , comprising measuring enzyme activity by radiometric detection.
11. The method of claim 1 , wherein enzyme activity is measured by incorporation of a radiolabel-containing moiety into product molecules or loss of a radiolabel-containing moiety into substrate molecules.
12. The method of claim 2 , wherein an acid is selected from acetic acid, phosphoric acid, or hydrochloric acid.
13. A method for identifying compounds that modulate enzyme activity in a reaction comprising
combining an enzyme, radiolabeled substrate(s) and test compounds;
adding a phase-partition scintillation fluid;
incubating the reaction for at least two hours; and
detecting enzyme activity,
wherein said reaction does not require mixing following addition of the phase-partition scintillation fluid and wherein said reaction is conducted in a single reaction vessel.
14. The method of claim 13 , further comprising
terminating the reaction by acidification.
15. The method of claim 13 , wherein the reaction is conducted in organic resistant multiwell plates.
16. The method of claim 15 , wherein the multiwell plate comprises up to 96 wells.
17. The method of claim 15 , wherein the multiwell plate comprises greater than 96 wells.
18. The method of claim 15 , wherein the multiwell plate comprises up to 384 wells.
19. The method of claim 15 , wherein the multiwell plate comprises greater than 384 wells.
20. The method of claim 13 , wherein said method is used for high throughput screening.
21. The method of claim 13 , wherein said enzyme is selected from fatty acid synthase; acetyl CoA carboxylase; diacylglycerol acetyltransferase; farnesyl diphosphate synthase; glycerol-3-phosphate O-acyltransferase; camitine O-palmitoyltransferase 1; serine C-palmitoyltransferase; or phospholipase C gamma.
22. The method of claim 13 , comprising measuring enzyme activity by radiometric detection.
23. The method of claim 13 , wherein enzyme activity is measured by incorporation of a radiolabel-containing moiety into product molecules or loss of a radiolabel-containing moiety into substrate molecules.
24. The method of claim 14 , wherein an acid is selected from acetic acid, phosphoric acid, or hydrochloric acid.