反应详情

To 100 μL of a buffer D (40 mmol/L Tris-HCl, pH: 7.4, 10 mmol/L MgCl2, 1 mM DTT, 2 μM cGMP) solution containing [3H]-cGMP (0.5 pa/mL), 10 μL of a compound solution for evaluation (a solution in which a compound was dissolved in DMSO and diluted so that the DMSO concentration became 5%) and 90 μL of a solution prepared by diluting the PDE9 enzyme solution prepared in the above with a buffer E (40 mmol/L Tris-HCl, pH: 7.4, 10 mmol/L MgCl2, 1 mM DTT, 1 mmol/L EGTA) were added under ice cooling. The resultant mixed solution was incubated at 30° C. for 10 min, and thereafter heated for 2 min in boiled water to stop the enzyme reaction of the PDE9. Then, the resultant was returned to room temperature; 50 μL of 5′-Nucleotidase (Biomol GmbH, 10 units/mL) was added thereto; and the resultant was incubated at 30° C. for 10 min to thereby convert [3H]-5′-GMP formed in the previous reaction to [3H]-guanosine. 500 μL of an anion exchange resin (Bio-Rad AG1-X2 resin, mesh size: 200-400, H2O:resin=2:1) was added to the resultant reaction liquid, and allowed to stand for 10 min, and thereafter centrifuged (2,000 rpm, 10 min); and a supernatant in which the [3H]-guanosine was present was transferred to a LumaPlate (PerkinElmer, Inc.), and the radioactivity was measured by a TopCount NXT microplate scintillation and luminescence counter (PerkinElmer, Inc.).

1. customprepared
2. additionby diluting the PDE9 enzyme solution
3. additionwere added under ice cooling
4. additionThe resultant mixed solution
5. customthe enzyme reaction of the PDE9
6. customwas returned to room temperature
7. addition50 μL of 5′-Nucleotidase (Biomol GmbH, 10 units/mL) was added
8. waitand the resultant was incubated at 30° C. for 10 min