Trichloroacetic Acid → Phosphoric Acid

A microsomal fraction prepared in accordance with the method of Sachs, et al. [J. Bio., Chem., 251, 7690 (1976)] by homogenizing a fresh gastric mucosal layer of swine and then subjecting the homogenate to density gradient ultra centrifugation was employed as a proton potassium-adenosine triphosphatase preparation. A solution (10 μl) of a test compound dissolved in dimethyl sulfoxide was added to 0.75 ml of a 70 mM tris-hydrochloric acid buffer (5 mM magnesium chloride, 20 mM potassium chloride, pH=6.85) containing 30 to 80 μg/ml, in terms of a protein concentration, of an enzyme preparation. The mixture was incubated with 200 times/min of agitation at 37° C. for 45 minutes. The enzymatic reaction was started by adding 0.25 ml of a 8 mM solution of disodium adenosine triphosphate. After this enzymatic reaction was continued for 20 minutes, 1 ml of a 10% trichloroacetic acid-activated charcoal (100 mg) solution was added to terminate the reaction. The reaction mixture was centrifuged (at 4° C. and 3000 rpm) for 15 minutes. Inorganic phosphoric acid formed by the hydrolysis of adenosine triphosphate in the supernatant was subjected to colorimetry by the method of Yoda, et al. [Biochem. Biophys, Res. Commun., 40, 880 (1970)]. The amount of inorganic phosphoric acid in a reaction mixture free from potassium chloride was also measured. By subtracting this amount from the amount of inorganic phosphoric acid in the presence of potassium chloride, proton potassium-adenosine triphosphatase activity was determined. An inhibition ratio (%) was determined from the active value of the control and the active value of the test compound at each concentration, whereby a 50% inhibitory concentration (IC50 μg/ml) against proton-potassium-adenosine triphosphatase was determined. As a result, the compound of Example 1 had a 50% inhibitory concentration (IC50) not greater than 0.1 μg/ml, exhibiting excellent activity.

1. customA microsomal fraction prepared in accordance with the method of Sachs, et al. [J. Bio., Chem., 251, 7690 (1976)]
2. additioncontaining 30 to 80 μg/ml
3. concentrationin terms of a protein concentration
4. customof an enzyme preparation
5. customThe enzymatic reaction
6. customAfter this enzymatic reaction
7. waitwas continued for 20 minutes
8. customto terminate
9. customthe reaction
10. custom(at 4° C. and 3000 rpm)
11. customfor 15 minutes

ORD-Eintrag

ord-4133a7b5f7da424f936dfab3a28529fa

Quelldatensatz

uspto-grants-2003_12

DOI

10.6084/m9.figshare.5104873.v1

Patentnummer

US06670360B2