Hydrochloric 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 the protein concentration, of the 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 an 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, protonpotassium-adenosine triphosphatase activity (H+.K+-ATPase) 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 protonpotassium-adenosine triphosphatase was determined. As a result, the compound of Example 2 had a 50% inhibitory concentration (IC50) of 0.015 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. additionwas added to 0.75 ml of a 70 mM
3. additioncontaining 30 to 80 μg/ml
4. concentrationin terms of the protein concentration
5. customof the enzyme preparation
6. customThe enzymatic reaction
7. customAfter this enzymatic reaction
8. waitwas continued for 20 minutes
9. customto terminate
10. customthe reaction
11. custom(at 4° C. and 3000 rpm for 15 minutes)

ORDレコード

ord-273158902ca44484b2e05efc4af559e2

出典データセット

uspto-grants-2004_05

DOI

10.6084/m9.figshare.5104873.v1

特許番号

US06734181B2