Hydrochloric Acid → Phosphoric Acid

Participants

Reaction Steps

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.

Provenance & Evidence