Acetylsalicylate hydrolase of rabbit gastric mucosa. Isolation and purification.

The mechanisn of hydrolysis of acetylsalicylate during absorption from the gastrointestinl tract has been investigated by identification, quantitation, and purification of a hydrolase from gastric mucosal homogenates. The hydrolase was found to be a soluble, cytosolic enzyme with a pH optimum in the slightly alkaline range, pH 8.6 Acetylsalicylate hydrolase activity was purified from the 100,000 g supernatant fraction by differential (NH4)2SO4 fractionation followed by DEAE-cellulose ion-exchange chromatography and Sephadex or Sephacryl gel filtration. The activity could also be fractionated on hydroxylapatite. The Sephadex-purified fraction containing peak enzyme activity gave a single protein band on SDS-polyacrylamide gel electrophoresis. The molecular weight of the acetylsalicylate hydrolase was 66,400 based on SDS-polyacrylamide gel electrophoresis of the Sephadex-purified enzyme and 59,000 based on gel filtration. By use of the technique described, acetylsalicylate hydrolase can be purified over 100-fold to a specific activity of 10.6 mumol . mg-1 . min-1.