Localization and characterization of rat small intestinal aminopeptidase P and its role in prolyl peptide digestion

Abstract The distribution of aminopeptidase P was examined in rat small intestinal epithelial cells using the substrate Gly-Pro-Hyp. Soluble activity accounted for 65% of the total activity in these cells while 27% was membrane bound and localized to the brush border membrane fraction. Soluble and brush border membrane associated activities differed with respect to their pH optima, kinetic constants, and thermostability indicating that they are due to different enzymes. Both activities, however, exhibited similar sensitivity to several types of inhibitors. Brush border membrane aminopeptidase P was readily inhibited by metal chelating agents and was reactivated by addition of increasing concentrations of Mn 2+ . The mode of membrane association was investigated by detergent solubilization and enzymatic release of aminopeptidase P. A phosphatidylinositol-specific phospholipase C was the most effective in releasing aminopeptidase P, suggesting that the enzyme is anchored via a glycosyl-phosphatidylinositol moiety similar to alkaline phosphatase. These results indicate that aminopeptidase P is a major intestinal brush border membrane enzyme and probably plays an important role in conjunction with other intestinal prolyl peptidases in the digestion of proline containing peptides and proteins.