LOCALIZATION AND CHARACTERIZATION OF RAT SMALL-INTESTINAL AMINOPEPTIDASE-P AND ITS ROLE IN PROLYL PEPTIDE DIGESTION

The distribution of aminopeptidase P was examined in rat small intesti nal 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. So luble and brush border membrane associated activities differed with re spect to their pH optima, kinetic constants, and thermostability indic ating 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 chelat ing agents and was reactivated by addition of increasing concentration s of Mn2+. The mode of membrane association was investigated by deterg ent solubilization and enzymatic release of aminopeptidase P. A phosph atidylinositol-specific phospholipase C was the most effective in rele asing aminopeptidase P, suggesting that the en;yme is anchored via a g lycosyl-phosphatidylinositol moiety similar to alkaline phosphatase. T hese results indicate that aminopeptidase P is a major intestinal brus h border membrane enzyme and probably plays an important role in conju nction with other intenstinal prolyl peptidases in the digestion of pr oline containing peptides and proteins.