Purification and characterization of pig kidney aminopeptidase P. A glycosyl-phosphatidylinositol-anchored ectoenzyme.

摘要: Aminopeptidase P (EC 3.4.11.9) was solubilized from pig kidney membranes with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) and then purified by a combination of anion-exchange hydrophobic-interaction chromatographies. Contaminating peptidase activities were removed selective affinity chromatography. The enzyme apparently homogeneous on SDS/PAGE an Mr 91,000. Enzymic deglycosylation revealed that aminopeptidase is glycoprotein, up to 25% weight the protein being due presence N-linked sugars. phospholipase-solubilized recognized antiserum cross-reacting determinant (CRD) characteristic glycosyl-phosphatidylinositol anchor. This recognition abolished mild acid treatment or deamination HNO2, indicating CRD exclusively inositol 1,2-cyclic phosphate ring epitope generated action PI-PLC. activity inhibited chelating agents stimulated Mn2+ Co2+ ions, confirming metallo-enzyme nature this peptidase. Selective inhibitors other aminopeptidases (actinonin, amastatin, bestatin puromycin) had little no inhibitory effect.