Purification and characterization of pig kidney aminopeptidase P. A glycosyl-phosphatidylinositol-anchored ectoenzyme.
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 and hydrophobic-interaction chromatographies. Contaminating peptidase activities were removed by selective affinity chromatography. The purified enzyme was apparently homogeneous on SDS/PAGE with an Mr of 91,000. Enzymic deglycosylation revealed that aminopeptidase P is a glycoprotein, with up to 25% by weight of the protein being due to the presence of N-linked sugars. The phospholipase-solubilized aminopeptidase P was recognized by an antiserum to the cross-reacting determinant (CRD) characteristic of the glycosyl-phosphatidylinositol anchor. This recognition was abolished by mild acid treatment or deamination with HNO2, indicating that the CRD was due exclusively to the inositol 1,2-cyclic phosphate ring epitope generated by the action of PI-PLC. The activity of aminopeptidase P was inhibited by chelating agents and was stimulated by Mn2+ or Co2+ ions, confirming the metallo-enzyme nature of this peptidase. Selective inhibitors of other aminopeptidases (actinonin, amastatin, bestatin and puromycin) had little or no inhibitory effect.
Hooper
N M
NM
M.R.C. Membrane Peptidase Research Group, Department of Biochemistry, University of Leeds, U.K.
Hryszko
J
J
Turner
A J
AJ
D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D002413:Cations, Divalent; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006017:Glycolipids / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D010716:Phosphatidylinositols / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D012995:Solubility; D013552:Swine
D002413:Cations, Divalent; D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D010716:Phosphatidylinositols; D011480:Protease Inhibitors; D000626:Aminopeptidases; C022750:X-Pro aminopeptidase