Membrane-bound aminopeptidase P from bovine lung : its purification, properties, and degradation of bradykinin

摘要: The membrane-bound form of aminopeptidase P (aminoacylprolyl-peptide hydrolase) (EC 3.4.11.9) was purified to apparent homogeneity from bovine lung microsomes. enzyme solubilized using phosphatidylinositol-specific phospholipase C (Bacillus thuringiensis), indicating that amino-peptidase is attached membranes via a glycosylphosphatidylinositol anchor. 1900-fold with yield 25% by chromatography on decyl-agarose, omega-aminodecyl-agarose, second decylagarose column, DEAE-Sephacel, and an ultrafiltration step. Native gradient polyacrylamide gel electrophoresis revealed single stained protein band whose position in the corresponded cleavage Arg1-Pro2 bond bradykinin. Mr 360,000 permeation 95,000 reducing sodium dodecyl sulfate-polyacrylamide electrophoresis. substrate specificity determined approximately 50 peptides proline position. could hydrolyze lower NH2-terminal homologs bradykinin, including Arg-Pro-Pro, which used as routine rapid fluorescence assay performed absence added Mn2+. Some having amino acids other than arginine were also cleaved. Aminopeptidase appeared favor had 2 residues or analogs positions 3 substrate. In general, tripeptides residue poor substrates. inhibited series peptides, 3-8 long, Pro-Pro sequence. metal-chelating agents, 2-mercaptoethanol (4 mM), p-chloromercuribenzenesulfonic acid, NaCl at concentrations greater equal 0.25 M. pH optimum 6.5-7.0 most stable basic range. A role for pulmonary inactivation circulating bradykinin proposed.