Abstract
The membrane-bound form of aminopeptidase P (aminoacylprolyl-peptide hydrolase) (EC 3.4.11.9) was purified to apparent homogeneity from bovine lung microsomes. The enzyme was solubilized using phosphatidylinositol-specific phospholipase C (Bacillus thuringiensis), indicating that bovine lung amino-peptidase P is attached to membranes via a glycosylphosphatidylinositol anchor. The enzyme was purified 1900-fold with a yield of 25% by chromatography on decyl-agarose, omega-aminodecyl-agarose, a second decylagarose column, DEAE-Sephacel, and an ultrafiltration step. Native gradient polyacrylamide gel electrophoresis revealed a single stained protein band whose position in the gel corresponded to cleavage of the Arg1-Pro2 bond of bradykinin. The Mr was 360,000 by gel permeation chromatography and 95,000 by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The substrate specificity of aminopeptidase P was determined using approximately 50 peptides with proline in the second position. The enzyme could hydrolyze lower NH2-terminal homologs of bradykinin, including Arg-Pro-Pro, which was used as the routine substrate in a rapid fluorescence assay performed in the absence of added Mn2+. Some peptides having NH2-terminal amino acids other than arginine were also cleaved. Aminopeptidase P appeared to favor peptides that had 2 proline residues or proline analogs in positions 2 and 3 of the substrate. In general, tripeptides having a single proline residue in position 2 were poor substrates. Aminopeptidase P was inhibited by a series of peptides, 3-8 residues long, having an NH2-terminal Pro-Pro sequence. The enzyme was also inhibited by metal-chelating agents, 2-mercaptoethanol (4 mM), p-chloromercuribenzenesulfonic acid, and NaCl at concentrations greater than or equal to 0.25 M. The purified enzyme had a pH optimum of 6.5-7.0 and was most stable in the basic pH range. A role for membrane-bound aminopeptidase P in the pulmonary inactivation of circulating bradykinin is proposed.
Highlights
From the Departmentof Molecular and Cellular Biochemistry, Loyola University of Chicago, Stritch School of Medicine, Maywood, Illinois 60153
The substrate specificity of aminopeptidase aminopeptidase P was originally studied in porcine kidney cortex [7] and was recently purified from this source [8].To date, there are no reports on the purification and characterization of aminopeptidase P from lung tissue
Because aminopeptidase P apparently requires a proline residue in the second position of a peptide substrate, this enzyme has the potential to be highly specific for the pulmonary inactivation of bradykinin (Argl-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-PheRArg)
Summary
4.4 8.5 25 a Phosphatidylinositol-specific phospholipase C from B. thuringiensis. Purification -fold 1 deoxycholate [4]. Relative and purified membrane-bound aminopeptidase P prepararesidual activity was determined using Arg-Pro-Pro as tions from bovine lung and pig kidney cortex, there seem the substrate. The pig kidney enzyme, like the bovine lung enzyme [4], was stimulated by Mn2+when GlyPro-Hyp was used as a substrateand was inhibited by metalchelating agents, thiol compounds such as 2-mercaptoethanol, and thiol-reactive agents such as o-hydroxymercuribenzenebound, soluble, and possibly even the circulating forms of aminopeptidase P may represent members of a multigene family or at least differentially modified geneproducts Along these lines, E. coli has recently been shown to possess two aminopeptidase P-like enzymes that have somewhat different specificities [33]. Typing the not yet been reported whether the pig kidney enzyme can cleave peptides such as Arg-Pro-Proequally well in thepres-
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