title: Purification and characterization of guinea pig serum aminoacylproline hydrolase (aminopeptidase P).
abstract: Aminoacylproline hydrolase (EC 3.4.11.9) of guinea pig serum has been obtained as two apparently homogeneous isoforms. Dialyzed serum was chromatographed successively on Affi-gel blue, hydroxyapatite, DE-cellulose, phenyl-Sepharose, an affinity matrix for angiotensin converting enzyme and concanavalin-Sepharose. On the latter matrix, 68% of the enzyme activity was eluted with alpha-methyl mannoside at 10 and 100 mM, and 29% was eluted with alpha-methyl glucoside, 500 mM, at 56 degrees C. The two fractions ('biantennary' and 'high mannose' fractions, respectively) were concentrated and then chromatographed separately on Sephacryl S-200HR. Both fractions were eluted as expected for a globular protein of Mr 217,000. On SDS-PAGE, under reducing and non-reducing conditions, each of the concanavalin-Sepharose fractions was separated into two protein bands, Mr 89,000 and Mr 81,500. Each of the bands was found to be N-blocked when N-terminal amino acid sequencing was attempted. The reaction of the 'biantennary' fraction with the synthetic substrate Arg-Pro-Pro-[3H]benzylamide was characterized in part: Km 0.7 microM, kcat 124.6 min-1, kcat/Km 1.78.10(8) M-1 min-1. Hydrolysis of the substrate was strongly inhibited by bradykinin and those of its lower homologs that contain two adjacent proline residues. Cu2+ was strongly inhibitory. Co2+ at 30 microM activated the enzyme, as did Mn2+, Mg2+ and Ca2+ at higher concentrations. Sulfhydryl compounds, including captopril, inhibited the enzyme as did 1,10-phenanthroline. Iodoacetamide and N-ethylmaleimide had no effects, but 4-hydroxymercuribenzoate conferred a partial inhibition over a remarkably wide concentration range: 0.34-1400 microM. Amastatin and bestatin did not inhibit the enzyme. Aminoacylproline hydrolase of guinea pig serum appears to be a heterogeneous, glycosylated metallo-enzyme with a high affinity for bradykinin and related peptides in which the sequence Pro-Pro, Xaa-Pro-Pro or Xaa-Pro-Hyp is N-terminal.
authors.affiliation: Department of Medicine, University of Miami School of Medicine, FL.
authors.forename: J W
authors.initials: JW
authors.lastname: Ryan
authors.forename: F
authors.initials: F
authors.lastname: Valido
authors.forename: P
authors.initials: P
authors.lastname: Berryer
authors.forename: A Y
authors.initials: AY
authors.lastname: Chung
authors.forename: J E
authors.initials: JE
authors.lastname: Ripka
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000097:blood / Q000302:isolation & purification*; D000818:Animals; D001920:Bradykinin / Q000378:metabolism; D002413:Cations, Divalent; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006023:Glycoproteins / Q000302:isolation & purification; D006168:Guinea Pigs; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight
chemical_list: D000596:Amino Acids; D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D006023:Glycoproteins; D008670:Metals; D000626:Aminopeptidases; C022750:X-Pro aminopeptidase; D001920:Bradykinin