Purification and characterization of guinea pig serum aminoacylproline hydrolase (aminopeptidase P).
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.
Ryan
J W
JW
Department of Medicine, University of Miami School of Medicine, FL.
Valido
F
F
Berryer
P
P
Chung
A Y
AY
Ripka
J E
JE
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
D000596:Amino Acids; D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D006023:Glycoproteins; D008670:Metals; D000626:Aminopeptidases; C022750:X-Pro aminopeptidase; D001920:Bradykinin