title: Purification and characterization of l,(l/d)-aminopeptidase from Guinea pig serum.
abstract: Mammalian sera contain enzymes that catalyze the hydrolytic degradation of peptidoglycans and molecules of related structure and are relevant for the metabolism of peptidoglycans. We now report on a novel L,(L/D)-aminopeptidase found in human and mammalian sera. The enzyme hydrolyses the pentapeptide L-Ala-D-iso-Gln-meso-DAP(omegaNH(2))-D-Ala-D-Ala yielding the free L-alanine and the respective tetrapeptide (K(M) 18 mM). L,(L/D)-aminopeptidase from guinea pig serum was highly purified in four chromatographic steps, up to 700-fold. Molecular weight of the enzyme was estimated by HPLC to be approximately 175,000. The configuration of alanine obtained by hydrolysis of the pentapeptide was determined by oxidation with L-amino acid oxidase. The amino acids sequence in the respective tetrapeptide was deduced from the results of mass spectrometry. The novel L,(L/D)-aminopeptidase also hydrolyzed alanine-4-nitroanilide (K(M)=0.6 mM) and several peptides comprising L-amino acids. Peptides containing D-amino acid at the amino end and L-Asp-L-Asp were not the substrates for this enzyme. The purified enzyme also exhibited enkephalin degrading activity, hydrolyzing enkephalins comprising L,L- and L,D-peptide bonds. The enzyme was inhibited strongly by metal chelating agents, bestatin and amastatin.
authors.affiliation: Institute of Immunology Inc., Zagreb, Croatia. marina.krstanovic@belupo.hr
authors.forename: Marina
authors.initials: M
authors.lastname: Krstanović
authors.forename: Marija
authors.initials: M
authors.lastname: Brgles
authors.forename: Beata
authors.initials: B
authors.lastname: Halassy
authors.forename: Ruza
authors.initials: R
authors.lastname: Frkanec
authors.forename: Anto
authors.initials: A
authors.lastname: Vrdoljak
authors.forename: Karmen
authors.initials: K
authors.lastname: Branović
authors.forename: Jelka
authors.initials: J
authors.lastname: Tomasić
authors.forename: Fabio
authors.initials: F
authors.lastname: Benedetti
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification*; D000818:Animals; D006168:Guinea Pigs / Q000097:blood*; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009842:Oligopeptides / Q000737:chemistry; D010084:Oxidation-Reduction; D010457:Peptidoglycan / Q000737:chemistry* / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity
chemical_list: D009842:Oligopeptides; D010457:Peptidoglycan; D011480:Protease Inhibitors; D000626:Aminopeptidases; D000409:Alanine