Purification and characterization of l,(l/d)-aminopeptidase from Guinea pig serum.
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.
Krstanović
Marina
M
Institute of Immunology Inc., Zagreb, Croatia. marina.krstanovic@belupo.hr
Brgles
Marija
M
Halassy
Beata
B
Frkanec
Ruza
R
Vrdoljak
Anto
A
Branović
Karmen
K
Tomasić
Jelka
J
Benedetti
Fabio
F
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
D009842:Oligopeptides; D010457:Peptidoglycan; D011480:Protease Inhibitors; D000626:Aminopeptidases; D000409:Alanine