Purification and Characterization of an Aminopeptidase from Bovine Leukocytes

Abstract

An aminopeptidase was purified about 4,000-fold from the clarified homogenate of bovine leukocytes by a series of column chromatographies on DEAE-cellulose, hydroxyapatite, Sephadex G-150, and DEAE-Toyopearl. The purified enzyme had a specific activity of 3.8 mumol X min-1 X mg-1 with arginine beta-naphthylamide (Arg-2-NNap) as substrate, and a minute amount of contaminating protein was found to be present by gel electrophoresis. The molecular weight of the enzyme was estimated to be 94,000 by gel filtration on Sephadex G-150. The enzyme had a broad substrate specificity and a pH optimum between 6.5 and 7.0 for the hydrolysis of alpha-aminoacyl beta-naphthylamides. It hydrolyzed beta-naphthylamides of basic, aliphatic, and aromatic amino acids, and also catalyzed the liberation of amino-terminal phenylalanine from phenylalanyl peptides. The enzyme was inhibited by bestatin, puromycin, 1,10-phenanthroline, sulfhydryl reagents, and a variety of heavy metal ions. Only the cobaltous ion stimulated the enzyme and the values of both Km and Vmax for Arg-2-NNap increased. In gross properties the present enzyme resembles porcine liver aminopeptidase reported previously (Kawata, S., et al. (1982) J. Biochem. 92, 1093-1101) very closely.

HPLC Purification and Characterization of Soluble Alanyl Aminopeptidase from Porcine Skeletal Muscle

“…The activity on alanyl-AMC derivative was maximal at pH 6.5 and is similar to that by Wagner et al (1981). Other authors have found maximal activity at pH 7.0 for other species (Schnebli et al, 1979;Kawata et al, 1982;Aratani et al, 1984;Ishiura et al, 1987;Hiroi et al, 1992). The enzyme activity sharply decreased to less than 10% of the maximal activity when assayed at acid (pH ) 5.0) or basic (pH ) 8.0) conditions.…”

Activity Staining and Inhibition Characterization of Dipeptidylpeptidase-III Enzyme from Goat Brain

“…Several other metalloenzymes having –SH group at the active site have also been reported earlier [12, 13]. …”

Basic Amino Acids Preferring Broad Specificity Aminopeptidase from Human Erythrocytes

“…Characteristics of its specificity are preference for Lys-and Arg-2NA but also an acceptance of Met-, Leu-and Phe-2NA as substrate. Broad specificity, Co 2 *-activated APs having similar Mr, pH optimum and patterns of sensitivity toward inhibitors and specificity toward aminoacyl-2NAs (estimated on the basis of V |nax /K m ratios) were isolated from the cytosol of bovine leukocytes [4], porcine liver [13] and skeletal muscle [14]. Broad specificity, Co 2 *-activated APs having similar Mr, pH optimum and patterns of sensitivity toward inhibitors and specificity toward aminoacyl-2NAs (estimated on the basis of V |nax /K m ratios) were isolated from the cytosol of bovine leukocytes [4], porcine liver [13] and skeletal muscle [14].…”

“…Reports on several APs from blood cells, able to hydrolyze Arg-2NA [1][2][3][4][5][6], rose a question of their similarity and belonging to one of the already established groups of APs [7], To clear dilemmas concerning AP from human red blood cells which we have previously described [2], a new isolation procedure for this enzyme was developed and the study of its properties was extended. Therefore more extensive data on catalytic properties and specificity are needed to compare and classify these enzymes properly.…”