An aminopeptidase B from porcine liver was purified about 2,000-fold by ammonium sulfate fractionation and a series of chromatographies on hydroxyapatite, DEAE-cellulose, Sephadex G-150, hydroxyapatite and DEAE-Sepharose columns. The purified preparation was homogeneous on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was about 58,000 as determined by gel filtration on Sephadex G-100 and disc gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme exhibited maximum activity at pH 7.5 for the hydrolysis of L-arginine beta-naphthylamide at 25 degrees C. The enzyme was labile to prolonged warming and freezing. The enzyme was markedly stimulated by chloride ion, and was inhibited by Cu2+, Zn2+, Cd2+, Hg2+, Pb2+, and metal chelating agents. p-Chloromercuribenzoate was an uncompetitive inhibitor of the enzyme with a Ki value of 1.8 X 10(-6) M. The enzyme was inhibited by 1,10-phenanthroline and the inhibition was of the mixed type with a K1 value of 1.9 X 10(-4) M but activity was restored by the addition of Zn2+, Co2+, and Fe2+.
A proline iminopeptidase was purified about 18,000-fold from apricot seeds (Prunus armeniaca LINN.) by a five-step procedure comprised of extraction from seeds, ammonium sulfate fractionation, DEAE-cellulose chromatography, CM-Sepharose chromatography, and rechromatography on CM-Sepharose. The purified enzyme had a molecular weight of 220,000 by gel filtration and 55,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This indicates that the native enzyme may be composed of four identical subunits. The isoelectric point was 6.2 as determined by gel electrofocusing. The pH optimum for L-proline beta-naphthylamide was between pH 7.5 and 8.0, and the enzyme was stable in the pH 6.5-to-8.0 region and up to 40 degrees C. The enzyme was specific for L-proline beta-naphthylamide among various amino acid beta-naphthylamides, and it also hydrolzyed L-prolylglycine and L-prolylglycylglycine. The enzyme was strongly inhibited by p-chloromercuribenzoate, 5,5'-dithiobis(2-nitrobenzoic acid), N-ethylmaleimide, and heavy metal ions, but was not activated significantly by thiol compounds. Moreover, the enzyme was inactivated by diethyl pyrocarbonate, p-bromophenacyl bromide, and photooxidation, but was not affected by diisopropyl fluorophosphate, phenylmethanesulfonyl fluoride, bestatin, puromycin, or metal chelating agents. No activation of the enzyme was observed on addition of metal ions. These results suggest that the enzyme is not classifiable as a metalloenzyme, and that cysteine and histidine residues may participate in the enzyme activity.
An aminopeptidase was purified about 700-fold from porcine liver homogenate by ammonium sulfate fractionation and a series of column chromatographies on DEAE-cellulose, Sephadex G-150, DEAE-Sepharose, and hydroxyapatite. The purified enzyme had a specific activity of 4.6 mumol X min-1 X mg-1 using L-leucine beta-naphthylamide as the substrate. The molecular weight of the enzyme was about 96,000 as determined by Sephadex G-150 column chromatography. The enzyme had a broad specificity and a pH optimum between 6.5 and 7.0 for hydrolysis of alpha-aminoacyl-beta-naphthylamines, and it hydrolyzed the beta-naphthylamides of aliphatic, basic, and aromatic amino acids. The enzyme also hydrolyzed peptide substrates with phenylalanine residues as their amino-termini, but it did not hydrolyze L-phenylalanyl-L-proline. The enzyme was inhibited by metal-chelating agents, sulfhydryl reagents, heavy metals, bestatin, and puromycin. Activity of the enzyme inhibited by sulfhydryl-reactive reagents was restored by the addition of sulfhydryl compounds. The enzyme was activated by cobaltous ion and the values of both Km and Vmax increased. The activation was pH-dependent and above pH 7.5 cobalt ion behaved as an inhibitor of the enzyme. No metal ions other than cobaltous ion stimulated the enzyme appreciably.
An aminopeptidase was purified about 1,700-fold from seeds of Japanese apricot (Prunus mume Sieb.) by a seven-step procedure comprising extraction from seeds, ammonium sulfate fractionation, DEAE-cellulose chromatography, first and second DEAE-Sepharose chromatography, hydroxyapatite chromatography, and Sephadex G-200 gel filtration. The purified enzyme was shown to be homogeneous on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be about 56,000 by gel filtration on Sephadex G-200 and the isoelectric point was 4.9. The pH optimum for L-leucine beta-naphthylamide was between pH 6.5 and 7.0, and the enzyme was stable in the pH 5.0 to 8.3 region and up to 50 degrees C. The enzyme hydrolyzed a variety of aminopeptidase substrates with a free alpha-amino group. Of the amino acid beta-naphthylamides, the enzyme was highly specific for substrates with a hydrophobic side chain in the amino terminal residue. The enzyme was strongly inhibited by p-chloromercuribenzoate, heavy metals, diethyl pyrocarbonate, and photooxidation with methylene blue, but was not affected by thiol compounds, peptidase inhibitors of microbial origin, such as bestatin and puromycin, or metal chelating agents. No activation of the enzyme by metal ions was observed. These results suggest that the enzyme is a true aminopeptidase in which cysteine and histidine residues participate in the catalytic process, and is not classifiable as a metalloenzyme.
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.
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