title: HPLC purification and characterization of porcine muscle aminopeptidase B.
abstract: An aminopeptidase B from porcine skeletal muscle was successfully purified by ammonium sulphate fractionation and HPLC anion-exchange. The purified aminopeptidase B eluted at 0.18 M NaCl, had a relative molecular mass of 76,000 Da and was markedly stimulated in the presence of 0.2 M chloride anion. The enzyme exhibited maximum activity for the hydrolysis of the arginine-aminoacyl bond at pH 6.5 and 37 degrees C. Other substrates consisting of phenylalanine, proline and alanine-aminoacyl bonds were cleaved at 5.9, 5.1 and 2.5% of the maximum activity with the arginine-aminoacyl bond. The enzyme did not show endopeptidase activity and was very stable at pH above 6 and temperatures below 35 degrees C. However, the enzyme inactivated very fast when incubated at pH 5 or at 50-65 degrees C. Bestatin (50 microM) completely inhibited the aminopeptidase B activity while EDTA (5 mM) only inhibited 40% of its activity. However, 0.5 mM of E-64 did not cause any inhibition while 0.05 mM amastatin and 1 mM puromycin only inhibited 11% of the enzyme activity.
authors.affiliation: Instituto de Agroquímica y Tecnología de Alimentos (CSIC), Valencia, Spain.
authors.forename: M
authors.initials: M
authors.lastname: Flores
authors.forename: M C
authors.initials: MC
authors.lastname: Aristoy
authors.forename: F
authors.initials: F
authors.lastname: Toldrá
mesh_terms: D000626:Aminopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D001120:Arginine / Q000378:metabolism; D005591:Chemical Fractionation; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity; D013552:Swine; D013696:Temperature
chemical_list: D012965:Sodium Chloride; D001120:Arginine; D000626:Aminopeptidases; C019721:aminopeptidase B