Purification and characterisation of an alanine aminopeptidase from bovine skeletal muscle

A monomeric alanine aminopeptidase was purified to a single band in SDS–PAGE from bovine skeletal muscle by using a procedure including ammonium sulphate fractionation, adsorption on DEAE–cellulose, gel filtration on Ultrogel ACA 34, and adsorption on hydroxyapatite. Molecular weight determination by gel filtration and sodium dodecyl sulphate–polyacrylamide gel electrophoresis yielded a molecular size of 60 kDa. The aminopeptidase activity was optimal at pH 8.0 and 37 °C. It was totally abolished by Co2+ and Zn2+ ions, and almost completely inhibited by bestatin and Mn2+. The activity was strongly inactivated by phenylmethansulfonyl fluoride, Mg2+, and Fe3+ ions but stimulated by pepstatin and EDTA. However the activity was not affected by Ca2+, puromycin and iodoacetate. When compared with its activity toward Ala-β-naphthylamides (Ala-βNA), the enzyme exhibited 15–17% as much activity toward Pro- and Leu-βNA, 4–6% activity toward Met- and Arg-βNA, and negligible or no activity toward Glu- and Ser-βNA.