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

A soluble alanyl aminopeptidase (EC 3.4.11.14) from porcine skeletal muscle was purified by ammonium sulfate fractionation and anion-exchange HPLC. The enzyme eluted at 0.31 M NaCl, had a relative molecular mass of 106 000 (by SDS−polyacrylamide gel electrophoresis), and was markedly stimulated by sulfhydryl compounds, Co2+ and Ca2+. The enzyme exhibited maximum activity at pH 6.5 and 50 °C and showed a broad substrate specificity hydrolyzing aromatic, aliphatic, and basic aminoacyl bonds, but did not show endopeptidase activity. The affinity of the enzyme toward dipeptides was increased in the presence of an aromatic amino acid and the C-terminal side. Inhibition of enzyme activity was obtained in the presence of metal-chelating agents, sulfhydryl reagents, bestatin, amastatin, and puromycin. The enzyme was stable at temperatures below 15 °C and had a higher stability, 60% of initial activity after 3.5 months of incubation at −25 °C, in the presence of 50% ethylene glycol. Keywords: Alanyl aminopeptidase...