HPLC PURIFICATION AND CHARACTERIZATION OF SOLUBLE ALANYL AMINOPEPTIDASE FROM PORCINE SKELETAL-MUSCLE

A soluble alanyl aminopeptidase (EC 3.4.11.14) from porcine skeletal m uscle was purified by ammonium sulfate fractionation and anion-exchang e HPLC. The enzyme eluted at 0.31 M NaCl, had a relative molecular mas s of 106 000 (by SDS-polyacrylamide gel electrophoresis), and was mark edly stimulated by sulfhydryl compounds, Co2+ and Ca2+. The enzyme exh ibited maximum activity at pH 6.5 and 50 degrees C and showed a broad substrate specificity hydrolyzing aromatic, aliphatic, and basic amino acyl bonds, but did not show endopeptidase activity. The affinity of t he enzyme toward dipeptides was increased in the presence of an aromat ic amino acid and the C-terminal side. Inhibition of enzyme activity w as obtained in the presence of metal-chelating agents, sulfhydryl reag ents, bestatin, amastatin, and puromycin. The enzyme was stable at tem peratures below 15 degrees C and had a higher stability, 60% of initia l activity after 3.5 months of incubation at -25 degrees C, in the pre sence of 50% ethylene glycol.