PURIFICATION AND PROPERTIES OF AN INTRACELLULAR LEUCINE AMINOPEPTIDASE FROM THE FUNGUS, PENICILLIUM-CITRINUM STRAIN IFO-6352

An intracellular leucine aminopeptidase (LAP) from Penicillium citrinu m (IFO 6352) was purified to homogeneity using three successive purifi cation steps. The enzyme has a native molecular mass of 63 kDa using H PLC gel filtration analysis and a molecular mass of 65 kDa when using SDS-polyacrylamide gel electrophoresis. This monomeric aminopeptidase showed maximum enzyme activity at pH 8.5. An optimum temperature was 4 5-50 degrees C when L-Leu-p-nitroanilide (pNA) was the substrate, and enzyme activity drastically decreased above 60 degrees C. The Michaeli s-Menten constants for L-Leu-pNA and L-Met-pNA were 2.7 mM and 1.8 mM, respectively. When the enzyme reacted with biosynthetic methionyl hum an growth hormone, it showed high specificity for N-terminal methionin e residue and recognized a stop sequence (Xaa-Pro). The aminopeptidase was inactivated by EDTA or 1,10-phenanthroline, indicating that it is a metallo-exoprotease. Enzyme activity was restored to 90% of maximal activity by addition of Co2+ ions. The activity of EDTA-treated enzym e was restored by addition of Zn2+, but reconstitution with Ca2+, Mg2 or Mn2+ restored some enzyme activity. It is likely that Co2+ ions pl ay an important role in the catalysis or stability of the Penicillium citrinum aminopeptidase, as zinc plays a similar function in other leu cine aminopeptidases.