[A membrane-bound alanine aminopeptidase from Acinetobacter calcoaceticus. 1. Isolation and purification of the...

The alanine aminopeptidase of Acinetobacter calcoaceticus was found to be bound to the inner membranes only. The enzyme was solubilized by Triton X-100 and purified approximately 480-fold by gel filtration and affinity chromatography on alanine methyl ketone-AH-Sepharose 4B. The purified alanine aminopeptidase has a molecular mass of 212 kDa, estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme selectively catalyses the hydrolysis of N-terminal alanine residues of peptides. The enzyme is inhibited by p-hydroxy-mercuribenzoate, 1,10-phenanthroline, and puromycin, but was activated by CO2(+)-ions.