ALANINE AMINOPEPTIDASE OF GUINEA-PIG BRAIN - A BROAD-SPECIFICITY CYTOPLASMIC ENZYME CAPABLE OF HYDROLYZING SHORT AND INTERMEDIATE LENGTH PEPTIDES

Alanine aminopeptidase is reported to be a broad specificity aminopept idase acting on peptides of different lengths. In this study we wish t o define the properties of the activity from guinea-pig brain and comp are these properties with previous findings. Alanine amino-peptidase w as purified from cytoplasm of guinea-pig brain by a four-step procedur e involving chromatography on DE-52, hydroxylapatite, Sephacryl S-200 and DEAE-Sephacryl. Relative molecular mass was determined by chromato graphy on Sephacryl S-200 column and subunit size determined by SDS-PA GE under denaturing conditions. Cations which reactivate the enzyme we re determined with EDTA treated enzyme. Substrate specificity was dete rmined by TLC and kinetic parameters were derived from Lineweaver-Burk plots. A 216-fold purification was achieved by the above procedures. The purified enzyme was found to consist of one polypeptide chain with a relative molecular mass of 104,000. Its activity was inhibited by c helating agents, sulphydryl reactive agents, puromycin, bestatin and a mastatin but stimulated over 6-fold by dithiothreitol. Some dipeptides and all tripeptides and longer peptides containing up to 16 amino aci ds tested were hydrolysed provided neither Glp or Pro occurred at the N-terminus or that Pro did not occur in the penultimate position from the N-terminus. The enzyme preferred bulky non-polar residues at the N -terminal and penultimate positions and was found to hydrolyse three d ipeptidyl methyl coumarin amides used in detecting dipeptidyl aminopep tidases. Alanine aminopeptidase is thus a broad specificity amino-pept idase acting on short and intermediate length peptides whose affinity for substrates increases with increasing peptide length. Its propertie s are well suited to a role in peptide turnover in brain cytoplasm.