Alanine aminopeptidase of guinea-pig brain: a broad specificity cytoplasmic enzyme capable of hydrolysing short and intermediate length peptides.
Alanine aminopeptidase is reported to be a broad specificity aminopeptidase acting on peptides of different lengths. In this study we wish to define the properties of the activity from guinea-pig brain and compare these properties with previous findings. Alanine amino-peptidase was purified from cytoplasm of guinea-pig brain by a four-step procedure involving chromatography on DE-52, hydroxylapatite, Sephacryl S-200 and DEAE-Sephacryl. Relative molecular mass was determined by chromatography on Sephacryl S-200 column and subunit size determined by SDS-PAGE under denaturing conditions. Cations which reactivate the enzyme were determined with EDTA treated enzyme. Substrate specificity was determined 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 chelating agents, sulphydryl reactive agents, puromycin, bestatin and amastatin but stimulated over 6-fold by dithiothreitol. Some dipeptides and all tripeptides and longer peptides containing up to 16 amino acids 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 dipeptidyl methyl coumarin amides used in detecting dipeptidyl aminopeptidases. Alanine aminopeptidase is thus a broad specificity amino-peptidase acting on short and intermediate length peptides whose affinity for substrates increases with increasing peptide length. Its properties are well suited to a role in peptide turnover in brain cytoplasm.
Smyth
M
M
Department of Biochemistry, University College Galway, Ireland.
O'Cuinn
G
G
D000595:Amino Acid Sequence; D000804:Angiotensin II / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000201:enzymology*; D018826:CD13 Antigens / Q000302:isolation & purification / Q000378:metabolism*; D002845:Chromatography; D006168:Guinea Pigs; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000378:metabolism*; D013379:Substrate Specificity
D010455:Peptides; D000804:Angiotensin II; D018826:CD13 Antigens