Purification and characterization of a neuropeptide-degrading aminopeptidase from human brain.

The major aminopeptidase from human post-mortem brain (occipital cortex) was purified to homogeneity (as judged by polyacrylamide gel electrophoresis) by anion-exchange chromatography (two steps) and gel filtration (two steps). The molecular weight of the enzyme was estimated as 105,000 from gel filtration. Maximum activity was obtained in the presence of 0.5 mM Ca2+ and 1 mM 2-mercaptoethanol at pH 7.3. Enzyme activity was lost on freezing and thawing or on lyophilization. The enzyme was inhibited by metal-ion chelating agents, sulphydryl blocking agents, bestatin, and puromycin. A series of amino acyl-7-amido-4-methylcoumarins was hydrolysed by the enzyme, with the alanyl derivative being hydrolysed most rapidly (Km 170 microM). Specificity studies with a series of alanine dipeptides suggested that a hydrophobic second residue favoured hydrolysis. Several naturally occurring neuropeptides, including Leu5-enkephalin (Km 180 microM), cholecystokinin octapeptide, and Arg8-vasopressin, were hydrolysed by the aminopeptidase. In a series of opioid peptides, increasing chain length led to decreased susceptibility to hydrolysis. Sulphation of the Tyr1 residue of Leu5-enkephalin and the Tyr2 residue of cholecystokinin octapeptide made the peptides more resistant to hydrolysis.