Abstract
A specific aminopeptidase localized exclusively in neurons of the central nervous system was identified with an automated continuous-flow aminopeptidase analyzer developed recently in this laboratory. The enzyme was purified from rat brain 4933-fold to homogeneity with 9.3% recovery by ammonium sulfate fractionation, followed by column chromatography successively on phenyl-Sepharose, Sephadex G-200, and twice on Mono Q FPLC. The purified single-chain enzyme was estimated to be 110 kDa in molecular mass. It has a pI of 5.25 and a pH optimum of 7.0. Only Mg(II) restores the activity of the apoenzyme. The neutral aminopeptidase hydrolyzes beta-naphthylamides of amino acids with aliphatic, polar uncharged, positively charged, or aromatic side chains. It has a Km of 95 microM and a kcat of 7.8 s-1 on methionine-enkephalin, releasing only the N-terminal tyrosine. The thiol-dependent metallo-enzyme is most sensitive to amastatin inhibition with a Ki of 0.04 microM, and is the aminopeptidase most sensitive to puromycin. Its properties are different from those of the ubiquitous puromycin-sensitive aminopeptidase obtained from the same enzyme preparation. The blocked N terminus, substrate and inhibitor specificity, hydrolytic coefficiency, metal effects, pI, molecular weight, and catalytic site show that this enzyme is distinct from all other known aminopeptidases. Its enrichment in the synaptosomes suggests that this first reported neuron-specific peptidase plays a role in neurotransmission and synaptic differentiation.
Highlights
A specific aminopeptidase localized exclusively in neurons of the central nervous system was identified with an automated continuous-flow aminopeptidase analyzer developed recently in this laboratory
Aminopeptidases (EC 3.4.11.-) are classified according to the number of amino acids cleaved from the N terminus, the relative efficiency with which residues are removed, the location of the aminopeptidase, the susceptibility to inhibitors, the metal ion content or the residues that link the metal to the enzyme, and the pH for maximal activity
Using our newly developed FPLC aminopeptidase analyzer [10], we identified a neural aminopeptidase enriched in brain synaptosomes
Summary
(Received for publication, April 8, 1998, and in revised form, September 2, 1998). From the Peptide Research Laboratory, Neurochemistry Division, Nathan S. A specific aminopeptidase localized exclusively in neurons of the central nervous system was identified with an automated continuous-flow aminopeptidase analyzer developed recently in this laboratory. Neuropeptides are an essential class of chemical messengers in the central nervous system (CNS), yet the role of specific peptidases in regulating their formation and disposition is equivocal. Transcripts of the ubiquitous puromycin-sensitive aminopeptidase (PSA), could be detected by in situ hybridization in rat CNS neurons, but not in surrounding glial cells or in blood vessels [9]. Despite all these compelling findings, a neuronspecific peptidase has not been identified. The purified enzyme degrades enkephalins, and its properties are distinct from all known aminopeptidases
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