A NOVEL NEURON-SPECIFIC AMINOPEPTIDASE IN RAT-BRAIN SYNAPTOSOMES - ITS IDENTIFICATION, PURIFICATION, AND CHARACTERIZATION

A specific aminopeptidase localized exclusively in neurons of the cent ral nervous system was identified with an automated continuous-flow am inopeptidase analyzer developed recently in this laboratory, The enzym e was purified from rat brain 4933-fold to homogeneity with 9.3% recov ery by ammonium sulfate fractionation, followed by column chromatograp hy 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 M g(II) restores the activity of the apoenzyme. The neutral aminopeptida se hydrolyzes beta-naphthylamides of amino acids with aliphatic, polar uncharged, positively charged, or aromatic side chains, It has a K-m of 95 mu M and a k(cat) of 7.8 s(-1) on methionine-enkephalin, releasi ng only the N-terminal tyrosine, The thiol-dependent metallo-enzyme is most sensitive to amastatin inhibition with a K-i of 0.04 mu M, and i s the aminopeptidase most sensitive to puromycin, Its properties are d ifferent from those of the ubiquitous puromycin-sensitive aminopeptida se obtained from the same enzyme preparation. The blocked N terminus, substrate and inhibitor specificity, hydrolytic coefficiency, metal ef fects, pI, molecular weight, and catalytic site show that this enzyme is distinct from all other known aminopeptidases. Its enrichment in th e synaptosomes suggests that this first reported neuron-specific pepti dase plays a role in neurotransmission and synaptic differentiation.