A novel neuron-specific aminopeptidase in rat brain synaptosomes. Its identification, purification, and characterization.
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.
Hui
K S
KS
Peptide Research Laboratory, Neurochemistry Division, Nathan S. Kline Institute for Psychiatric Research, Orangeburg, New York 10962, USA.
Saito
M
M
Hui
M
M
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002413:Cations, Divalent / Q000494:pharmacology; D004745:Enkephalins / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009281:Naphthalenes / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism; D009474:Neurons / Q000201:enzymology*; D011480:Protease Inhibitors / Q000494:pharmacology; D011691:Puromycin / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013379:Substrate Specificity; D013574:Synaptosomes / Q000201:enzymology*; D014018:Tissue Distribution
D002413:Cations, Divalent; D004745:Enkephalins; D009281:Naphthalenes; D009419:Nerve Tissue Proteins; D011480:Protease Inhibitors; C072587:2-naphthylamide; D011691:Puromycin; D000626:Aminopeptidases; C025579:enkephalin degrading enzyme