A new type of neuron-specific aminopeptidase NAP-2 in rat brain synaptosomes.
A novel neutral aminopeptidase (NAP-2) was found exclusively in the rat central nervous system (CNS). It was separated from the ubiquitous puromycin-sensitive aminopeptidase (PSA) and the neuron-specific aminopeptidase (NAP) by an automated FPLC-aminopeptidase analyzer. The activity of the neuronal aminopeptidase enriched in the synaptosomes is different from NAP and PSA in distribution and during brain development. The enzyme was purified 2230-fold to apparent homogeneity from rat brain cytosol with 4% recovery by ammonium sulfate fractionation, followed by column chromatography successively on Phenyl-Sepharose, Q-Sepharose, Sephadex G-200, and Mono Q. The single-chain enzyme with a molecular mass of 110kDa has an optimal pH of 7.0 and a pI of 5.6. It splits beta-naphthylamides of amino acid with aliphatic, polar uncharged, positively charged, and aromatic side chain. Leucyl beta-naphthylamide (Leu betaNA) is the best substrate with the highest hydrolytic coefficiency followed by Met betaNA=Arg betaNA=Lys betaNA>Ala betaNA>Tyr betaNA>Phe betaNA. The cysteine-, metallo-, glyco-aminopeptidase releases the N-terminal Tyr from Leu-enkephalin with a K(m) 82microM and a k(cat) of 1.08s(-1), and Met-enkephalin with a K(m) of 106microM and a k(cat) of 2.6s(-1). The puromycin-sensitive enzyme is most susceptible to amastatin with an IC(50) of 0.05microM. The data indicate that the enzyme is a new type of NAP found in rodent. Its possible function in neuron growth, neurodegeneration, and carcinomas is discussed.
Hui
Maria
M
Neurochemistry Division, Nathan S. Kline Institute for Psychiatric Research, Orangeburg, NY 10962, USA. hui@nki.rfmh.org
Hui
Koon-Sea
KS
D000626:Aminopeptidases / Q000737:chemistry* / Q000302:isolation & purification; D000818:Animals; D001921:Brain / Q000201:enzymology* / Q000648:ultrastructure; D002417:Cattle; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D019743:Chemokines, CXC; D002645:Chickens; D002845:Chromatography; D003600:Cytosol / Q000201:enzymology; D006801:Humans; D008297:Male; D051379:Mice; D008970:Molecular Weight; D010455:Peptides / Q000494:pharmacology; D017729:Presynaptic Terminals / Q000201:enzymology* / Q000648:ultrastructure; D011480:Protease Inhibitors / Q000494:pharmacology; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013045:Species Specificity
D019743:Chemokines, CXC; D010455:Peptides; C493799:Ppbp protein, rat; D011480:Protease Inhibitors; C016977:amastatin; D000626:Aminopeptidases