Observations on Nα‐Deacetylation of Model Amino Acids and Peptides: Distribution and Purification of a Specific N‐Acyl Amino Acid Releasing Enzyme in Rat Brain

Abstract

N.alpha.-Acyl amino acid releasing enzyme (NAARE), an enzyme cleaving acetylMet-Ala at the Met-Ala bond was purified from rat brain cytosol to apparent homogeneity by salt precipitation, gel filtration and several steps of ion exchange. Levels of NAARE exceeded acylase measured with acetylmethionine in all brain regions and subcellular fractions examined: 60% was associated with cytosol and the remainder with debris or the crude nuclear and mitochondrial-synaptosomal subfractions. Activity was highest in pituitary and was .apprx. 0.5-0.6 that of liver or kidney. The purified enzyme preferentially hydrolyzed acetylmethionyl peptides: Km for acetylMEt-Ala was 0.93; Vmax, 3.5 nmol-1 (kcat [catalytic constant] 1185) with pH optimum of 8.9 as compared with 8.2 for acylases measured in cytosol. The purified enzyme was devoid of acylase and common exo- and endopeptidase contamination. Structure-activity relationships examined with synthetic formylated or acetylated peptides indicated no significant effects for di- or tripeptides if the 2nd substituent was Ala, Ser, Asn or Thr, but the activity was reduced 0.5-fold for Leu, a branched-chain amino acid. No hydrolysis was observed for polypeptides with > 5 residues having N-terminal acetylated Tyr (enkephalin) or Ser (.alpha.-melanocyte-stimulating hormone, thymosin.alpha.1), supporting the notion that the enzyme plays a role only in turnover of smaller peptides formed perhaps as a result of endopeptidase cleavage of proteins or polypeptides containing acetylated Met at the N terminus.