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
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
复制标题
模型氨基酸和肽的 Nα-脱乙酰化观察：特定 N-酰基氨基酸释放酶在大鼠脑中的分布和纯化
DOI：
10.1111/j.1471-4159.1983.tb13670.x
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发表时间：
1983
影响因子：
4.7
通讯作者：
W. Danho
中科院分区：
文献类型：
--
作者：
N. Marks;Ee‐Sing Lo;F. Stern;W. Danho
摘要：N α-酰基氨基酸释放酶（NAARE）是一种在Met-Ala键处切割乙酰Met-Ala的酶，通过盐沉淀、凝胶过滤和几个步骤的离子交换从大鼠脑细胞质中纯化至表观均一。在检查的所有脑区和亚细胞组分中，NAARE水平超过了用乙酰甲硫氨酸测量的酰化酶：60%与胞质溶胶相关，其余与碎片或粗核和线粒体-突触体亚组分相关。垂体中的活性最高，约为肝脏或肾脏的0.5-0.6。纯化的酶优先水解乙酰-甲硫氨酰肽：乙酰蛋氨酸-丙氨酸的Km为0.93; Vmax为3.5 nmol−1（kcat，1185），最适pH为8.9，而在胞质溶胶中测量的酰化酶为8.2。纯化的酶没有酰化酶和常见的外肽酶和内肽酶污染。用合成的甲酰化或乙酰化肽检查的结构-活性关系表明，如果第二个取代基是Ala、Ser、Asn或Thr，则对二肽或三肽没有显著影响，但对Leu（一种支链氨基酸）的活性降低0.5倍。对于具有5个或更多个N-末端乙酰化Tyr（脑啡肽）或Ser（α-黑素细胞刺激激素，胸腺素α1）残基的多肽，未观察到水解，支持该酶仅在较小肽的周转中起作用的观点，所述较小肽可能是由于内肽酶切割N-末端含有乙酰化Met的蛋白质或多肽而形成的。
Abstract: N α–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 approximately 0.5–0.6 that of liver or kidney. The purified enzyme preferentially hydrolyzed acetyl‐methionyl peptides: Km for acetylMet‐Ala was 0.93; Vmax, 3.5 nmol−1 (kcat, 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 second 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 five or more residues having N‐terminal acetylated Tyr (enkephalin) or Ser (α‐melanocyte‐stimulating hormone, thymosinα1), 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.