Purification and properties of aminoendopeptidase from rat epidermis.
Purification and properties of aminoendopeptidase from rat epidermis.
复制标题
大鼠表皮氨基内肽酶的纯化和性质。
DOI：
10.1111/1523-1747.ep12340333
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发表时间：
1984
期刊：
影响因子：
--
通讯作者：
Epstein,WL
中科院分区：
文献类型：
--
作者：
Ito,Y;Fukuyama,K;Yabe,K;Epstein,WL
采用硫酸铵分离、DE-52柱层析、Sephadex G-200凝胶过滤、CM-52和DEAE-Sepharose 6B柱层析等方法纯化2日龄大鼠表皮的氨基内肽酶，使其具有明显的同质性。酶活性仅在巯基化合物存在时表现出来，并在添加5 mM EDTA时进一步增强。对邻苯二甲酸氯脲、其他巯基阻断剂和邻菲罗啉均有抑制作用。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析，该酶的单体形态为Mr= 52,000±2,300，而天然形态为Mr= 400,000±26,000，等电点pH为5.25。在合成底物中，该酶水解氨基酸2-萘酰胺衍生物和L-亮氨酸胺（L- LeuNH2）的效率最高。N-α-苯甲酰- dl -精氨酸-2-萘酰胺（BANA）是该酶唯一的内肽酶底物，也是其氨基肽酶活性的竞争性抑制剂。蛋白质底物尚未发现。最佳pH值为7.5，在pH 6.5-7.5范围内，在37°C下保持30分钟稳定，但在50°C下失去约50%的活性。
An aminoendopeptidase isolated from 2-day-old rat epidermis was purified to apparent homogeneity by the procedures of ammonium sulfate fractionation, DE-52 column chromatography, Sephadex G-200 gel filtration, and CM-52 and DEAE-Sepharose 6B column chromatography. Enzymatic activity was exhibited only in the presence of sulfhydryl compounds and further enhanced by addition of 5 mM EDTA. It was inhibited byp-chloromercuribenzoate, other sulfhydryl blocking reagents, ando-phenanthroline. The monomer form of the enzyme is Mr= 52,000 ± 2,300 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, but a native form was considered to be Mr= 400,000 ± 26,000 having an isoelectric point of pH 5.25. Among synthetic substrates the enzyme hydrolyzed amino acid 2-naphthylamide derivatives and L-leucine amine (L- LeuNH2) most effectively. N-α-benzoyl-DL-arginine-2-naphthylamide (BANA) was the only endopeptidase substrate for the enzyme and a competitive inhibitor for its aminopeptidase activity. Protein substrates have not yet been found. The pH optimum is 7.5 and in a range of pH 6.5–7.5 it is stable at 37°C for 30 min but loses about 50% of its activity at 50°C.