A novel assay method for aminopeptidase P and partial purification of two types of the enzyme in Escherichia coli
A novel assay method for aminopeptidase P and partial purification of two types of the enzyme in Escherichia coli
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大肠杆菌中氨肽酶 P 的新测定方法及两种酶的部分纯化
DOI：
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发表时间：
1988
期刊：
影响因子：
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通讯作者：
D. Tsuru
中科院分区：
文献类型：
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作者：
T. Yoshimoto;N. Murayama;D. Tsuru
通过使用 Gly-Pro-色原（例如 Gly-Pro-β-萘酰胺、Gly-Pro-p-硝基苯胺或 .Gly-Pro-4-甲基香豆素酰胺）作为底物与脯氨酸亚氨基肽酶偶联，建立了一种新的氨肽酶 P 测定方法。对于每种底物，酶量与由于释放的色原而产生的显色或荧光之间建立了线性关系。该测定方法不受培养液中物质的干扰。通过使用该测定方法，通过 DEAE-Sephadex 色谱和高效液相色谱 (HPLC) 从大肠杆菌 HB101 中部分纯化氨肽酶 P。在DEAE-Sephadex柱色谱上，观察到氨肽酶P的两个峰，分别命名为APP-I和APP-II。使用DEAE-5PW和Phenyl-5PW柱通过HPLC进一步纯化APP-I。 APP-I 和 APP-II 的最适 pH 值分别为 8.0 和 9.0。与 APP-I 在 pH 10 左右稳定相比，APP-II 在 pH 8 至 9 下稳定。在 pH 8.0 下孵育 30 分钟后，在 60°C 和 50°C 下观察到 APP-I 和 APP-II 剩余活性的 50%。通过添加 5 和 30μM Mn2+，APP-I 和 APP-II 被激活 3 倍。它们被 EDTA 抑制，并通过添加 Mn2+ 重新激活。 APP-I和APP-II的分子量分别为350, 000和210, 000。当脯氨酸位于倒数第二个位置时，每种酶都释放氨基末端氨基酸。除 Pro-Pro 键外，大多数肽的 X-Pro 键（X=氨基酸）的酶水解速度没有显着差异。 APP-II 以比 APP-I 高得多的速率水解五-(Pro-Pro-Gly)，表明 Yaron 和 Mlynar (BBRC, 32, 658 (1968)) 报道的氨肽酶 P 是 APP-II。
A new assay for aminopeptidase P was established by coupling with proline iminopeptidase using Gly-Pro-chromogen (e.g. Gly-Pro-β-naphthylamide, Gly-Pro-p-nitroanilide, or .Gly-Pro-4-methyl coumarin amide) as the substrate. With each substrate, a linear relationship was established between the enzyme amounts and color development or fluorescence due to the chromogen released. This assay method did not suffer from interference by materials in culture broth. By using this assay method, aminopeptidase P was partially purified from Escherichia coli HB101 by chromatographies on DEAE-Sephadex and high performance liquid chromatography (HPLC). On the chromatogram with a DEAE-Sephadex column, two peaks of aminopeptidase P were observed and were named APP-I and APP-II. APP-I was further purified by HPLC using DEAE-5PW and Phenyl-5PW columns. Optimum pHs of APP-I and APP-II were 8.0 and 9.0, respectively. In contrast to APP-I which was stable around pH 10, APP-II was stable at pH 8 to 9. After incubation for 30min at pH 8.0, fifty percent of the remaining activity of APP-I and APP-II were observed at 60°C and 50°C. APP-I and APP-II were activated 3-fold by the addition of 5 and 30μM Mn2+. They were inhibited by EDTA, and reactivated by adding Mn2+. The molecular weights of APP-I and APP-II were 350, 000 and 210, 000, respectively. Each enzymes released the ammo terminal amino acid when proline is at the penultimate position. The velocity of hydrolysis by the enzymes was not significantly different for most X-Pro bonds (X=amino acid) of peptides except for Pro-Pro bond. APP-II hydrolyzed penta-(Pro-Pro-Gly) at a much higher rate than APP-I, suggesting the aminopeptidase P reported by Yaron and Mlynar (BBRC, 32, 658 (1968)) to be APP-II.