Isolation of a neuropeptide-degrading endopeptidase from the leech Theromyzon tessulatum.
Isolation of a neuropeptide-degrading endopeptidase from the leech Theromyzon tessulatum.
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从水蛭 Theromyzon tessulatum 中分离出神经肽降解内肽酶。
DOI：
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发表时间：
1995
期刊：
影响因子：
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通讯作者：
M. Salzet
中科院分区：
文献类型：
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作者：
V. Laurent;M. Salzet
从水蛭Theromyzon tessulatum制备的头部提取物水解合成的[D-Ala 2，Leu 5]脑啡肽的Gly 3-Phe 4键和苯甲酰-Gly-His-Leu的Gly-His键。苯甲酰-Gly-His-Leu的代谢被卡托普利完全抑制，与血管紧张素转化酶活性一致。这种酶最近已从T. tessulatum。然而，通过卡托普利（100 μ M）的脑啡肽水解被抑制至最大70%。水解脑啡肽的残留活性被phosphoramidon抑制，这与内肽酶-24.11（一种参与神经肽代谢的哺乳动物酶）的存在一致。该酶分离使用四个步骤的纯化，包括凝胶渗透和阴离子交换色谱法，然后通过反相HPLC。这种神经肽内肽酶（分子量约为45 kDa）在pH 7和37 ℃下水解合成的[D-Ala 2，Leu 5]脑啡肽的Gly 3-Phe 4键和血管紧张素I的Phe 8-His 9键。[D-Ala 2，Leu 5]脑啡肽的切割分别产生比活性为29 nmol蛋白-1（Km 95 μ M）的Tyr-D-Ala-Gly和Phe-Leu肽。Tyr-D-Ala-Gly.min-1.mg血管紧张素I的水解产生血管紧张素II和具有1.2 nmol血管紧张素蛋白-1（Km 330 μ M）的比活性的二肽His-Leu。min-1.mg这些肽的代谢被磷酰胺完全抑制。因此，本研究提供了生化证据的神经肽降解内肽酶在水蛭。
Extracts of head parts prepared from the leech Theromyzon tessulatum hydrolyse the Gly3-Phe4 bond of synthetic [D-Ala2, Leu5]enkephalin and the Gly-His bond of benzoyl-Gly-His-Leu. The metabolism of benzoyl-Gly-His-Leu was completely inhibited by captopril, consistent with an angiotensin-converting enzyme activity. Such an enzyme has recently been isolated from T. tessulatum. However, the enkephalin hydrolysis by captopril (100 microM) was inhibited to a maximum of 70%. The residual activity hydrolyzing enkephalin was inhibited by phosphoramidon, consistent with the presence of endopeptidase-24.11, a mammalian enzyme implicated in the metabolism of neuropeptides. This enzyme was isolated using four steps of purification including gel-permeation and anion-exchange chromatographies followed by reverse-phase HPLC. This neuropeptide endopeptidase (of approximate molecular mass 45 kDa) hydrolyses, at pH 7 and 37 degrees C, both the Gly3-Phe4 bond of synthetic [D-Ala2, Leu5]enkephalin and the Phe8-His9 bond of angiotensin I. Cleavage of [D-Ala2, Leu5]enkephalin yields, respectively, the Tyr-D-Ala-Gly and Phe-Leu peptides with a specific activity of 29 nmol Tyr-D-Ala-Gly.min-1.mg protein-1 (Km 95 microM). The hydrolysis of angiotensin I yields angiotensin II and the dipeptide His-Leu with a specific activity of 1.2 nmol angiotensin min-1.mg protein-1 (Km 330 microM). The metabolism of these peptides was totally inhibited by phosphoramidon. This study therefore provides biochemical evidence for neuropeptide-degrading endopeptidases in leeches.