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Purification of -amidating enzyme and elucidation of its reaction mechanism during the maturation process of amidated peptide hormones

酰胺化肽激素成熟过程中α-酰胺化酶的纯化及其反应机制的阐明

基本信息

批准号：
62580143
负责人：
NOGUCHI Masato
金额：
$ 1.28万
依托单位：
TOHOKU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

项目摘要

A number of bioactive peptides possess a C-terminal -amide, the presence of Which in most cases is essential for their optimal bioactivities. An enzymeactivity catalyzing the -amidation reaction, peptidylglycine -amidating enzyme, was first detected in porcine pituitary in 1982 by Bradbury; now the activity is thought to be physiologically involved in the C-taminal amide formation of peptide hormones. The purposes of this study were to purify the enzyme from rat, to characterize its properties and to clarify its reaction mechanism. We preliminarily characterized the -amidating activities from rat pituitary, brain and gut, and found that these tissues, though their specific activities were different, had activities capable converting of the glycine-extended to corresponding -amidated peptides. Enzymes from these tissues had similar properties in respects of cofactor requirements and Km values fot substrates, indicating that similar enzymes are functioning in these tissues.But the crude enzymes from these tissues showed pH profile with two pH optimal peaks at neutral (6.5-7.5) and alkaline pH (8.5-9.0). Analyses by DEAE-cellulose and gel chromatographies revealed that the alkaline pH activity was due to an enzyme species of Mr of 36K (36K enzyme), on the other hand, the neutral pH activity could be elicited by combining the 36K enzyme with a protein of Mr of 41K (41K protein) which apparently showed almost no or only marginal activity at either pH 7 or 8.5. Thus, the two pH optima seen with crude enzyme were due to the presence of the two proteins at an appropriate ratio. They are found to be co-localized in the secretory vesicles wherein -amidation occurs, suggesting the combined action by these proteins being of physiological significance. The pH optimum of the -amidating enzyme has been a matter of controversy. Our finding hopefully sheds light on the problem.

许多生物活性肽都具有 C 末端酰胺，在大多数情况下，其存在对于其最佳生物活性至关重要。 1982年，Bradbury首次在猪垂体中检测到一种催化β-酰胺化反应的酶——肽基甘氨酸-酰胺化酶；现在，该活性被认为在生理学上参与了肽激素的C-塔米那酰胺的形成。本研究的目的是从大鼠体内纯化该酶，表征其性质并阐明其反应机制。我们初步表征了大鼠垂体、大脑和肠道的β-酰胺化活性，发现这些组织虽然其具体活性不同，但具有能够将甘氨酸延伸转化为相应的β-酰胺化肽的活性。来自这些组织的酶在辅因子要求和底物 Km 值方面具有相似的特性，表明相似的酶在这些组织中发挥作用。但是来自这些组织的粗酶显示出 pH 曲线，在中性 (6.5-7.5) 和碱性 pH (8.5-9.0) 处有两个 pH 最佳峰值。 DEAE-纤维素和凝胶色谱分析表明，碱性pH活性是由36K的Mr酶种类（36K酶）引起的，另一方面，中性pH活性可以通过将36K酶与41K的Mr蛋白质（41K蛋白质）组合来引发，该蛋白质在pH 7或8.5下显然几乎没有或仅表现出边际活性。因此，用粗酶观察到的两个最佳pH值是由于两种蛋白质以适当的比例存在所致。发现它们共定位于发生酰胺化的分泌囊泡中，表明这些蛋白质的联合作用具有生理意义。 β-酰胺化酶的最适pH值一直存在争议。我们的发现希望能够阐明这个问题。