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

许多生物活性肽具有c端-酰胺，在大多数情况下，其存在对其最佳生物活性是必不可少的。1982年，Bradbury首次在猪垂体中发现了一种催化-酰胺化反应的酶——肽基甘氨酸-酰胺化酶；现在这种活性被认为在生理上参与了肽激素的c -胺酰胺的形成。本研究的目的是从大鼠体内纯化该酶，对其性质进行表征，并阐明其反应机理。我们初步鉴定了大鼠垂体、脑和肠道的-酰胺化活性，发现这些组织虽然具体活性不同，但都具有将甘氨酸延伸转化为相应-酰胺化肽的活性。来自这些组织的酶在辅因子需求和底物Km值方面具有相似的特性，表明类似的酶在这些组织中起作用。但这些组织中粗酶的pH分布有两个最优pH峰，分别为中性（6.5 ~ 7.5）和碱性（8.5 ~ 9.0）。deae -纤维素和凝胶色谱分析表明，36K酶的碱性活性是由一种Mr of 36K的酶（36K酶）引起的，另一方面，36K酶与一种Mr of 41K的蛋白（41K蛋白）结合可引起中性pH活性，该蛋白在pH 7或8.5时几乎没有活性或只有边际活性。因此，粗酶的两个最佳pH值是由于两种蛋白质以适当的比例存在。它们被发现在发生-酰胺化的分泌囊泡中共定位，表明这些蛋白质的联合作用具有生理意义。氨基化酶的最佳pH值一直是一个有争议的问题。我们的发现有望对这个问题有所帮助。

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.