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-末端酰胺，在大多数情况下，其存在对于其最佳生物活性是必不可少的。1982年，Bradbury在猪垂体中首次发现了一种催化β-酰胺化反应的酶活性，即肽基甘氨酸-酰胺化酶，现在认为该活性在生理上参与肽激素的C-氨基酰胺形成。本研究的目的是从大鼠中分离纯化该酶，并对其性质进行表征，阐明其反应机理。我们初步表征了大鼠脑垂体、脑和肠的-酰胺化活性，发现这些组织虽然比活性不同，但都具有将甘氨酸延伸肽转化为相应-酰胺化肽的活性。这些组织中的酶在对辅因子的需求量和对底物的Km值方面具有相似的性质，表明这些组织中的酶具有相似的功能，但这些组织中的粗酶的pH分布有两个pH最适峰，分别为中性（6.5 - 7.5）和碱性（8.5 - 9.0）。通过DEAE-纤维素和凝胶色谱分析表明，碱性pH活性是由于36K的Mr的酶物种（36K酶），另一方面，中性pH活性可以通过将36K酶与41K的Mr的蛋白质（41K蛋白质）结合而引起，该蛋白质在pH 7或8.5下显然几乎没有或仅显示出边缘活性。因此，用粗酶观察到的两个最适pH是由于两种蛋白质以适当的比例存在。他们被发现是共同定位在分泌囊泡，其中-酰胺化发生，这表明这些蛋白质的生理意义的联合行动。酰胺化酶的最适pH值一直是一个有争议的问题。我们的发现有望阐明这个问题。

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.