INTERACTIONS OF PEPTIDES & IONS WITH GASTRIC H/K ATPASE

胃H/K-ATPase(E.C.1.2.1.36)是一种依赖于镁的K-ATPase。 依赖的，以及负责HCI的H转运酶 壁细胞的分泌物。一类天然的和合成的 多肽是一种新的、特异的和有效的血管紧张素转换酶抑制剂 H/K-ATPase已被鉴定。这些是两亲性螺旋 多肽，包括蜂毒毒素、蜂毒素和 具有相似三维结构的合成多肽和 理化性质。值得注意的是，生理上的相关性 阳离子，包括钾，可以防止抑制。我们还有 鉴定出一种内源性壁细胞蛋白，显示出 与蜂毒素有共同的决定因素，这种蛋白质似乎 表现出刺激依赖性与H/K-ATPase的相关性。这个 广泛的、长期的目标是定义 是调节胃酸分泌的基础。治疗控制 胃溃疡疾病依赖于控制HCI的干预 分泌物。这项建议的具体目标是 了解这个多肽结合位点的结构 H/K-ATPase；占用该站点导致 抑制力；如何实现阳离子保护；以及 这些发现的生理学意义。具体目标 1)比较和对比使用的适宜性 含对苯甲酰基苯丙氨酸的合成多肽 [125-I]叠氮水杨基蜂毒素光亲和探针的研究 多肽受体序列测定的目的 H/K-ATPase。纯化的碎片将用于2) H/K-ATPase相互作用残基的鉴定 用氨基末端分析和测定多肽 氨基酸组成。这些序列将被映射到 光标记的氨基酸将在已知的初级蛋白质上被识别 H/K-ATPase的结构。3)我们将检查功能 占据两亲性多肽结合的后果 通过蜂毒素，并定义了生理上 相关的阳离子保护酶不受抑制。结果是 运输和三磷酸腺苷水解的研究将与 H/K-ATPase构象的荧光研究结构性 并将开发和测试动力学模型。4)内生性 含有蜂毒素类决定簇的多肽将通过以下方法纯化 高效液相色谱法进行免疫亲和纯化。约束性研究， 光亲和标记、构象研究和功能 该蛋白与纯化的H/K相互作用的后果 将对ATPase进行调查。我们的假设是阿尔法 H/K-ATPase的亚基含有一个多肽结合部位 蜂毒素和这种内源蛋白结合在一起。类蜂毒素 蛋白质可能在HCI的调节中发挥生理学作用 分泌物。这些研究代表了一个重要的新方向 结构/功能关系的研究 胃H/K-ATPase的调节功能。

The gastric H/K ATPase (E.C. 1.2.1.36) is a Mg2+-dependent, K+- dependent, and H+ transporting enzyme which is responsible for HCI secretion by the parietal cell. A class of natural and synthetic polypeptides which are novel, specific, and potent Inhibitors of the H/K ATPase have been Identified. These are amphipathic helical polypeptides, and include the bee venom toxin, melittin and synthetic polypeptides of similar 3-dimensional structure and physiochemical properties. Significantly, physiologically relevant cations, including K+, protect against inhibition. We have also Identified an endogenous parietal cell protein which exhibits determinants In common with melittin, and this protein appears to exhibit stimulus-dependent association with the H/K ATPase. The broad, long term objectives are to define the mechanisms which underlie regulated gastric HCI secretion. Therapeutic control of gastric ulcer disease relies upon interventions which control HCI secretion. The specific aims of this proposal are directed toward understanding the structure of this polypeptide binding site on the H/K ATPase; the mechanism whereby occupation of this site causes inhibition; how protection by cations is achieved; and the physiological significance of these findings. The specific aims are: 1) To compare and contrast the suitability of using p-benzoylphenylalanine-containing synthetic polypeptide photoaffinity probes with [125-I]azidosalicylyl melittin for the purposes of sequence determination of the polypeptide receptor on the H/K ATPase. Purified fragments will be used for 2) identification of the residues of the H/K ATPase which interact with polypeptides by NH2 terminal analysis and determination of amino acid composition. These sequences will be mapped ana the photolabeled amino acids will be identified on the known primary structure of the H/K ATPase. 3) We will examine the functional consequences of occupation of the amphipathic polypeptide binding site by melittin, and define the mechanisms whereby physiologically relevant cations protect the enzyme from inhibition. The results of transport and ATP hydrolysis studies will be correlated with fluorescent studies of conformation of the H/K ATPase. Structural and kinetic models will be developed and tested. 4) The endogenous polypeptide with melittin-like determinants will be purified by immunoaffinity purification by HPLC. Binding studies, photoaffinity labeling, conformational studies, and the functional consequences of interaction of this protein with purified H/K ATPase will be investigated. It is our hypothesis that the alpha subunit of the H/K ATPase contains a polypeptide binding site to which melittin and this endogenous protein bind. The melittin-like protein may play a physiological role in regulation of HCI secretion. These studies represent an important new direction for the study of structure/function relationships which underlie regulated function of the gastric H/K ATPase.