INTERACTIONS OF PEPTIDES & IONS WITH GASTRIC H/K ATPASE

胃H/K ATP酶（E.C. 1.2.1.36 依赖性和H+转运酶，负责HCI 由壁细胞分泌。 一类天然的和合成的 本发明提供了一种多肽，其是新的、特异性的和有效的抑制剂， H/K ATP酶已被鉴定。 这些是两亲性螺旋 多肽，并且包括蜂毒毒素、蜂毒肽和 具有相似三维结构的合成多肽， 理化性质 重要的是，生理相关 包括K+在内的阳离子防止抑制。 我们还 鉴定了一种内源性壁细胞蛋白， 与蜂毒素一样，这种蛋白质似乎 显示与H/K ATP酶的刺激依赖性关联。 的 广泛的长期目标是确定机制， 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的干预措施 分泌物 本提案的具体目标是 了解这种多肽结合位点的结构， H/K ATP酶;占据该位点导致 抑制;如何实现阳离子的保护;以及 这些发现的生理意义。 具体目标 主要有：1）比较和对比使用 含对苯甲酰苯丙氨酸的合成多肽 [125-I]叠氮水杨酰蜂毒肽光亲和探针 多肽受体序列测定的目的 H/K ATP酶。 纯化的片段将用于2） H/K ATP酶相互作用的残基的鉴定 用多肽通过NH2末端分析和测定 氨基酸组成 这些序列将被映射为 光标记的氨基酸将在已知的主要氨基酸上被鉴定。 H/K ATP酶的结构。3)我们将研究功能 两亲性多肽结合的占据的结果 通过蜂毒肽定位，并确定生理学上 相关的阳离子保护酶不受抑制。 结果 运输和ATP水解研究将与 H/K ATP酶构象的荧光研究。 结构 并将开发和测试动力学模型。4)内生 具有蜂毒肽样决定簇的多肽将被纯化， 通过HPLC进行免疫亲和纯化。 结合研究， 光亲和标记，构象研究，和功能 这种蛋白质与纯化的H/K相互作用的结果 将研究ATP酶。 我们假设阿尔法狼 H/K ATP酶的亚基含有多肽结合位点， 蜂毒肽和内源性蛋白质结合。 蜂毒肽样 蛋白质可能在HCI的调节中起生理作用 分泌物 这些研究代表了重要的新方向， 结构/功能关系的研究， 调节胃H/K ATP酶的功能。

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.