REGULATION OF GASTRIN POST-TRANSLATIONAL PROCESSING

肽激素是提供媒介的化学信使 身体的一个部分与另一部分之间的交流，从而发挥 对机体的综合功能起着至关重要的作用。 研究的 因此，肽激素生物合成的调节可以提供 对主要生理功能的控制具有重要意义 健康和疾病。 与大多数蛋白质和肽一样， 激素可以发生在转录、翻译和水平 分泌。 除了这些经过充分研究的过程之外，在 肽激素还有另一个潜在的调节水平，即 翻译后处理。 在各种加工反应中， 最重要的是羧基末端酰胺化反应 肽基甘氨酰α-酰胺化单加氧酶 (PAM)，因为它赋予 对超过一半的已知肽激素具有生物活性 胃泌素翻译后加工中的限速步骤。 拟议的研究将侧重于生物化学和 PAM 在调节胃泌素合成中的生理意义 它与胃酸分泌的功能控制有关。 初步 研究已经描述了胃窦和垂体中的 PAM 以及 在开发过程中以及 PAM 活动的相关变化 胃泌素翻译后加工和胃酸分泌。 的 该提案的首要目标是描述以下方面的具体差异： PAM 可以解释组织特异性差异 胃泌素在胃窦和垂体中的翻译加工。 第二， 聚合酶链式反应将用于扩增逆转录 胃肠道中的 PAM mRNA 序列。 放大后的全 大鼠 PAM cDNA 的长度随后将被克隆和测序，并且可以 用作探针测量 PAM 序列、合成 PAM 多肽 将产生片段以产生抗PAM抗体。 这种抗体 然后将用于通过蛋白质印迹法定量 PAM 蛋白并 通过免疫组织化学将 PAM 定位在胃肠道中。 第四，利用上述工具，体内检测PAM表达， 在发育过程中，在原代细胞中分离的产生胃泌素的 G 细胞中 培养物以及含有内分泌肿瘤细胞系的 PAM 中。 最后，它 PAM 基因表达的相关变化至关重要， 合成和/或活性不仅与翻译后的变化有关 胃泌素的加工，也改变生理功能，例如 如胃酸分泌、胃泌素受体结合和胃肠道 细胞生长。 密歇根大学，特别是 T. Yamada 博士的 实验室，为拟议项目提供了理想的环境 所有必要的设备和技术资源人员以及 合作者随时可用。

Peptide hormones are the chemical messengers that provide the medium of communication between one part of the body and another and thus play a crucial role in the integrative function of the body. Study of the regulation of peptide hormone biosynthesis, therefore, can provide important insight into the control of major physiological functions in health and disease. As with most proteins and peptides, the regulation of hormones can occur at the level of transcription, translation, and secretion. In addition to these well studied processes, in the case of peptide hormones there is another potential level of regulation, that of post-translational processing. Of the various processing reactions, the most important is the carboxyl terminal amidation reaction catalyzed by the enzyme Peptidyl-glycyl Alpha-amidating Monooxygenase (PAM) since it confers biological activity to more than half of the known peptide hormones and is the rate-limiting step in the post-translational processing of gastrin. The proposed studies will focus on the exploration of the biochemical and physiological implications of PAM in regulating the synthesis of gastrin as it relates to functional control of gastric acid secretion. Preliminary studies have characterized PAM in the antrum and pituitary as well as during development and related alterations in PAM activity to changes in gastrin post-translational processing and gastric acid secretion. The first aim of the proposal will be to characterize specific differences in PAM can account for the tissue -specific differences in the post- translational processing of gastrin in the antrum and pituitary. Second, the polymerase chain reaction will be used to amplify reversed transcribed PAM mRNA sequences in the gastrointestinal tract. The amplified full length rat PAM cDNA will subsequently be cloned and sequenced and can be used as a probe to measure PAM at sequence, synthetic PAM polypeptide fragments will be made to generate an anti-PAM antibody. This antibody will then be used to quantitate PAM protein by western blots and to localize PAM in the gastrointestinal tract with immunohistochemistry. Fourth, utilizing the above tools, PAM expression will be examined in vivo, during development, in isolated gastrin producing G cells in primary culture, and in PAM containing endocrine tumor cell lines. Finally, it will be of critical importance of relate changes in PAM gene expression, synthesis, and/or activity not only to changes in the post-translational processing of gastrin but also to alterations of physiologic functions such as gastric acid secretion, gastrin receptor binding, and gastrointestinal cell growth. The University of Michigan, and specifically Dr. T. Yamada's laboratory, provides an ideal environment for the proposed project since all of the necessary equipment and technical resource personnel and collaborators are readily available.