REGULATION OF GASTRIN POST-TRANSLATIONAL PROCESSING

多肽激素是一种化学信使，它提供了 身体的一个部位和另一个部位之间的交流，从而起到 在身体的综合功能中起着至关重要的作用。对中国传统文化的研究 因此，调节多肽激素的生物合成可以提供 对人体主要生理功能控制的重要洞察 健康和疾病。与大多数蛋白质和多肽一样，调节 荷尔蒙可以在转录、翻译和 分泌物。除了这些经过充分研究的过程外，在 肽激素还有另一个潜在的调节水平，那就是 翻译后处理。在各种加工反应中， 最重要的是羧基末端的酰胺化反应。 肽-甘氨酰α-酰胺化单加氧酶(PAM) 对一半以上已知的多肽荷尔蒙具有生物活性 胃泌素翻译后加工中的限速步骤。 拟议的研究将集中在探索生物化学和 PAM在调节胃泌素AS合成中的生理意义 这与胃酸分泌的功能调控有关。初步 研究表明，PAM存在于胃窦和脑垂体以及 在发育过程中PAM活性的相关变化与 胃泌素翻译后加工与胃酸分泌。这个 该提案的第一个目标将是描述以下方面的具体差异 PAM可以解释组织特异性差异在后 胃窦部和垂体部胃泌素的翻译加工。第二, 聚合酶链式反应将被用来扩增逆转录 胃肠道中的PAM基因序列。放大后的Full 随后将克隆和测序长度的大鼠PAM基因，并可以 用来作为探针测定PAM的序列，合成的PAM多肽 这些片段将被制成抗PAM抗体。这种抗体 然后用免疫印迹法对PAM蛋白进行定量，并 用免疫组织化学方法定位PAM在胃肠道中的分布。 第四，利用上述工具，将在体内检测PAM的表达， 在发育过程中，在分离的胃泌素产生G细胞在原代 培养，并在PAM中含有内分泌肿瘤细胞系。最后，它 将对PAM基因表达的相关变化至关重要， 合成和/或活动不仅对翻译后的变化有影响 对胃泌素的加工，也对生理功能的改变，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 实验室，为拟议的项目提供了理想的环境，因为 所有必要的设备和技术资源人员以及 合作者随手可得。

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.