Role of glucokinase localization in regulating activity

说明书(申请人提供)：葡萄糖磷酸化酶， 葡萄糖激酶(GK)在葡萄糖刺激的胰岛素中作为葡萄糖感受器 从胰岛B细胞释放。尽管酶制剂 GK的特征已经被广泛地表征，GK的机制 激活仍然难以捉摸。最近，GK的电子显微镜照片 胰岛的分布表明GK位于胰岛素上。 分泌颗粒。鉴于其他组织中的GK活性是受调节的 通过本地化的变化，建议将GK与 胰岛素分泌颗粒可能与GK活性有直接关系。为了 验证这一假设，GK与胰岛素颗粒的关联动力学将 在表达荧光标记的GK嵌合体的细胞中进行评估。量化 漂白研究将用于表征GK与 胰岛素颗粒及特异性检测葡萄糖-GK相互作用的作用 在触发GK重新分配到细胞质中。GK中的蛋白质结构域 参与将OK靶向胰岛素颗粒的人将通过以下方式确定 缺失突变。识别GK目标域将允许 定点突变对GK靶向的特异性干扰 GK易位对糖代谢影响的研究进展 B细胞。这些研究将进一步加深我们对GK在 胰岛及GK区划在葡萄糖刺激中的作用 胰岛素分泌。

DESCRIPTION (provided by applicant): The glucose phosphorylating enzyme, glucokinase (GK),acts as the glucose sensor in glucose stimulated insulin release from the pancreatic B-islet cells. Although the enzymatic characteristics of GK have been characterized extensively, the mechanism of GK activation has remained elusive. Recently, electron micrographs of GK distribution in pancreatic islets have revealed that GK is located on insulin secretory granules. Given that GK activity in other tissues is regulated through changes in localization, it is proposed that GK association with insulin secretory granules may be directly related to GK activity. In order to test this hypothesis, the dynamics of GK association with insulin granules will be assessed in cells expressing a fluorescently tagged GK chimera. Quantitative bleaching studies will be used to characterize the association of GK with insulin granules and specifically examine the role of glucose-GK interactions in triggering GK redistribution to the cytoplasm. The protein domains in GK that participate in targeting OK to insulin granules will be identified by deletion mutagenesis. Identification of the GK targeting domain will allow specific disruption of GK targeting by site-directed mutagenesis and examination of the importance of GK translocation to glucose metabolism in the B-cell. These studies will further our understanding of GK regulation in the pancreatic islet and the role of GK compartmentalization in glucose stimulated insulin secretion.