Role of glucokinase localization in regulating activity

描述（由申请人提供）：葡萄糖磷酸化酶， 葡萄糖激酶（GK），在葡萄糖刺激的胰岛素中充当葡萄糖传感器 从胰岛B细胞释放。虽然酶 GK的特性已被广泛表征，GK的机制 激活仍然难以捉摸。最近，GK的电子显微照片 在胰岛中的分布表明GK位于胰岛素上， 分泌颗粒鉴于GK在其他组织中的活性受到调节， 通过本地化的变化，建议GK与 胰岛素分泌颗粒可能与GK活性直接相关。为了 为了验证这一假设，GK与胰岛素颗粒的结合动力学将 在表达荧光标记的GK嵌合体的细胞中进行评估。定量 漂白研究将用于表征GK与 胰岛素颗粒，并特别检查葡萄糖-GK相互作用的作用 触发GK重新分布到细胞质中。GK中的蛋白质结构域 参与将OK靶向至胰岛素颗粒的细胞将通过 缺失诱变GK靶向结构域的鉴定将允许 通过定点诱变特异性破坏GK靶向， 检查GK易位对葡萄糖代谢的重要性， B细胞这些研究将进一步加深我们对GK调控的理解， 葡萄糖刺激的胰岛细胞及GK区室化的作用 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.