GLYCOPROTEINS CONTAINING PHOSPHOGLUCOSE IN NEURAL RETINA

我们最近描述了一种62 kDa的胞质磷酸糖蛋白 (Pgp62)似乎以两种状态存在，一种状态仅包含O- 内衬的人双糖和另一个还含有， 磷酸二酯连接的GLC。我们还描述了负责的酶 用于去除和添加GLC-1-P。我们的数据支持 假设pgp62密切参与了导致 突触囊泡释放GLC-1-P的移除和添加是一种 胞外过程中不可或缺的一部分。我们打算对此进行批判性的测试 假设并确定该发现是否可被利用来提供 活动突触前终末的高分辨率解剖标记物 视网膜和外侧膝状核中的神经元。的具体目标 这项建议包括： 1.继续研究pgp62及其相关酶的特性 监管。这包括制备哺乳动物的特异性抗体。 Pgp62，确定其亚细胞形态、k及其克隆和测序 它。此外，似乎与其相关的两种酶 调节，GLC-1-P磷酸二酯酶和Glc磷酸转移酶，将 还在继续研究中。 2.检验突触小泡释放伴随着 GLC-1-P在pgp62上的周转及其生理功能的测定 这一修改的意义在于。这包括持续的生化 PC-12细胞葡萄糖和磷酸盐代谢特性的研究 突触小体及其通透性细胞分析方法的发展 突触体将使我们能够评估分离蛋白的作用， 抗体，以及分泌机制中的抑制因子。 3.在完整的神经元中确定刺激对 从标记的GLC或2-脱氧葡萄糖掺入到大分子中 确定是否可以利用这种差异来开发高分辨率 视网膜和外侧活跃的突触前终末的标志物 膝状核。

We have recently described a cytoplasmic phosphoglycoprotein of 62 kDa (pgp62) that appears to exist in two states, one containing solely an O- lined Man disaccharide and the other containing, in addition, phosphodiester-linked Glc. We have also described the enzymes responsible for the removal and addition of the glc-1-P. Our data support the hypothesis that pgp62 is intimately involved in the mechanism leading to synaptic vesicle release that the removal and addition of the Glc-1-P is an integral part of the exocytic process. We intend to critically test this hypothesis and to determine if the finding can be exploited to provide a high-resolution anatomical marker for active pre-synaptic terminals of neurons in the retina and lateral geniculate nucleus. The specific aims of this proposal are: 1.To continue the characterization of pgp62 and the enzymes involved in its regulation. This includes preparing antibodies specific for mammalian pgp62, determining its subcellular topography,k and cloning and sequencing it. In addition, the two enzymes that appear to be involved in its regulation, glc-1-P phosphodiesterase and glc phosphotransferase, will continue to be studied. 2.To test the hypothesis that synaptic vesicle release is accompanied by turnover of glc-1-P on pgp62 and to determine what the physiological significance of this modification is. This includes continuing biochemical characterization of glucose and phosphate metabolism in PC-12 cells and in synaptosomes and the development of an assay with permeabilized cells or synaptosomes that will allow us to assess the roles of isolated proteins, antibodies, and inhibitory factors n the secretory mechanism. 3.To determine in intact neurons the effects of stimulation on incorporation into macromolecules from labeled glc or 2-deoxyglucose and to determine if such differences can be exploited to develop a high-resolution marker for active pre-synaptic terminals in the retina and lateral geniculate nucleus.