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 （pgp 62）似乎存在于两个国家，一个只包含一个O- lined Man二糖和另一种含有，此外， 磷酸二酯连接的Glc. 我们还描述了负责的酶 删除和添加glc-1-P。我们的数据支持 假设pgp 62密切参与导致 突触囊泡释放Glc-1-P的去除和添加是 胞吐过程的组成部分。 我们打算严格检验这一点 假设，并确定是否可以利用该发现提供一个 高分辨率解剖学标记物，用于活性突触前末梢 视网膜和外侧膝状体中的神经元。 的具体目标 这些建议是： 1.继续研究pgp 62及其相关酶的特性， 调控 这包括制备针对哺乳动物的特异性抗体， pgp 62的亚细胞形态学测定、K值测定及克隆测序 了 此外，两种酶似乎参与了其 调节，glc-1-P磷酸二酯酶和glc磷酸转移酶，将 继续被研究。 2.验证突触囊泡释放伴随着 pgp 62上glc-1-P的周转，并确定 这一修改的意义在于。 这包括持续的生物化学 在PC-12细胞中和在细胞中的葡萄糖和磷酸盐代谢的表征 突触体和开发透化细胞试验，或 突触体将使我们能够评估分离蛋白质的作用， 抗体和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-脱氧葡萄糖掺入大分子中， 确定是否可以利用这些差异来开发高分辨率 视网膜和侧膜中活性突触前末梢的标记物 膝状体核

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.