GLYCOPROTEINS CONTAINING PHOSPHOGLUCOSE IN NEURAL RETINA

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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.