Molecular and biochemical characterization of NP185, a neuronal-specific, synapse-enriched, and clathrin coated-vesicle associated phosphoprotein.
Item
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Title
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Molecular and biochemical characterization of NP185, a neuronal-specific, synapse-enriched, and clathrin coated-vesicle associated phosphoprotein.
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Identifier
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AAI9510682
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identifier
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9510682
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Creator
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Li, Shengwen.
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Contributor
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Adviser: Saul Puszkin
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Date
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1994
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Molecular | Biology, Neuroscience | Biology, Cell | Chemistry, Biochemistry
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Abstract
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NP185 was previously discovered as a neuronal protein associated with brain clathrin-coated vesicles (CCVs) using two monoclonal antibodies (mAbs), 8G8 and 6G7. Further biochemical studies displayed that the NP185 binds to synaptic vesicles (SVs), synaptosomal plasma membranes (SPM), decoated vesicles, clathrin light chains (CLCs) and tubulin, and its binding affinity for tubulin and CLCs are regulated by two different protein kinases. Developmental expression of the NP185 molecules follows a pattern consistent with avian cerebellar synaptogenesis.;This thesis demonstrates the presence of NP185 not only in the central nervous system, but also in the peripheral nervous system, particularly in the neuromuscular junction of avian striated muscle, by using indirect rhodamine immunofluorescence of NP185 mAb for NP185 localization combined with fluorescein-{dollar}\alpha{dollar}-bungarotoxin to mark the postsynaptic site of acetylcholine receptors. That was further supported by SDS-PAGE and Western blot analyses using the sample of immunoprecipitation of NP185 protein from avian striated muscle.;Molecular analyses establish the identity of NP185 and its relation to other known proteins. The internal amino acid sequence microsequenced from the purified NP185 showed clear homologies to internal amino acid sequences of a mouse brain protein, F1-20. The NP185 mAbs, as well as F1-20 mAb react with a CCV protein with molecular mass of 185-190 kDa by immunoblot analysis of SDS-PAGE. While F1-20 mAb binds to immunoaffinity purified NP185; NP185 mAbs recognize the GST-F1-20 fusion protein expressed in E. coli. Functional analyses of the bacterially expressed protein showed that GST-F1-20 induces assembly of clathrin into cage. In addition, the bacterially expressed GST-F1-20 protein binds to brain tubulin. The tubulin binding domain was mapped to 124 residues, and epitopes for both 8G8 and 6G7 mAbs mapped to 60 residues of deduced amino acid sequence by truncation and PCR domain amplification.;Taken together, the data implicate NP185 as a multifunctional protein in synaptic terminals involved in docking, signalling, and molecular adaptor function. A testable model is introduced.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
