Biochemical and immunological properties of NP185: A brain clathrin-coated vesicle-associated protein.
Item
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Title
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Biochemical and immunological properties of NP185: A brain clathrin-coated vesicle-associated protein.
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Identifier
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AAI9020813
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identifier
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9020813
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Creator
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Su, Borcherng.
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Contributor
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Adviser: Saul Puszkin
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Date
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1990
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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, Neuroscience
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Abstract
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A neuronal protein, NP185, associated with clathrin coated vesicles (CCVs), was recently identified and partially characterized with two monoclonal antibodies (mAbs): 8G8 & 6G7 (Kohtz & Puszkin, 1988), In this thesis I describe a method I developed to extract NP185 from brain CCVs, present a procedure for its purification by affinity chromatography and describe new binding properties for other coat proteins. After elution from a mAb 8G8 conjugated affinity column and from a hydroxylapatite column, the NP185 was found associated with clathrin light chains (LCs). When phosphorylated and non-phosphorylated clathrin LCs were crosslinked to Sepharose 4B beads, NP185 molecules did not bind to the phosphorylated LCs. The NP185 however, became tightly bound to the non-phosphorylated LCs and was eluted by 50% ethylene glycol pH 11.5, which weakens binding through hydrophobic domains. Similarly, NP185 retained on the mAb 8G8 affinity column did not bind the phosphorylated LCs from solution, while the non-phosphorylated LCs were bound by NP185 and were eluted as a complex with 0.15 M glycine buffer pH 2.5. The complex of NP185 with clathrin LCs had casein kinase II activity which phosphorylated the clathrin LCs in the presence of polylysine. The data suggest that in brain the functions of NP185 may in part depend on their association to clathrin LCs in CCVs. When CCVs were partially proteolyzed, the clathrin LCs were degraded but the smallest NP185 fragment (65 kD), recognized by mAb 8G8, remained bound to the vesicle membrane. Nerve endings CCVs contained in addition to clathrin molecule, NP185, assembly polypeptides (APs), synaptophysin and two protein kinases, entities similar to those of CCVs from whole brain. The NP185 molecules, linked to an 8G8 affinity column, retained salt pre-treated synaptics vesicles, decoated CCVs, synaptosomal membrane, tubulin and clathrin molecules. NP185 induced polymerization of clathrin cages. The resulting pellet which passed through a 10% sucrose barrier contained NP185. These results suggest that NP185 is involved in membrane homeostatis in nerve endings.
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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.
