Abstract
We have performed the recombinant expression and purification of the reductase domain of endothelial nitric oxide synthase (eNOS) and used it as a bait in search for interacting proteins present in endothelial cells. Using mass spectrometry of the bound proteins run in a PAGE-SDS gel, we were able to identify the ryanodine receptor (RyR) as a novel eNOS-binding partner. This interaction was confirmed through immunoprecipitation of both RyR and eNOS from endothelial cells and cardiac myocytes. Immunofluorescence data indicated that a subpopulation of eNOS associates with RyR in perinuclear regions of the cell, where eNOS might be responsible for the known nitrosylation of RyR.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Cattle
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Cells, Cultured
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Fluorescent Antibody Technique
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Immunoprecipitation
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Mass Spectrometry
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Nitric Oxide Synthase / chemistry*
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Nitric Oxide Synthase / genetics
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Nitric Oxide Synthase / isolation & purification
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Nitric Oxide Synthase / metabolism*
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Nitric Oxide Synthase Type III
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Oxidoreductases / chemistry*
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Oxidoreductases / genetics
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Oxidoreductases / isolation & purification
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Oxidoreductases / metabolism*
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Protein Binding
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Protein Structure, Tertiary
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / metabolism
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Ryanodine Receptor Calcium Release Channel / chemistry
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Ryanodine Receptor Calcium Release Channel / metabolism*
Substances
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Recombinant Proteins
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Ryanodine Receptor Calcium Release Channel
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Oxidoreductases
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Nitric Oxide Synthase
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Nitric Oxide Synthase Type III