title: Direct interaction between the reductase domain of endothelial nitric oxide synthase and the ryanodine receptor.
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.
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Facultad CC. Químicas, Universidad Complutense, 28040 Madrid, Spain.
authors.forename: Mónica
authors.initials: M
authors.lastname: Martínez-Moreno
authors.forename: Alberto
authors.initials: A
authors.lastname: Alvarez-Barrientos
authors.forename: Fernando
authors.initials: F
authors.lastname: Roncal
authors.forename: Juan Pablo
authors.initials: JP
authors.lastname: Albar
authors.forename: Francisco
authors.initials: F
authors.lastname: Gavilanes
authors.forename: Santiago
authors.initials: S
authors.lastname: Lamas
authors.forename: Ignacio
authors.initials: I
authors.lastname: Rodríguez-Crespo
mesh_terms: D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D010088:Oxidoreductases; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III