Direct interaction between the reductase domain of endothelial nitric oxide synthase and the ryanodine receptor.
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.
Martínez-Moreno
Mónica
M
Departamento de Bioquímica y Biología Molecular, Facultad CC. Químicas, Universidad Complutense, 28040 Madrid, Spain.
Alvarez-Barrientos
Alberto
A
Roncal
Fernando
F
Albar
Juan Pablo
JP
Gavilanes
Francisco
F
Lamas
Santiago
S
Rodríguez-Crespo
Ignacio
I
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*
D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D010088:Oxidoreductases; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III