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Citrulline‐nitric oxide cycle protein‐protein interactions
Author(s) -
Flam Brenda R,
Samreen Naziya,
Strom Joel A.,
Solomonson Larry P.,
Eichler Duane C.
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a624-c
Subject(s) - citrulline , argininosuccinate synthase , nitric oxide , enos , arginine , chemistry , biochemistry , nitric oxide synthase , nitric oxide synthase type iii , microbiology and biotechnology , biology , enzyme , amino acid , organic chemistry
Background: Nitric oxide (NO), a key vasorelaxant, is formed when arginine is converted to NO and citrulline by endothelial nitric oxide synthase (eNOS). Citrulline is regenerated to arginine by argininosuccinate synthase (AS) followed by argininosuccinate lyase (AL). These reactions constitute the citrulline‐NO cycle, which plays an essential role in endothelial NO production. Citrulline‐NO cycle enzymes are associated with caveolae but there is no evidence for direct protein interactions between these components. Methods: AS and AL were cloned and expressed in E. coli with an N‐terminal hexahistidine‐T7 (6xHis‐T7) tag. Bovine aortic endothelial cells (BAEC) were either left in the basal state or stimulated with a calcium agonist. Endothelial cell lysates were prepared and used to test for protein‐protein interactions using in vitro interaction assays with bacterially expressed fusion proteins. Results: AS and AL with 6xHis‐T7 tags pulled down eNOS and caveolin‐1 from a BAEC lysate. Conclusion: These results provide, for the first time, evidence for a physical association between AS, AL, eNOS and caveolin‐1, consistent with the important functional role of the citrulline‐NO cycle in endothelial NO production. This work was supported by American Heart Association FL Affiliate Grants 9950864V/0455228B and the University of South Florida Foundation—Mary and Walter Traskiewicz Memorial Fund.