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Use of Chimeric Forms of Neuronal Nitric‐Oxide Synthase as Dominant Negative Mutants
Author(s) -
Phung Yume T.,
Black Stephen M.
Publication year - 1999
Publication title -
iubmb life
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.132
H-Index - 113
eISSN - 1521-6551
pISSN - 1521-6543
DOI - 10.1080/713803520
Subject(s) - mutant , heme , chemistry , nitric oxide , fusion protein , dimer , nitric oxide synthase , neuronal nitric oxide synthase , transfection , microbiology and biotechnology , atp synthase , biochemistry , biophysics , enzyme , biology , recombinant dna , gene , organic chemistry
Because the functional form of neuronal nitric‐oxide synthase (nNOS) is a homodimer, we investigated whether we could disrupt dimer formation with inactive nNOS chimeras acting as dominant negative mutants. To test this hypothesis, we either expressed the heme and reductase regions of rat nNOS as single domains or produced fusion proteins between the rat nNOS heme domain and various other electron‐shuttling proteins. A dominant negative potential of these constructs was demonstrated by their ability to reduce NOS activity when transfected into a cell line stably expressing rat nNOS. In the presence of these nNOS mutant proteins, cellular levels of inactive nNOS monomers were significantly increased, indicating that their mechanism of action is through the disruption of nNOS dimer formation. These dominant negative mutants should prove valuable in analyzing the role of nNOS in biological systems.