title: Use of chimeric forms of neuronal nitric-oxide synthase as dominant negative mutants.
abstract: 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.
authors.affiliation: Department of Pediatrics, University of California, San Francisco 94143-0106, USA.
authors.forename: Y T
authors.initials: YT
authors.lastname: Phung
authors.forename: S M
authors.initials: SM
authors.lastname: Black
mesh_terms: D000818:Animals; D019556:COS Cells; D005799:Genes, Dominant; D009154:Mutation; D019001:Nitric Oxide Synthase / Q000096:biosynthesis / Q000235:genetics*; D052248:Nitric Oxide Synthase Type I; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics*; D014162:Transfection
chemical_list: D011993:Recombinant Fusion Proteins; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat