Effect of redox-cycling agents on nitric oxide synthase activity in vitro and modulation of protein expression in vivo: NO nitric oxide synthase in mitochondria
This dissertation describes studies conducted in three areas dealing with the interaction of redox-active compounds with the flavin- and heme-containing enzyme, neuronal nitric oxide synthase (NOS). They are as follows: (I) Studies were initiated using extracts from several varieties of tobacco to identify constituent(s) that could interact with nNOS. Results from [14C]- L-arginine to [14C]-L-citrulline conversion assays to which tobacco extracts were individually added, indicate that there are nNOS inhibitors present in tobacco and they are non-polar, lipophilic and non-volatile in nature. In addition, results from assays in which the tobacco-derived redox-active compound, 2,3,6-trimethyl-1,4-naphthoquinone or 2-methyl-1,4-naphthoquinone were individually incorporated into the nNOS activity assays indicate that single compounds can produce either stimulation or inhibition of L-citrulline production depending on the concentration of the quinone to which nNOS is exposed as well as the compound's electrochemical characteristics. (II) Data, both for and against the presence of a mitochondrial NOS isoform (mtNOS) is in the refereed literature. However, irrefutable evidence, either for or against the existence of a mtNOS isoform has not been forthcoming. Therefore, studies were initiated at our laboratory to investigate whether any NOS isoform resides in ultra pure rat liver mitochondria, utilizing 3 independent techniques. Results from activity assays, mass spectrometry (MS) analyses and immunochemical detection for NOS, independently, as well as collectively, refute the claim that a NOS isoform exists within rat liver mitochondria, at least down to the limit of detection. (III) Mitochondria are thought to be involved in modulating the toxicity of redox-active compounds. In order to understand the effect of 1, 1-dimethyl-4,4-bipyridinium ion (paraquat), a redox-active compound, on levels of protein expression in whole liver homogenate as well as rat liver mitochondria during the early stage of paraquat exposure (40 mg/kg, i.p), proteomic analyses were performed 3 hrs after paraquat administration. Mass spectrometric analyses of paraquat-treated rats identified 37 proteins in rat liver mitochondria as well as in whole rat liver homogenate that were either down- and/or up-regulated, at least 2 folds above control levels. GO ontology and Interpro scan analyses identified that the modulated proteins in rat liver mitochondria were oxidoreductases and transferases. In whole rat liver homogenate, the modulated proteins were mainly oxidoreductases, transferases as well as proteins involved in protein binding functions. Additionally, rat liver proteins involved in ubiquitination and the proteolytic pathway were modulated. In addition to these studies, a cost-effective method was designed and validated for obtaining and purifying the chicken antibodies against the stable, reactive nitrogen species biomarker, 3-nitrotyrosine.
Venkatakrishnan, Priya, "Effect of redox-cycling agents on nitric oxide synthase activity in vitro and modulation of protein expression in vivo: NO nitric oxide synthase in mitochondria" (2008). ETD Collection for University of Texas, El Paso. AAI3341654.