Superoxide dismutases (SODs) are metalloenzymes that catalytically deactivate superoxide radicals (O2-) generated in all aerobic organisms. The deleterious effects of O2- on macromolecules, subcellular components, cells and tissues suggest that O2- can be an initiating or contributing factor in disease (Bannister 1984). Superoxide-related pathology may result from several factors including an increased production of O2- during exposure to hyperoxia or from decreased activity of SOD (Bannister et a/. 1987). Understanding of how SOD activation is regulated by oxygen tension (pO2) is biomedically relevant because CuZnSOD and MnSOD reduce and prevent oxidative stress and damage in humans (Strauss et al. 1980). Three SODs (CuZnSOD, MnSOD, FeSOD) have been characterized in both eucaryotes and procaryotes. A model of MnSOD induction in Escherichia coli by metal ions, redox-cycling compounds and oxygen has been proposed (Touati 1988a) which involves multiregulation at the transcriptional and posttranslational levels. However, in eucaryotes, the mechanisms regulating the increased activity of SODs at elevated pO2 are not known, particularly at the transcriptional and translational levels. As a result, no eucaryotic model exists to explain the induction of SODs by pO2. The long term objective of this project is to develop a model for eucaryotes to determine how elevated pO2 regulates the activation and/or induction of SODs in vivo. We will use the eucaryotic dinoflagellate Symbidiodinium microadriaticum as an experimental organism because this species contains the three SODs and is frequently exposed to extremes of oxygen (0-78% pO2). Specifically, three hypotheses will be tested. These are: 1) SOD activation is regulated by the direct action of oxygen. This will be studied by exposing cell-free extracts from normoxic (21% pO2) cultures to hyperoxia (63% pO2) and then measuring the specific activity of SOD with time relative to controls. 2) SOD induction is regulated at the transcriptional level. Cells will be labelled with [NaH-14C]O3 in the presence and absence of rifampicin (a transcriptional inhibitor), lysed, polypeptides separated electrophoretically and probed with SOD polyclonal antibodies in order to determine whether SOD induction is regulated at the level of RNA. 3) SOD induction is regulated at the translational level. Cycloheximide, a translational inhibitor will be used as described in hypothesis 2, to determine whether SOD induction is regulated at the level of protein synthesis. In addition, the influence of pO2 on levels of translatable SOD mRNA will be studied by means of in vitro translation and immunoprecipitation studies. Baseline studies will be initially performed to purity CuZnSOD, MnSOD and FeSOD to homogeneity and to raise polyclonal antibodies against each of the three proteins. Limited N-terminal amino acid sequences will be obtained for these proteins in order to synthesize oligonucleotide probes. These probes will provide the basis to study molecular mechanisms of how elevated pO2 regulates the induction of SOD in eucaryotes.
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