Efffects of Calmodulin Site II and IV Mutations on the Activity of Inductible Nitric Oxide Synthase
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Nitric oxide is a free radical, whose roles in the human body include such important functions as neurotransmission, blood pressure control, and immune system response. However, if overproduced, nitric oxide can be detrimental, especially when it combines with other reactive species, chemically changing bio macromolecules. Due to the oxidative stress that it causes, nitric oxide has been implicated in serious diseases, such as neurodegenerative disorders and autoimmune disorders. Nitric oxide synthase exists in three distinct isoforms. All three are activated by calmodulin, which is the focus of this study. As it turns out, calmodulin binds to two NOS isoforms only at high micromolar concentrations of intracellular calcium (for which it has four binding sites), and it seems to bind the third - inducible NOS (or iNOS) - even if intracellular calcium is low. This fact can be used to further investigate how calmodulin interacts with NOS and how the regulation of this isoform takes place. Dr. Stevens-Truss has done previous work on iNOS and calmodulin interaction. It turned out that inhibition of calcium-binding sites of calmodulin had a differential effect on the iNOS activity depending on where the impaired site was located. This project focused on the effect that deactivated calcium-binding sites II and IV of two calmodulin mutants had on iNOS activity. Successful expression, purification, and testing yielded the following results: inactive site II of calmodulin decreased iNOS activity to 39% of the maximum value, whereas inactive site IV allowed iNOS to retain 76% maximal activity.