| Summary: | Calmodulin (CaM) is a calcium-binding protein that has promiscuous regulatory interactions with over three hundred intracellular protein targets. The focus of this study was to characterize the functional role of phosphorylated CaM in vitro and calcium-deficient CaM (Apo-CaM) ex vivo. In the in vitro study, the effect of phosphorylated CaM on the binding and activation of CaM target proteins was analyzed using mammalian Nitric Oxide Synthase (NOS). NOS is an enzyme that catalyzes the conversion of L-arginine to L-citrulline and •NO. In addition, the activation of NOS by modified CaM proteins was also analyzed in the presence of a CaM binding peptide, PEP-19.
Protein trafficking experiments were performed ex vivo to extend our understanding of Apo-CaM’s functional role in mammalian cells. The cell lines that were used in this investigation include mouse Embryonic Stem Cells (mESC), Human Umbilical Vein Endothelia Cells (HUVEC) and Human Neuronal Glioma Cells (HNGC).
The major finding of this projects are: phosphorylation of selective CaM residues can attenuated NOS activity, electrostatic interactions are important in the activation of iNOS by CaM, and the activation of iNOS by CaM occurs in a calcium-dependent manner
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