Lobe-Specific Calcium Binding in Calmodulin Regulates Endothelial Nitric Oxide Synthase Activation

BACKGROUND: Human endothelial nitric oxide synthase (eNOS) requires calcium-bound calmodulin (CaM) for electron transfer but the detailed mechanism remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: Using a series of CaM mutants with E to Q substitution at the four calcium-binding sites, we found that single mutation at any calcium-binding site (B1Q, B2Q, B3Q and B4Q) resulted in ∼2-3 fold increase in the CaM concentration necessary for half-maximal activation (EC50) of citrulline formation, indicating that each calcium-binding site of CaM contributed to the association between CaM and eNOS. Citrulline formation and cytochrome c reduction assays revealed that in comparison with nNOS or iNOS, eNOS was less stringent in the requirement of calcium binding to each of four calcium-binding sites. However, lobe-specific disruption with double mutations in calcium-binding sites either at N- (B12Q) or at C-terminal (B34Q) lobes greatly diminished both eNOS oxygenase and reductase activities. Gel mobility shift assay and flavin fluorescence measurement indicated that N- and C-lobes of CaM played distinct roles in regulating eNOS catalysis; the C-terminal EF-hands in its calcium-bound form was responsible for the binding of canonical CaM-binding domain, while N-terminal EF-hands in its calcium-bound form controlled the movement of FMN domain. Limited proteolysis studies further demonstrated that B12Q and B34Q induced different conformational change in eNOS. CONCLUSIONS: Our results clearly demonstrate that CaM controls eNOS electron transfer primarily through its lobe-specific calcium binding

Search for polyisoprenoids in the flowers and fruits of selected coastal plants using two-dimensional thin layer chromatography

Coastal plants are recognized to yield secondary metabolites including polyisoprenoid alcohols. Coastal plants have been shown to have biological and phytochemical activities. The present study reports the search for polyisoprenoids composition from flowers and fruits of selected coastal plants, Amorphophallus paeoniifolius, Guettarda speciosa, and Jatropha curcas. A two-dimensional thin layer chromatography (2D-TLC) was used to analyse the composition and occurrence of polyisoprenoid alcohols (polyprenols and dolichols) in coastal plants. The distribution of polyprenols and dolichols in the flowers and fruits were detected and classified into one type only, type-II. Type-II, having the presence of both polyprenols and dolichols, was found in all samples investigated: in the flowers and fruits of A. paeoniifolius, G. spiciosa, and J. curcas. It is interesting to note that no dominating dolichols over polyprenols (type-I) or predominance polyprenol over dolichols (type-III) detected in this study. The present study, therefore, suggested the diversity of polyisoprenoids in the generative tissues of tropical coastal plants

Search for polyisoprenoids in the flowers and fruits of selected coastal plants using two-dimensional thin layer chromatography

Coastal plants are recognized to yield secondary metabolites including polyisoprenoid alcohols. Coastal plants have been shown to have biological and phytochemical activities. The present study reports the search for polyisoprenoids composition from flowers and fruits of selected coastal plants, Amorphophallus paeoniifolius, Guettarda speciosa, and Jatropha curcas. A two-dimensional thin layer chromatography (2D-TLC) was used to analyse the composition and occurrence of polyisoprenoid alcohols (polyprenols and dolichols) in coastal plants. The distribution of polyprenols and dolichols in the flowers and fruits were detected and classified into one type only, type-II. Type-II, having the presence of both polyprenols and dolichols, was found in all samples investigated: in the flowers and fruits of A. paeoniifolius, G. spiciosa, and J. curcas. It is interesting to note that no dominating dolichols over polyprenols (type-I) or predominance polyprenol over dolichols (type-III) detected in this study. The present study, therefore, suggested the diversity of polyisoprenoids in the generative tissues of tropical coastal plants