國家衛生研究院 NHRI:Item 3990099045/6507
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    Please use this identifier to cite or link to this item: http://ir.nhri.org.tw/handle/3990099045/6507


    Title: Lobe-specific calcium binding in calmodulin regulates endothelial nitric oxide synthase activation
    Authors: Wu, PR;Kuo, CC;Yet, SF;Liou, JY;Wu, KK;Chen, PF
    Contributors: Institute of Cellular and Systems Medicine
    Abstract: 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.
    Date: 2012-06
    Relation: PLoS ONE. 2012 Jun;7(6):Article number e39851.
    Link to: http://dx.doi.org/10.1371/journal.pone.0039851
    JIF/Ranking 2023: http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=NHRI&SrcApp=NHRI_IR&KeyISSN=1932-6203&DestApp=IC2JCR
    Cited Times(WOS): https://www.webofscience.com/wos/woscc/full-record/WOS:000305892100112
    Cited Times(Scopus): http://www.scopus.com/inward/record.url?partnerID=HzOxMe3b&scp=84863088955
    Appears in Collections:[Kenneth Kun-Yu Wu] Periodical Articles
    [Jun-Yang Liou] Periodical Articles
    [Shaw-Fang Yet] Periodical Articles
    [Cheng-Chin Kuo] Periodical Articles

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