Nitric oxide synthases (NOS) contain three subdomains, the N-terminal monooxygenase domain, the C-terminal reductase domain and the connecting canonical calmodulin(CaM)- binding domain. Neuronal NOS(nNOS) activity is Ca+/CaM-dependen, whereas inducible NOS (iNOS) activity is Ca2+independent presumably due to tightly bound CaM. To study the mechanism responsible for iNOS Ca2+-independent activity and nNOS Ca2+-dependent activity, a series of chimeric NOSs derived from nNOS and iNOS were transiently expressed in COS-7 cells. Ca2+-dependent NOS activities were measured in cell lysates in the presence of CaM. Results indicate that Ca2+independent activity ofiNOS results primarily from its monooxygenase domain and the canonical CaM-binding region acting in concert. However, neither the iNOS monooxygenase domain nor the canonical CaM-binding region alone confers Ca2+-independent NOS activity. Replacement of CaM-binding sequence of nNOS with that of iNOS does not confer Ca2+-independent NOS activity. Replacement of CaM-binding sequence of iNOS with that of nNOS changes the activity to Ca2+-dependent. In both cases, there is increased Ca2+sensitivity compared to nNOS. Additionally, the reductase domain of iNOS increases the Ca2+-sensitivity of a chimeric NOS containing nNOS CaM-binding sequence and the nNOS monoxygenase domain. The Ca2+-independent activity of iNOS appears to be mediated through different regions of the molecule, rather than the canonical CaM-binding region alone.
|Original language||English (US)|
|State||Published - Dec 1 1997|
ASJC Scopus subject areas
- Molecular Biology