Spratt Donald E, Taiakina Valentina, Guillemette J Guy
Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada.
Biochim Biophys Acta. 2007 Oct;1774(10):1351-8. doi: 10.1016/j.bbapap.2007.07.019. Epub 2007 Aug 19.
The nitric oxide synthase (NOS) enzymes are bound and activated by the Ca(2+)-binding protein, calmodulin (CaM). We have utilized CaM mutants deficient in binding Ca(2+) with mutations in the N-lobe (CaM(12)), the C-lobe (CaM(34)), or both lobes of CaM (CaM(1234)) to determine their effect on the binding and activation of the Ca(2+)-dependent neuronal (nNOS) and Ca(2+)-independent inducible NOS (iNOS) isoforms. Four different kinetic assays were employed to monitor the effect of these CaM mutants on electron transfer rates in NOS. Protein-protein interactions between CaM and NOS were studied using steady-state fluorescence and spectropolarimetry to monitor the binding of these CaM mutants to nNOS and iNOS CaM-binding domain peptides. The CaM mutants were unable to activate nNOS, however, our CD results show that the C-terminal lobe of CaM is capable of binding to nNOS peptide in the presence of Ca(2+). Our results prove for the first time without the use of chelators that apo-CaM is capable of binding to iNOS peptides and holoenzymes.
一氧化氮合酶（NOS）酶与钙结合蛋白钙调蛋白（CaM）结合并被其激活。我们利用了在N端叶（CaM(12)）、C端叶（CaM(34)）或CaM的两个叶（CaM(1234)）中存在结合Ca(2+)缺陷突变的CaM突变体，以确定它们对Ca(2+)依赖性神经元型（nNOS）和Ca(2+)非依赖性诱导型NOS（iNOS）同工型的结合和激活的影响。采用四种不同的动力学测定方法来监测这些CaM突变体对NOS中电子转移速率的影响。利用稳态荧光和旋光光谱法研究了CaM与NOS之间的蛋白质-蛋白质相互作用，以监测这些CaM突变体与nNOS和iNOS的CaM结合域肽段的结合情况。CaM突变体无法激活nNOS，然而，我们的圆二色性结果表明，在存在Ca(2+)的情况下，CaM的C末端叶能够与nNOS肽段结合。我们的结果首次在不使用螯合剂的情况下证明，脱辅基CaM能够与iNOS肽段和全酶结合。
