Narayanasami R, Nishimura J S, McMillan K, Roman L J, Shea T M, Robida A M, Horowitz P M, Masters B S
Department of Biochemistry, University of Texas Health Science Center at San Antonio 78284-7760, USA.
Nitric Oxide. 1997 Feb;1(1):39-49. doi: 10.1006/niox.1996.0103.
Changes in flavin and protein fluorescence of neuronal nitric oxide synthase (nNOS) and its flavoprotein module were studied in the presence of urea and compared with those previously reported for cytochrome P450 reductase (CPR) [R. Narayanasami, P. M. Horowitz, and B. S. S. Masters (1995) Arch. Biochem. Biophys. 316, 267-274]. As in the case of CPR, FMN was relatively loosely bound to nNOS and the flavoprotein module, but FAD remained bound at concentrations of up to 2 M urea Protein fluorescence increased progressively with increasing urea concentration, but could not be correlated with changes in flavin binding. NADPH-cytochrome c reductase activity of both nNOS and the flavoprotein module, but not that of CPR, was stimulated at early time points by both urea and guanidine hydrochloride (GnHCl), with levels of initial activity returning to baseline values within 60 min after addition of the chaotropic agent. Thus, at 3-4 M urea, enhancements of reductase activities of 20- and 5-fold with nNOS and the flavoprotein module, respectively, were obtained. Comparable enhancements of 12- and 6- to 7-fold, respectively, were obtained with calmodulin (CaM)/ CaCl2 and 0.5 M GnHCl. Thus, the effects of urea and GnHCl mimicked the stimulating effects of CaM. Separate preincubations of nNOS and cytochrome c with urea or GnHCl prior to initiation of the reductase assay showed that sensitivity to chaotropic agent under these conditions was a property of nNOS and not of cytochrome c. Moreover, when the nonprotein electron acceptor 2,6-dichlorophenolindophenol was employed in place of cytochrome c, comparable stimulation of reductase activity was observed in the presence of either urea or GnHCl. Fluorescence of 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfate in the presence of either nNOS or the flavoprotein module was increased optimally between 3 and 4 M urea, consistent with simultaneous exposure of hydrophobic regions of both proteins to solvent and optimization of reductase activity. FMN release from nNOS, but not from the flavoprotein module, was enhanced by CaM. Addition of FMN or FMN + FAD to nNOS, in the presence or absence of urea, brought about a doubling of initial cytochrome c reductase activity, but did not prevent the eventual decline in activity to basal levels. These data are consistent with conformational changes which favor increased electron transfer similar to that achieved with nNOS in the presence of CaM.
在有尿素存在的情况下，研究了神经元型一氧化氮合酶（nNOS）及其黄素蛋白模块的黄素和蛋白质荧光变化，并与先前报道的细胞色素P450还原酶（CPR）的情况进行了比较[R. 纳拉亚纳斯米、P. M. 霍洛维茨和B. S. S. 马斯特斯（1995年）《生物化学与生物物理学报》316卷，267 - 274页]。与CPR的情况一样，FMN与nNOS和黄素蛋白模块结合相对较松，但在高达2 M尿素浓度下FAD仍保持结合状态。蛋白质荧光随尿素浓度增加而逐渐增强，但与黄素结合的变化无关。nNOS和黄素蛋白模块的NADPH - 细胞色素c还原酶活性，而非CPR的该活性，在早期时间点受到尿素和盐酸胍（GnHCl）的刺激，在加入离液剂后60分钟内初始活性水平恢复到基线值。因此，在3 - 4 M尿素时，nNOS和黄素蛋白模块的还原酶活性分别增强了20倍和5倍。用钙调蛋白（CaM）/CaCl2和0.5 M GnHCl分别获得了12倍和6至7倍的类似增强。因此，尿素和GnHCl的作用模拟了CaM的刺激作用。在还原酶测定开始前，将nNOS和细胞色素c分别与尿素或GnHCl预孵育，结果表明在这些条件下对离液剂的敏感性是nNOS的特性而非细胞色素c的特性。此外，当使用非蛋白质电子受体2,6 - 二氯酚靛酚代替细胞色素c时，在有尿素或GnHCl存在的情况下观察到了对还原酶活性的类似刺激。在有nNOS或黄素蛋白模块存在的情况下，4,4'-二苯胺基 - 1,1'-联萘 - 5,5'-二硫酸盐的荧光在3至4 M尿素之间最佳增强，这与两种蛋白质的疏水区域同时暴露于溶剂中以及还原酶活性的优化一致。CaM增强了FMN从nNOS的释放，但未增强从黄素蛋白模块的释放。在有或没有尿素存在的情况下，向nNOS中添加FMN或FMN + FAD会使初始细胞色素c还原酶活性加倍，但并未阻止活性最终降至基础水平。这些数据与构象变化一致，这种构象变化有利于增加电子传递，类似于在有CaM存在时nNOS所实现的情况。
