Evidence for a bidomain structure of constitutive cerebellar nitric oxide synthase.
Evidence for a bidomain structure of constitutive cerebellar nitric oxide synthase.
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发表时间：
1994-05
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通讯作者：
Essam A. Sheta;Kirk McMillan;B. Masters
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作者：
Essam A. Sheta;Kirk McMillan;B. Masters
一氧化氮合酶(NOS)催化L精氨酸和分子氧合成NO和瓜氨酸的过程依赖于NADPH和钙调素。一氧化氮合酶NH2-末端的血红素结合共同序列和COOH-末端的核苷酸结合序列(FMN和FAD)的定位表明了一种双核蛋白结构。此外，在酶的血红素结合域和黄素结合域之间存在一个假定的钙调蛋白结合序列，这表明钙调蛋白在调节这些结构域的空间取向方面发挥了作用，这是催化活性所必需的。首先，为了确定钙调素的作用和分离结构域的功能，用荧光猝灭法测定了钙/钙调素结合引起的一氧化氮合酶的构象变化，由此计算出钙调素的结合常数约为1 nM。其次，对各种人工受体的电子传递进行了测量。Ca~(2+)/CaM的加入使细胞色素c的还原速度从10-15倍增加到10-15倍，而对2，6-二氯苯酚和铁氰化钠的还原速度仅有轻微的刺激作用。钙调素对NOS的刺激导致NADPH介导的对超氧化物歧化酶敏感的细胞色素c的还原，以及乙酰化的细胞色素c的减少，而非刺激的NOS只能微弱地还原细胞色素c。因此，这种被激发的活性可能是由超氧阴离子介导的。第三，在没有钙调蛋白的情况下，NOS的有限蛋白分解导致细胞色素C还原酶活性的时间依赖性增加，而超氧化物歧化酶不能抑制这种活性，并降低了对NO生成的催化作用。胰酶的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法分析表明，形成了大约89 kDa和79 kDa的片段。多肽的序列分析证实，从Ala728开始，胰酶在假定的钙调蛋白结合区切割酶。这一区域通过添加钙/钙调蛋白来保护其不被蛋白质降解。分离的NH2-末端结构域具有结合血红素的特征光谱，而COOH-末端结构域具有结合黄素的特征光谱。在钙调蛋白存在的情况下，还获得了其他的切割模式。这些数据表明，一氧化氮合酶的血红素和黄素结合域可以以功能完整的形式分离出来。
Nitric oxide synthase (NOS) catalyzes the NADPH-dependent, Ca2+/calmodulin-dependent formation of NO and citrulline from L-arginine and molecular oxygen. The localization of the heme-binding consensus sequence in the NH2-terminal half of NOS and of the binding sequences for nucleotides (FMN and FAD) in the COOH-terminal half suggests a bidomain structure. In addition, the presence of a putative calmodulin-binding sequence between the heme- and flavin-binding domains of the enzyme suggests a role for calmodulin in modulating a spatial orientation of these domains that is required for catalytic activity. First, to determine the effects of calmodulin and the functionality of the separated domains, Ca2+/calmodulin binding-induced conformational changes in NOS were measured by fluorescence quenching, from which a binding constant of approximately 1 nM for calmodulin was calculated. Second, electron transport to various artificial acceptors was measured. The addition of Ca2+/calmodulin increased cytochrome c reduction from 10-15-fold while stimulating the rate of 2,6-dichlorophenolindophenol and ferricyanide reduction only slightly, if at all. Calmodulin stimulation of NOS results in NADPH-mediated cytochrome c reduction, which is sensitive to superoxide dismutase, and the reduction of acetylated cytochrome c, which is only weakly reducible by unstimulated NOS. Thus, this stimulated activity is presumably superoxide anion-mediated. Third, limited proteolysis of NOS in the absence of calmodulin resulted in a time-dependent increase in cytochrome c reductase activity, which was not inhibitable by superoxide dismutase, and a decrease in catalysis of NO formation. SDS-polyacrylamide gel electrophoresis analysis of the tryptic digest demonstrated the formation of approximately 89- and approximately 79-kDa fragments. Sequence analysis of the peptides confirmed that trypsin cleaves the enzyme in the putative calmodulin-binding region beginning with Ala728. This region was protected from proteolysis by the addition of Ca2+/calmodulin. The separated NH2-terminal domain exhibited the characteristic spectrum of bound heme, while the COOH-terminal domain showed the characteristic spectrum of bound flavins. Other cleavage patterns were obtained in the presence of calmodulin. The data demonstrate that the heme- and flavin-binding domains of NOS can be isolated in functionally intact forms.