Chen P F, Tsai A L, Berka V, Wu K K
Vascular Biology Research Center, University of Texas Health Science Center at Houston, Houston, Texas 77030, USA.
J Biol Chem. 1996 Jun 14;271(24):14631-5.
A baculovirus system was used to express the oxygenase and reductase domains of human endothelial nitric-oxide synthase (ecNOS) as distinct proteins. The oxygenase domain (residues 1-491) was expressed using a vector containing a His6 tag at the N terminus. The purified oxygenase domain had an apparent molecular mass of approximately 54 kDa, and retained the ability to bind L-arginine and form the ferrous CO complex. The purified reductase domain (residues 492-1244) had an apparent molecular mass of approximately 82 kDa and retained the ability to catalyze NADPH-dependent cytochrome c reduction, which was enhanced 10-fold by the presence of Ca2+/calmodulin. Both purified domains exhibited immunoreactivity to rabbit anti-ecNOS IgG. The NOS activity was successfully reconstituted by mixing the two domains. These results demonstrate for the first time that the two domains of ecNOS are catalytically intact and can be reconstituted in vitro.
利用杆状病毒系统将人内皮型一氧化氮合酶（ecNOS）的加氧酶结构域和还原酶结构域表达为不同的蛋白质。加氧酶结构域（第1至491位氨基酸残基）使用在N端含有His6标签的载体进行表达。纯化后的加氧酶结构域表观分子量约为54 kDa，并保留了结合L-精氨酸和形成亚铁一氧化碳复合物的能力。纯化后的还原酶结构域（第492至1244位氨基酸残基）表观分子量约为82 kDa，并保留了催化NADPH依赖性细胞色素c还原的能力，在Ca2+/钙调蛋白存在的情况下，该能力增强了10倍。两个纯化后的结构域均对兔抗ecNOS IgG表现出免疫反应性。通过将两个结构域混合，成功地重建了NOS活性。这些结果首次证明ecNOS的两个结构域具有完整的催化活性，并且可以在体外重建。
