Moss D W, Wei X, Liew F Y, Moncada S, Charles I G
Wellcome Research Laboratories, Beckenham, Kent, UK.
Eur J Pharmacol. 1995 Mar 15;289(1):41-8. doi: 10.1016/0922-4106(95)90166-3.
A complementary DNA (cDNA) encoding murine inducible nitric oxide synthase was cloned from activated J774 macrophages. Expression of this cDNA in a baculovirus-insect cell system allowed comparison of the recombinant enzyme with the native homologue. Western blot analysis of activated J774 and baculovirus-infected insect cell cytosols demonstrated reactivity against a protein of 135 kDa. Kinetic studies on the recombinant and native enzymes revealed an absolute requirement for L-arginine and NADPH in order to achieve full activity. In addition, both enzymes were found to have similar maximum velocities and Km values for these two substrates. The nitric oxide synthase antagonists N-guanidino monomethyl L-arginine and N-iminoethyl L-ornithine inhibited both enzymes at a similar rate. Furthermore, comparable concentrations of inhibitor were required to achieve half maximal enzyme inhibition. These results indicate that recombinant inducible NO synthase appears to be pharmacologically indistinguishable from the native enzyme.
从活化的J774巨噬细胞中克隆出编码小鼠诱导型一氧化氮合酶的互补DNA（cDNA）。该cDNA在杆状病毒-昆虫细胞系统中的表达使得重组酶能够与天然同源物进行比较。对活化的J774和杆状病毒感染的昆虫细胞胞质溶胶进行的蛋白质免疫印迹分析表明，存在针对135 kDa蛋白质的反应性。对重组酶和天然酶的动力学研究表明，为了达到完全活性，绝对需要L-精氨酸和NADPH。此外，发现这两种酶对于这两种底物具有相似的最大速度和米氏常数（Km值）。一氧化氮合酶拮抗剂N-胍基单甲基L-精氨酸和N-亚氨基乙基L-鸟氨酸以相似的速率抑制这两种酶。此外，达到酶抑制半数最大值所需的抑制剂浓度相当。这些结果表明，重组诱导型一氧化氮合酶在药理学上似乎与天然酶没有区别。
