Klatt P, Schmidt K, Brunner F, Mayer B
Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Austria.
J Biol Chem. 1994 Jan 21;269(3):1674-80.
Nitric oxide (NO) is synthesized from L-arginine by different NO synthase isozymes, which are inhibited by the substrate analogs NG-methyl- and NG-nitro-L-arginine. We studied binding of 3H-labeled NG-nitro-L-arginine to purified brain NO synthase and compared the data with results obtained in enzyme kinetic experiments. Binding data revealed a single binding site for NG-nitro-L-[3H]arginine (KD = 0.17 microM). Binding was competitively antagonized by L-arginine (KI = 2.9 microM). The half-time of dissociation was remarkably slow (9.4 min) and closely correlated with the time necessary for surmounting NO synthase inhibition by dilution. Although an apparently less potent inhibitor, NG-methyl-L-arginine exhibited the same affinity for brain NO synthase as the nitro derivative (KI = 0.17 microM), and in initial rate experiments, almost equal KI values were obtained for NG-methyl-L-arginine (0.61 microM) and NG-nitro-L-arginine (0.53 microM). However, after prolonged incubation periods, NG-nitro-L-arginine induced a rapid inactivation of the enzyme, whereas the methyl derivative turned out to be a substrate of NO synthase, which was slowly converted into stoichiometric amounts of NO and L-citrulline.
一氧化氮（NO）由不同的一氧化氮合酶同工酶从L-精氨酸合成，这些同工酶被底物类似物NG-甲基-L-精氨酸和NG-硝基-L-精氨酸抑制。我们研究了3H标记的NG-硝基-L-精氨酸与纯化的脑一氧化氮合酶的结合，并将数据与酶动力学实验结果进行了比较。结合数据显示NG-硝基-L-[3H]精氨酸有一个单一结合位点（KD = 0.17微摩尔）。L-精氨酸竞争性拮抗结合（KI = 2.9微摩尔）。解离半衰期非常缓慢（9.4分钟），且与通过稀释克服一氧化氮合酶抑制所需的时间密切相关。尽管NG-甲基-L-精氨酸作为抑制剂的效力明显较低，但它对脑一氧化氮合酶的亲和力与硝基衍生物相同（KI = 0.17微摩尔），并且在初始速率实验中，NG-甲基-L-精氨酸（0.61微摩尔）和NG-硝基-L-精氨酸（0.53微摩尔）的KI值几乎相等。然而，长时间孵育后，NG-硝基-L-精氨酸会导致该酶快速失活，而甲基衍生物却是一氧化氮合酶的底物，它会缓慢转化为化学计量的NO和L-瓜氨酸。
