Yoneyama H, Kosaka H, Ohnishi T, Kawazoe T, Mizoguchi K, Ichikawa Y
Second Department of Physiology, Kagawa Medical University, Japan.
Eur J Biochem. 1999 Dec;266(3):771-7. doi: 10.1046/j.1432-1327.1999.00888.x.
A water-soluble iron complex with N-dithiocarboxysarcosine (Fe-DTCS) has been developed as an ESR spin-trapping agent for NO and successfully applied to ESR imaging of endogenous NO production in mice. We attempted to measure NO produced by purified neuronal NO synthase (nNOS) by this method, but could not detect NO. We speculated that Fe-DTCS inhibits NOS activity. In fact, it markedly inhibited NOS activity with an IC50 value of 9.7 +/- 0.7 microM in the citrulline-formation assay. DTCS alone did not inhibit the activity. An iron complex with N-methyl-D-glucamine dithiocarbamate, a similar spin-trapping agent for NO, also inhibited the activity, with an IC50 value of 25.1 +/- 2.9 microM. Fe-DTCS suppressed cytochrome c and ferricyanide reductase activities of nNOS, and markedly increased nNOS-mediated NADPH oxidation. Concomitantly, it accelerated oxygen consumption caused by activated nNOS. These results suggest that the ESR spin-trapping agent Fe-DTCS inhibits NO synthesis by interfering with the physiological electron flow from NADPH to nNOS heme iron.
一种与N - 二硫代羧基肌氨酸形成的水溶性铁络合物（Fe - DTCS）已被开发用作检测一氧化氮（NO）的电子顺磁共振（ESR）自旋捕获剂，并成功应用于小鼠体内内源性NO生成的ESR成像。我们试图用这种方法测量纯化的神经元型一氧化氮合酶（nNOS）产生的NO，但未能检测到。我们推测Fe - DTCS抑制了一氧化氮合酶的活性。事实上，在瓜氨酸生成试验中，它以9.7±0.7微摩尔的半数抑制浓度（IC50）显著抑制了一氧化氮合酶的活性。单独的二硫代羧基肌氨酸（DTCS）并没有抑制该活性。一种与N - 甲基 - D - 葡糖胺二硫代氨基甲酸盐形成的铁络合物，一种类似的用于检测NO的自旋捕获剂，也抑制了该活性，其IC50值为25.1±2.9微摩尔。Fe - DTCS抑制了nNOS的细胞色素c和铁氰化物还原酶活性，并显著增加了nNOS介导的NADPH氧化。同时，它加速了由活化的nNOS引起的氧气消耗。这些结果表明，ESR自旋捕获剂Fe - DTCS通过干扰从NADPH到nNOS血红素铁的生理电子流来抑制NO合成。
