Intracellular assembly of inducible NO synthase is limited by nitric oxide-mediated changes in heme insertion and availability
Intracellular assembly of inducible NO synthase is limited by nitric oxide-mediated changes in heme insertion and availability
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DOI：
10.1074/jbc.271.10.5414
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发表时间：
1996-03-08
影响因子：
4.8
通讯作者：
Stuehr, DJ
中科院分区：
文献类型：
--
作者：
Albakri, QA;Stuehr, DJ
细胞因子诱导小鼠巨噬细胞系RAW 264.7表达细胞因子诱导型一氧化氮合酶(INOS)，该酶仅以二聚体形式活跃。由于纯化的诱导型一氧化氮合酶亚基的二聚化需要四氢生物蝶呤、血红素和L精氨酸，我们调查了这些因素的可用性是否也影响诱导型一氧化氮合酶二聚体的细胞内组装。在细胞因子作用下，进一步培养16h后，iNOS蛋白几乎呈线性积累。相反，二聚体诱导型一氧化氮合酶的积累速度较慢，在培养过程中不断下降，导致16h时只有25%的聚合型诱导型一氧化氮合酶蛋白以二聚体的形式存在。进一步的实验证明，二聚体不稳定或L精氨酸和四氢生物蝶呤的可用性是限制一氧化氮合酶二聚体积累的因素。用N-奥米伽-硝基-L-精氨酸甲酯(L-NAME)阻断细胞内NO的合成，可显著增加iNOS二聚体的组装，提示NO合成限制了iNOS二聚体的形成。NO合成可阻止N-奥米伽-硝基-L-精氨酸甲酯处理的细胞中与诱导型一氧化氮合酶相关的可溶性血红素水平的增加，并减少血红素对诱导型一氧化氮合酶的插入。这些与NO相关的缺陷不能通过在激活的细胞培养中添加血红素前体或氯化血红素来逆转。从激活的细胞释放铁的测量表明，内源性NO合成显著增加了Fe-59向培养液中的释放。这些观察结果表明，iNOS二聚化在很大程度上受到iNOS NO合成的限制。NO似乎通过阻止血红素插入和降低血红素的可获得性来限制二聚体iNOS的细胞内组装。
Cytokines induce the mouse macrophage cell line RAW 264.7 to express cytokine-inducible nitric oxide synthase (iNOS), which is active only in dimeric form. Because dimerization of purified iNOS subunits requires tetrahydrobiopterin, heme, and L-arginine, we investigated if availability of these factors also influences intracellular assembly of dimeric iNOS. Following exposure to cytokines, iNOS protein was found to accumulate in a near linear manner over 16 h of further culture. In contrast, dimeric iNOS accumulated at a slower rate that continuously decreased during culture, resulting in only 25% of the accumulated iNOS protein being in dimeric form by 16 h. Further experiments argued against dimer instability or L-arginine and tetrahydrobiopterin availability as factors limiting NOS dimer accumulation. Blocking cellular NO synthesis with N-omega-nitro-L-arginine methyl ester (L-NAME) greatly increased iNOS dimer assembly, indicating NO synthesis limited iNOS dimerization. NO synthesis was found to prevent an increase in soluble heme level that was associated with iNOS induction in N-omega-nitro-L-arginine methyl ester-treated cells and also diminished heme insertion into iNOS. These NO-related defects were not reversed by adding heme precursors or hemin to the activated cell cultures. Measurement of iron release from activated cells demonstrated that endogenous NO synthesis substantially increased the release of Fe-59 to the medium. These observations suggest that iNOS dimerization is limited to a large extent by iNOS NO synthesis. NO appears to limit intracellular assembly of dimeric iNOS by preventing heme insertion and decreasing heme availability.