Synthesis of nitrogen oxides from L-arginine by macrophage cytosol: requirement for inducible and constitutive components.
Synthesis of nitrogen oxides from L-arginine by macrophage cytosol: requirement for inducible and constitutive components.
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DOI：
10.1016/0006-291x(89)92615-6
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发表时间：
1989-06
影响因子：
3.1
通讯作者：
D. Stuehr;Nyoun Soo Kwon;Steven S. Gross;Bonnie A. Thiel;Roberto Levi;Carl Nathan
中科院分区：
文献类型：
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作者：
D. Stuehr;Nyoun Soo Kwon;Steven S. Gross;Bonnie A. Thiel;Roberto Levi;Carl Nathan
由小鼠腹腔巨噬细胞和RAW 264巨噬细胞系制备的细胞溶胶催化L-精氨酸转化为不稳定的血管舒张剂一氧化氮及其累积终产物亚硝酸盐和硝酸盐。这种活性需要细胞预先暴露于干扰素-γ和细菌脂多糖。使用亚硝酸盐+硝酸盐的产生作为所有三种氮氧化物合成的指标，进一步表征氮氧化物合成酶活性。氮氧化物合成酶活性是热敏性的，NADPH依赖性的，并表现出底物立体专一性。亚硝酸盐+硝酸盐的形成与时间和胞液浓度成正比。然而，稀释降低了比活性，表明除NADPH外还需要辅因子。通过添加来自非活化巨噬细胞的胞质溶胶来恢复比活性，所述非活化巨噬细胞本身不产生一氧化氮。需要对照巨噬细胞胞质溶胶的高分子量和低分子量组分来恢复来自已被稀释或部分纯化的活化巨噬细胞的胞质溶胶的活性。因此，参与鼠巨噬细胞一氧化氮合成的酶系统由至少一种诱导型和两种组成型成分组成。
Cytosols prepared from murine peritoneal macrophages and the RAW 264 macrophage cell line catalyzed conversion of L-arginine to the labile vasorelaxant nitric oxide and its accumulating endproducts, nitrite and nitrate. This activity required previous exposure of the cells to interferon-γ and bacterial lipopolysaccharide. Nitrogen oxide synthetase activity was characterized further using nitrite + nitrate production as an indicator of the synthesis of all three nitrogen oxides. Nitrogen oxide synthetase activity was heat-sensitive, NADPH-dependent, and exhibited substrate stereospecificity. The nitrite + nitrate formation was proportional to time and concentration of cytosol. However, dilution decreased the specific activity, suggesting a cofactor requirement in addition to NADPH. Specific activity was restored by addition of cytosol from non-activated macrophages, which itself did not make nitric oxide. Both high and low molecular weight fractions of control macrophage cytosol were required to restore activity of cytosol from activated macrophages haat had been either diluted or partially purified. Thus, the enzymatic system involved in nitric oxide synthesis by murine macrophages consists of at least one inducible and two constitutive components.