PURIFICATION AND CHARACTERIZATION OF THE CYTOKINE-INDUCED MACROPHAGE NITRIC-OXIDE SYNTHASE - AN FAD-CONTAINING AND FMN-CONTAINING FLAVOPROTEIN
PURIFICATION AND CHARACTERIZATION OF THE CYTOKINE-INDUCED MACROPHAGE NITRIC-OXIDE SYNTHASE - AN FAD-CONTAINING AND FMN-CONTAINING FLAVOPROTEIN
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DOI：
10.1073/pnas.88.17.7773
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发表时间：
1991-09-01
影响因子：
11.1
通讯作者：
NATHAN, CF
中科院分区：
文献类型：
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作者：
STUEHR, DJ;CHO, HJ;NATHAN, CF
通过连续的阴离子交换、亲和和凝胶过滤色谱法，从用干扰素-γ和细菌脂多糖激活的小鼠巨噬细胞系中将可溶性一氧化氮 (NO) 合酶活性纯化了 426 倍。纯化的 NO 合酶的 SDS/PAGE 给出了 125 至 135 kDa 之间的三个间隔很近的银染蛋白条带。当在 L-精氨酸、NADPH、四氢生物蝶呤、FAD 和还原硫醇存在的情况下进行测定时，纯化的 NO 合酶的比活性为每分钟每毫克 1313 nmol NO2- 加 NO3-。该酶对 L-精氨酸和 NADPH 的表观 K(m) 分别为 2.8 和 0.3-mu-M。添加钙离子（含或不含钙调蛋白）不会增加纯化酶的活性，并且钙调蛋白抑制剂不会改变 NO 合成。凝胶过滤色谱表明，诱导的 NO 合酶作为几乎等于 250 kDa 的二聚体具有催化能力，但在不存在 L-精氨酸、FAD 和四氢生物蝶呤的情况下可以解离成几乎等于 130 kDa 的无活性单体。热变性后，NO合酶每摩尔130-kDa亚基释放1.1摩尔FAD和0.55摩尔FMN。因此，诱导型巨噬细胞NO合酶在几个方面与组成型NO合酶不同，并且是极少数同时含有FAD和FMN的真核酶之一。
A soluble nitric oxide (NO) synthase activity was purified 426-fold from a mouse macrophage cell line activated with interferon-gamma and bacterial lipopolysaccharide by sequential anion-exchange, affinity, and gel filtration chromatography. SDS/PAGE of the purified NO synthase gave three closely spaced silver-staining protein bands between 125 and 135 kDa. When assayed in the presence of L-arginine, NADPH, tetrahydrobiopterin, FAD, and reduced thiol, purified NO synthase had a specific activity of 1313 nmol of NO2- plus NO3-per min per mg. The apparent K(m) of the enzyme for L-arginine and NADPH was 2.8 and 0.3-mu-M, respectively. Addition of calcium ions with or without calmodulin did not increase the activity of the purified enzyme, and NO synthesis was not altered by calmodulin inhibitors. Gel filtration chromatography indicated that the induced NO synthase was catalytically competent as a dimer of almost-equal-to 250 kDa but could be dissociated into inactive monomers of almost-equal-to 130 kDa in the absence of L-arginine, FAD, and tetrahydrobiopterin. Upon heat denaturation, NO synthase released 1.1 mol of FAD and 0.55 mol of FMN per mol of 130-kDa subunit. Thus, inducible macrophage NO synthase differs in several respects from constitutive NO synthases and is one of very few eukaryotic enzymes containing both FAD and FMN.