MOLECULAR-CLONING AND EXPRESSION OF INDUCIBLE NITRIC-OXIDE SYNTHASE FROM HUMAN HEPATOCYTES
MOLECULAR-CLONING AND EXPRESSION OF INDUCIBLE NITRIC-OXIDE SYNTHASE FROM HUMAN HEPATOCYTES
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人肝细胞中诱导型一氧化氮合酶的分子克隆和表达
DOI：
10.1073/pnas.90.8.3491
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发表时间：
1993-04-15
影响因子：
11.1
通讯作者：
BILLIAR, TR
中科院分区：
文献类型：
--
作者：
GELLER, DA;LOWENSTEIN, CJ;BILLIAR, TR
一氧化氮是不同细胞类型的短寿命生物介质。脂多糖（LPS）和干扰素γ刺激小鼠巨噬细胞合成诱导型一氧化氮合酶（NOS）。在人肝细胞中，通过肿瘤坏死因子、白细胞介素1、干扰素γ和LPS的组合处理诱导NOS活性。我们现在报告的诱导型人肝细胞NOS（hep-NOS）cDNA的分子克隆和表达。hep-NOS与mac-NOS有80%的氨基酸同源性。与其他NOS亚型一样，存在FMN、FAD和NADPH的识别位点，以及共有钙调蛋白结合位点。在人293肾细胞转染hep-NOS cDNA的NOS活性减少的Ca 2+螯合和钙调素拮抗剂，反映了Ca 2+依赖性不明显的mac-NOS。北方印迹分析表明，在人肝细胞和主动脉平滑肌细胞与LPS和细胞因子刺激后，hep-NOS cDNA的4.5 kb的mRNA。人类基因组Southern印迹探测人类hep-NOS和人类内皮NOS cDNA克隆显示不同的基因组限制性内切酶片段，这表明不同的基因产物，这些NOS亚型。hep-NOS似乎是一种诱导型NOS，其不同于mac-NOS以及脑和内皮NOS同工酶。
Nitric oxide is a short-lived biologic mediator for diverse cell types. Synthesis of an inducible nitric oxide synthase (NOS) in murine macrophages is stimulated by lipopolysaccharide (LPS) and interferon gamma. In human hepatocytes, NOS activity is induced by treatment with a combination of tumor necrosis factor, interleukin 1, interferon gamma, and LPS. We now report the molecular cloning and expression of an inducible human hepatocyte NOS (hep-NOS) cDNA. hep-NOS has 80% amino acid sequence homology to macrophage NOS (mac-NOS). Like other NOS isoforms, recognition sites for FMN, FAD, and NADPH are present, as well as a consensus calmodulin binding site. NOS activity in human 293 kidney cells transfected with hep-NOS cDNA is diminished by Ca2+ chelation and a calmodulin antagonist, reflecting a Ca2+ dependence not evident for mac-NOS. Northern blot analysis with hep-NOS cDNA reveals a 4.5-kb mRNA in both human hepatocytes and aortic smooth muscle cells following stimulation with LPS and cytokines. Human genomic Southern blots probed with human hep-NOS and human endothelial NOS cDNA clones display different genomic restriction enzyme fragments, suggesting distinct gene products for these NOS isoforms. hep-NOS appears to be an inducible form of NOS that is distinct from mac-NOS as well as brain and endothelial NOS isozymes.