S-nitrosation and regulation of inducible nitric oxide synthase
S-nitrosation and regulation of inducible nitric oxide synthase
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DOI：
10.1021/bi0474463
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发表时间：
2005-03-29
期刊：
影响因子：
2.9
通讯作者：
Marletta, MA
中科院分区：
文献类型：
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作者：
Mitchell, DA;Erwin, PA;Marletta, MA
诱导型一氧化氮合酶（iNOS）和三个四硫代锌突变体（C104 A，C109 A和C104 A/C109 A）在大肠杆菌中表达和纯化。通过ICP-AES和锌特异性PAR比色测定发现突变体是无锌的，而野生型iNOS锌含量为0.38 +/- 0.01 mol Zn/mol iNOS二聚体。半胱氨酸突变体（C104 A和C109 A）的活性在野生型iNOS的误差范围内（2.24 +/- 0.12 μ mol NO min（-1）mg（-1）），但双半胱氨酸突变体的活性略有下降（1.75 +/- 0.14 μ mol NO min（-1）mg（-1））。为了确定NO是否可以刺激锌的释放和二聚体解离，允许野生型蛋白质与NO供体DEA/NO反应，然后进行缓冲液交换。用10 μ M DEA/NO处理的样品的ICP-AES显示锌含量降低（0.23 +/- 0.01至0.09 +/- 0.01摩尔Zn/摩尔iNOS二聚体），而没有血红素铁的损失。类似地处理的野生型iNOS的凝胶过滤导致与DEA处理的样品相比多20%的单体iNOS。与对照样品相比，只有野生型iNOS在与50 μ M DEA/NO反应后活性降低（42 +/-2%）。在sarne条件下使用blotin开关方法，只有野生型iNOS具有增加的S-生物素化水平。使用LysC消化和MALDIZ TOF/TOF MS将S-生物素化定位于野生型iNOS上的C104和C109。使用来自小鼠巨噬细胞系RAW-264.7的iNOS的免疫沉淀和生物素开关方法来确认iNOS的内-烯醇S-亚硝化，数据显示，四硫醇锌半胱氨酸的S-亚硝化导致锌从二聚体界面释放并形成无活性的单体，提示这种抑制模式可能发生在体内。
The inducible isoform of nitric oxide synthase (iNOS) and three zinc tetrathiolate mutants (C104A, C109A, and C104A/C109A) were expressed in Escherichia coli and purified. The mutants were found by ICP-AES and the zinc-specific PAR colorimetric assay to be zinc free, whereas the wild-type iNOS zinc content was 0.38 +/- 0.01 mol of Zn/mol of iNOS dimer. The cysteine mutants (C104A and C109A) had an activity within error of wild-type iNOS (2.24 +/- 0.12 mu mol of NO min(-1) mg(-1)), but the double cysteine mutant had a modestly decreased activity (1.75 +/- 0.14 mu mol of NO min(-1) mg(-1)). To determine if NO could stimulate release of zinc and dimer dissociation, wild-type protein was allowed to react with an NO donor, DEA/NO, followed by buffer exchange. ICP-AES of samples treated with 10 mu M DEA/NO showed a decrease in zinc content (0.23 +/- 0.01 to 0.09 +/- 0.01 mol of Zn/mol of iNOS dimer) with no loss of heme iron. Gel filtration of wild-type iNOS treated similarly resulted in similar to 20% more monomeric iNOS compared to a DEA-treated sample. Only wild-type iNOS had decreased activity (42 +/- 2%) after reaction with 50 mu M DEA/NO compared to a control sample. Using the blotin switch method under the sarne conditions, only wild-type iNOS had increased levels of S-biotinylation. S-Biotinylation was mapped to C104 and C109 on wild-type iNOS using LysC digestion and MALDIZ TOF/TOF MS. Immunoprecipitation of iNOS from the mouse rnacrophage cell line, RAW-264.7, and the biotin switch method were used to confirm endo-enOLIS S-nitrosation of iNOS, The data show that S-nitrosation of the zinc tetrathiolate cysteine results in zinc release frorn the dimer interface and formation of inactive monorners, suggesting that this mode of inhibition might occur in vivo.