Subcellular Localization and Characterization of Neuronal Nitric Oxide Synthase
Subcellular Localization and Characterization of Neuronal Nitric Oxide Synthase
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DOI：
10.1046/j.1471-4159.1994.62041524.x
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发表时间：
1994-04
影响因子：
4.7
通讯作者：
M. Hecker;A. Mülsch;R. Busse
中科院分区：
文献类型：
--
作者：
M. Hecker;A. Mülsch;R. Busse
摘要：与内皮细胞中主要参与的钙/钙调素依赖的一氧化氮合酶不同，相应的神经元同工酶被认为主要是可溶的，可能是因为缺乏翻译后肉豆蔻酸化。然而，来自该实验室和其他实验室的初步发现表明，神经元一氧化氮合酶活性的很大一部分实际上可能是膜结合的。因此，我们更详细地研究了这种酶在大鼠和兔小脑亚细胞部分中的分布。高达60%的NO合酶活性存在于颗粒组分中，密度梯度超速离心法显示，主要与内质网组分有关。可溶酶和颗粒酶在比活性、钙和pH依赖性、抑制敏感性和免疫反应性方面没有明显差异，表明大鼠和兔小脑都含有单一的钙/钙调蛋白依赖性的一氧化氮合酶。细胞色素P450抑制剂SKF-525A对这些亚细胞组份中NO合成酶活性的抑制(IC_(50)=90μM)以及哺乳动物细胞色素P450酶是内质网结合蛋白的事实支持了小脑NO合成酶是细胞色素P450型血红蛋白的观点。此外，上述发现表明，翻译后肉豆蔻酸化可能不是决定一氧化氮合酶在细胞内定位的唯一因素。
Abstract: In contrast to the predominantly participate, Ca2+/calmodulin‐dependent nitric oxide (NO) synthase in endothelial cells, the corresponding neuronal isoenzyme is considered to be mainly soluble, presumably owing to the lack of a posttranslational myristoylation. However, preliminary findings from this and other laboratories suggest that a substantial portion of the neuronal NO synthase activity may in fact be membrane bound. We have therefore investigated the distribution of this enzyme among subcellular fractions of the rat and rabbit cerebellum in more detail. Up to 60% of the total NO synthase activity was found in the particulate fraction and, according to density gradient ultracentrifugation, associated mainly with the endoplasmic reticulum fraction. There was no apparent difference between the soluble and particulate enzymes with respect to their specific activity, Ca2+ and pH dependency, inhibitor sensitivity, or immunoreactivity, suggesting that both rat and rabbit cerebella contain a single Ca2+/calmodulin‐dependent NO synthase. The inhibition by the cytochrome P450 inhibitor SKF‐525A of the NO synthase activity in these subcellular fractions (IC50= 90 μM) and the fact that mammalian cytochrome P450 enzymes are endoplasmic reticulum‐bound proteins support the notion that the cerebellar NO synthase is a cytochrome P450‐type hemoprotein. Moreover, the aforementioned findings suggest that posttranslational myristoylation may not be the only factor determining the intracellular localization of NO synthase.