Mizutani Y, Tamiya-Koizumi K, Nakamura N, Kobayashi M, Hirabayashi Y, Yoshida S
Laboratory of Cancer Cell Biology, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Showa-ku, Nagoya, 466-8550, Japan.
J Cell Sci. 2001 Oct;114(Pt 20):3727-36. doi: 10.1242/jcs.114.20.3727.
To examine the intracellular localization of neutral sphingomyelinase 1 (nSMase 1), a rabbit polyclonal antibody was raised against a recombinant form of the enzyme expressed in E. coli. It has been reported that, in rat liver or in ascites hepatoma AH7974, high activity of neutral sphingomyelinase (SMase) is found at the plasma membrane, with a lesser but significant amount in nucleus and cytoplasm. The biochemical properties, dithiothreitol requirement and high salt concentration dependency, of cloned and expressed nSMase 1 resemble those of previously described nuclear neutral SMase of AH7974. The present study was therefore focused on the nuclear localization of this enzyme. Western blotting of subcellular fractions using anti-rat nSMase 1 antibody revealed most nSMase 1 to be associated with the nuclei and some with microsomes, but not with plasma membranes. Consistently, neutral SMase activity in nuclear extract was immunoprecipitated by the antibody, while that of plasma membranes was not. The results indicate that nSMase 1 mainly resides in the nucleus and may thus differ from neutral SMase in plasma membrane. On gel-filtration column chromatography of nuclear extract, the profile of neutral SMase activity corresponded well with immunoreactive protein bands on western blotting, suggesting that a large part of nuclear neutral SMase may be nSMase 1. Removal of the nuclear envelope by treatment with Triton X-100 did not significantly decrease the amount of nuclear nSMase 1, and western blotting of subnuclear fractions (i.e. nuclear envelope, chromatin, and nuclear matrix) revealed nSMase 1 signal exclusively in the nuclear matrix. Immunocytochemistry with AH7974, as well as rat fibroblast cell line 3Y1, demonstrated nSMase 1 to be localized mainly in the nucleus, with some in the cytoplasm. Moreover, immuno-electron microscopy clearly showed the signal of nSMase 1 to be more dense in the nucleus than in the cytoplasm of AH7974.
为了研究中性鞘磷脂酶1（nSMase 1）的细胞内定位，制备了一种兔多克隆抗体，该抗体针对在大肠杆菌中表达的重组形式的该酶。据报道，在大鼠肝脏或腹水肝癌AH7974中，中性鞘磷脂酶（SMase）在质膜上具有高活性，在细胞核和细胞质中的活性较低但显著。克隆和表达的nSMase 1的生化特性、对二硫苏糖醇的需求和对高盐浓度的依赖性与先前描述的AH7974核中性SMase相似。因此，本研究聚焦于该酶的核定位。使用抗大鼠nSMase 1抗体对亚细胞组分进行蛋白质印迹分析显示，大多数nSMase 1与细胞核相关，一些与微粒体相关，但与质膜无关。一致地，细胞核提取物中的中性SMase活性可被该抗体免疫沉淀，而质膜的则不能。结果表明，nSMase 1主要存在于细胞核中，因此可能与质膜中的中性SMase不同。对细胞核提取物进行凝胶过滤柱层析时，中性SMase活性的图谱与蛋白质印迹上的免疫反应性蛋白条带良好对应，表明细胞核中性SMase的很大一部分可能是nSMase 1。用Triton X-100处理去除核膜并没有显著降低细胞核nSMase 1的量，对亚核组分（即核膜、染色质和核基质）进行蛋白质印迹分析显示，nSMase 1信号仅存在于核基质中。对AH7974以及大鼠成纤维细胞系3Y1进行免疫细胞化学分析表明，nSMase 1主要定位于细胞核中，一些存在于细胞质中。此外，免疫电子显微镜清楚地显示，AH7974细胞核中nSMase 1的信号比细胞质中更密集。
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