Ulmanen I, Peränen J, Tenhunen J, Tilgmann C, Karhunen T, Panula P, Bernasconi L, Aubry J P, Lundström K
Orion Corporation, Orion Pharma, Target Protein Laboratory, Helsinki, Finland.
Eur J Biochem. 1997 Jan 15;243(1-2):452-9. doi: 10.1111/j.1432-1033.1997.0452a.x.
The intracellular localization of soluble and membrane-bound isoforms of rat and human catechol O-methyltransferase (COMT) was studied by expressing the recombinant COMT proteins either separately or together in mammalian cell lines (HeLa and COS-7 cells) and in rat primary neurons. The distribution of soluble and membrane-bound COMT enzyme was visualized by immunocytochemistry. For comparison, the localization of native COMT was studied in rat C6 glioma cells by immunoelectron microscopy. Staining of cells expressing membrane-bound COMT with a COMT-specific antiserum revealed an immunofluorescence signal in intracellular reticular structures and in the nuclear membrane. Double-staining of the cells with antisera against proteins specific for the rough endoplasmic reticulum indicated that they colocalized with membrane-bound COMT, suggesting that it resided in the endoplasmic reticulum. Notably, no COMT-specific fluorescence of plasma membranes was detected. The signal in the endoplasmic reticulum was also evident in the cells expressing both recombinant COMT forms. Intracellular native COMT reaction was detected by immunoelectron microscopy in rat C6 glioma cells and an intense cytoplasmic signal was seen in the primary neurons infected with the recombinant Semliki Forest virus. The cells expressing recombinant soluble COMT revealed intense nuclear staining together with diffuse cytoplasmic immunoreactivity, suggesting that a part of soluble COMT is transported to nuclei. Western blotting from rat liver and brain revealed soluble COMT in the nuclei. Enzyme activity measurements from liver cytoplasmic and nuclear fractions suggested that about 5% of the soluble COMT resided in nuclei. The intracellular localization of both COMT forms implies that COMT acts in the cytoplasm and possibly also in the nuclear compartment, and that the physiological substrates of COMT enzymes may have to be internalized before their methylation by COMT.
通过在哺乳动物细胞系（HeLa和COS - 7细胞）以及大鼠原代神经元中单独或共同表达重组儿茶酚 - O - 甲基转移酶（COMT）蛋白，研究了大鼠和人类COMT可溶性及膜结合同工型的细胞内定位。通过免疫细胞化学观察可溶性和膜结合COMT酶的分布。为作比较，通过免疫电子显微镜研究了大鼠C6胶质瘤细胞中天然COMT的定位。用COMT特异性抗血清对表达膜结合COMT的细胞进行染色，在内质网结构和核膜中发现免疫荧光信号。用针对粗面内质网特异性蛋白的抗血清对细胞进行双重染色表明，它们与膜结合COMT共定位，提示其存在于内质网中。值得注意的是，未检测到质膜上的COMT特异性荧光。在表达两种重组COMT形式的细胞中，内质网中的信号也很明显。通过免疫电子显微镜在大鼠C6胶质瘤细胞中检测到细胞内天然COMT反应，在用重组Semliki森林病毒感染的原代神经元中观察到强烈的细胞质信号。表达重组可溶性COMT的细胞显示出强烈的核染色以及弥漫性的细胞质免疫反应性，提示一部分可溶性COMT被转运到细胞核。大鼠肝脏和大脑的蛋白质免疫印迹显示细胞核中有可溶性COMT。肝脏细胞质和细胞核组分的酶活性测量表明，约5%的可溶性COMT存在于细胞核中。两种COMT形式的细胞内定位表明，COMT在细胞质中起作用，可能也在细胞核区室中起作用，并且COMT酶的生理底物在被COMT甲基化之前可能必须被内化。
