Prescott A R, Lucocq J M, James J, Lister J M, Ponnambalam S
Department of Biochemistry, Medical Sciences Institute, University of Dundee, United Kingdom.
Eur J Cell Biol. 1997 Mar;72(3):238-46.
The Golgi proteins, TGN46 and GalT, were characterized in human HeLa cells using specific polyclonal and monoclonal antibodies. A bacterially expressed soluble recombinant TGN46 protein was used to raise rabbit polyclonal antibodies and used to probe HeLa cell extracts. Human TGN46 had an apparent molecular mass of 100 to 120 kDa which reflects extensive glycosylation. Epifluorescence light microscopy indicated substantial colocalization of TGN46 and GalT. However, confocal laser microscopy and three-dimensional reconstruction of double-labeled HeLa cells revealed large areas of colocalization but also specific differences in the distribution of these two proteins within the Golgi apparatus. Importantly, quantitative immunoelectron microscopy showed that there was little overlap between the distribution of GalT and TGN46. Approximately 75% of GalT was in the Golgi stack, whereas 80% of TGN46 was detected in tubules. Distinct GalT-positive regions within the Golgi cisternal stack were not labeled for TGN46.
利用特异性多克隆抗体和单克隆抗体对人宫颈癌细胞系HeLa细胞中的高尔基体蛋白TGN46和半乳糖基转移酶（GalT）进行了表征。使用细菌表达的可溶性重组TGN46蛋白制备兔多克隆抗体，并用于检测HeLa细胞提取物。人TGN46的表观分子量为100至120 kDa，这反映了其广泛的糖基化。落射荧光显微镜显示TGN46和GalT大量共定位。然而，共聚焦激光显微镜和双标记HeLa细胞的三维重建显示，这两种蛋白在高尔基体中存在大面积共定位，但分布也存在特定差异。重要的是，定量免疫电子显微镜显示GalT和TGN46的分布几乎没有重叠。约75%的GalT位于高尔基体堆叠中，而80%的TGN46在小管中被检测到。高尔基体扁平囊堆叠内不同的GalT阳性区域未被TGN46标记。
