Miller-Podraza H, Fishman P H
Biochemistry. 1982 Jul 6;21(14):3265-70. doi: 10.1021/bi00257a003.
A new method was developed to follow the translocation of gangliosides from their site of synthesis within the cell to the plasma membrane. Cultured mouse neuroblastoma N18 and rat glioma C6 cells were labeled for increasing times with D- [1-3H]galactose and then subjected to mild oxidation with NaIO4. Under the conditions chosen, oxidation was essentially restricted to cell-surface sialic acid residues, which were converted to derivatives with an aldehyde function. The labeled gangliosides were isolated from the cells and reacted with dinitrophenylhydrazine to form dinitrophenyl (DNP) derivatives of the oxidized gangliosides. The DNP-gangliosides then were separated from their unmodified counterparts by thin-layer chromatography. Thus, the rate of labeling of surface gangliosides was distinguished from the rate of labeling of total gangliosides. Our results indicated that the transfer of gangliosides from the site of synthesis to the cell surface required approximately 20 min and that newly synthesized gangliosides appeared to be transported to the plasma membrane at a constant rate. No essential differences were found in the rates of translocation of different ganglioside species by N18 cells or between gangliosides of N18 and C6 cells.
开发了一种新方法来追踪神经节苷脂从细胞内合成位点到质膜的转运过程。用D-[1-³H]半乳糖对培养的小鼠神经母细胞瘤N18细胞和大鼠胶质瘤C6细胞进行不同时长的标记，然后用高碘酸钠进行轻度氧化。在所选条件下，氧化基本仅限于细胞表面的唾液酸残基，这些残基被转化为具有醛功能的衍生物。从细胞中分离出标记的神经节苷脂，并与二硝基苯肼反应，形成氧化神经节苷脂的二硝基苯基（DNP）衍生物。然后通过薄层色谱将DNP-神经节苷脂与其未修饰的对应物分离。因此，表面神经节苷脂的标记速率与总神经节苷脂的标记速率得以区分。我们的结果表明，神经节苷脂从合成位点转移到细胞表面大约需要20分钟，并且新合成的神经节苷脂似乎以恒定速率转运到质膜。在N18细胞转运不同神经节苷脂种类的速率方面，以及在N18细胞和C6细胞的神经节苷脂之间，未发现本质差异。
