Byrne M C, Sbaschnig-Agler M, Aquino D A, Sclafani J R, Ledeen R W
Anal Biochem. 1985 Jul;148(1):163-73. doi: 10.1016/0003-2697(85)90641-4.
While several methods for ganglioside extraction and isolation have been described, relatively little attention has been given to the effectiveness of separation from peptides, phospholipids, and various low-molecular-weight contaminants. A procedure is described for isolation of gangliosides in high purity and good yield from 1- to 400-mg samples (wet wt). A key step was mild acidification following homogenization, designed to dissociate gangliosides from lipophilic peptides which coextracted into organic solvents. This has proved particularly helpful for myelin and myelin-containing tissues (e.g., white matter, nerve) whose proteins have presented special problems in ganglioside isolation. In this study isolation was effected by consecutive chromatographies on Sephadex LH-20, DEAE-Sephadex, and silica gel following the initial acidification. The method applied to bovine white matter gave tissue concentrations (calculated from yields and radiolabeled tracer recoveries) that were similar to those obtained with three previously described procedures; however, peptide contaminants were an order of magnitude lower. Removal of low-molecular-weight contaminants, including nucleotide sugars, was virtually complete. In addition to ganglioside isolation the method can be used to obtain neutral glycosphingolipids as well. It is believed to have broad applicability to a diversity of tissues.
虽然已经描述了几种神经节苷脂提取和分离的方法，但对于从肽、磷脂和各种低分子量污染物中分离的有效性关注相对较少。本文描述了一种从1至400毫克样品（湿重）中以高纯度和高收率分离神经节苷脂的方法。一个关键步骤是在匀浆后进行温和酸化，目的是使与共提取到有机溶剂中的亲脂性肽解离的神经节苷脂。这已被证明对髓磷脂和含髓磷脂的组织（如白质、神经）特别有帮助，这些组织的蛋白质在神经节苷脂分离中存在特殊问题。在本研究中，在初始酸化后，通过在Sephadex LH - 20、DEAE - Sephadex和硅胶上连续色谱法进行分离。应用于牛白质的方法得到的组织浓度（根据产量和放射性标记示踪剂回收率计算）与之前描述的三种方法得到的浓度相似；然而，肽污染物低了一个数量级。低分子量污染物，包括核苷酸糖的去除几乎是完全的。除了神经节苷脂分离外，该方法还可用于获得中性糖鞘脂。据信它对多种组织具有广泛的适用性。
