Izumi K, Sawada M T
Hokkaido National Industrial Research Institute, AIST, MITI, Sapporo, Japan.
Lipids. 2001 Jan;36(1):97-101. doi: 10.1007/s11745-001-0674-z.
Lysoglycosphingolipids consist of a sphingoid long-chain base and monosaccharide or complex sugar, and they lack the fatty acyl group present in native glycosphingolipids. Less than 1 pmol of lyso-Forssman glycolipid and lysoganglioside GM1 were detected on a thin-layer chromatogram by an enzyme-linked immunochemical coloration method with anti-Forssman glycolipid antibody (FOM-1) and cholera toxin B subunit, respectively. Each spot between 1 and 100 pmol lyso-Forssman glycolipid was immunostained as densely as that of the same amount of native Forssman glycolipid. The density of the lyso-Forssman glycolipid spots increased proportionally with increment in the amount of lysoglycolipid. The density of spots of 0.2-100 pmol lysoganglioside GM1 was also proportional to the amount of each lyso-GM1 spot. These results indicated that less than 1 to 100 pmol of deacylated glycosphingolipid was quantifiable by the immunochemical coloration method with sugar chain-specific antibodies. Glycosphingolipid deacylase, which cleaved an amide bond between the sphingoid long-chain base and fatty acyl chain in ceramide of glycosphingolipid, was assayed by detecting the lyso-Forssman glycolipid produced. Lipophilic compounds, recovered from an aliquot of the reaction mixture of Forssman glycolipid and crude enzyme at appropriate times, were analyzed by thin-layer chromatography. It was found that lyso-Forssman glycolipid was produced in the first 1-2 h by the enzyme and production increased with incubation time. This coloration method is more sensitive and specific than the visualization method with a nonspecific reagent such as orcinol-sulfuric acid reagent.
溶血糖鞘脂由一个鞘氨醇长链碱基和单糖或复合糖组成，并且它们缺乏天然糖鞘脂中存在的脂肪酰基。分别用抗福斯曼糖脂抗体（FOM-1）和霍乱毒素B亚基通过酶联免疫化学染色法在薄层色谱上检测到不到1皮摩尔的溶血福斯曼糖脂和溶血神经节苷脂GM1。1至100皮摩尔溶血福斯曼糖脂之间的每个斑点免疫染色的密度与相同量的天然福斯曼糖脂一样密集。溶血福斯曼糖脂斑点的密度随溶血糖脂量的增加成比例增加。0.2至100皮摩尔溶血神经节苷脂GM1斑点的密度也与每个溶血GM1斑点的量成比例。这些结果表明，通过使用糖链特异性抗体的免疫化学染色法可定量检测不到1至100皮摩尔的脱酰基糖鞘脂。通过检测产生的溶血福斯曼糖脂来测定糖鞘脂脱酰酶，该酶可裂解糖鞘脂神经酰胺中鞘氨醇长链碱基和脂肪酰链之间的酰胺键。在适当的时间从福斯曼糖脂和粗酶反应混合物的等分试样中回收的亲脂性化合物，通过薄层色谱进行分析。发现该酶在最初的1至2小时内产生溶血福斯曼糖脂，并且产量随孵育时间增加。这种染色方法比使用诸如间苯二酚 - 硫酸试剂等非特异性试剂的可视化方法更灵敏、更特异。
