Trinchera M, Ghidoni R
Department of Medical Chemistry and Biochemistry, Medical School, University of Milan, Italy.
J Biochem. 1990 Apr;107(4):619-23. doi: 10.1093/oxfordjournals.jbchem.a123096.
The demonstration of a precursor-product relationship in the course of GM1 and GD1a biosynthesis is described in the present paper. We injected rats with GM2 gangliosides [GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4Glc beta 1----1'Cer] of brain origin, which were isotopically radiolabeled on the GalNAc ([GalNAc-3H]GM2) or sphingosine ([Sph-3H]GM2) residue. We then compared the time-courses of GM1 and GD1a biosynthesis in the liver after the administration of each radiolabeled GM2 derivative. After the administration of [GalNAc-3H]GM2, GM1, and GD1a were both present as doublets, that could be easily resolved on TLC. The lower spot of each doublet was identified as a species having the typical rat brain ceramide moiety and represented gangliosides formed through direct glycosylation of the injected GM2. The upper spot of each doublet was identified as a species having the typical rat liver ceramide moiety and represented gangliosides formed through recycling of the [3H]GalNAc residue, released during ganglioside catabolism. After the administration of [Sph-3H]GM2, only ganglioside with the rat brain ceramide moiety were found, that represented the sum of ganglioside formed through direct glycosylation and those formed through recycling of some sphingosine-containing fragments. In each case, the time-course of GM1 and GD1a biosynthesis exhibited a precursor-product relationship. The curve obtained from the direct glycosylation showed a timing delay with respect to those obtained from recycling of GM2 fragments. These results are consistent with the hypothesis that the sequential addition of activated sugars to a sphingolipid precursor is a dissociative process, catalyzed by physically independent enzymatic activities.
本文描述了GM1和GD1a生物合成过程中前体-产物关系的证明。我们给大鼠注射了脑源性的GM2神经节苷脂[GalNAcβ1----4(NeuAcα2----3)Galβ1----4Glcβ1----1'Cer]，其在GalNAc（[GalNAc-3H]GM2）或鞘氨醇（[Sph-3H]GM2）残基上进行了同位素放射性标记。然后我们比较了每种放射性标记的GM2衍生物给药后肝脏中GM1和GD1a生物合成的时间进程。给予[GalNAc-3H]GM2后，GM1和GD1a均以双峰形式存在，在薄层层析上很容易分离。每个双峰的较低斑点被鉴定为具有典型大鼠脑酰基鞘氨醇部分的物质，代表通过注射的GM2直接糖基化形成的神经节苷脂。每个双峰的较高斑点被鉴定为具有典型大鼠肝脏酰基鞘氨醇部分的物质，代表通过神经节苷脂分解代谢过程中释放的[3H]GalNAc残基循环形成的神经节苷脂。给予[Sph-3H]GM2后，仅发现具有大鼠脑酰基鞘氨醇部分的神经节苷脂，其代表通过直接糖基化形成的神经节苷脂与通过一些含鞘氨醇片段循环形成的神经节苷脂之和。在每种情况下，GM1和GD1a生物合成的时间进程均表现出前体-产物关系。从直接糖基化获得的曲线相对于从GM2片段循环获得的曲线显示出时间延迟。这些结果与以下假设一致，即向鞘脂前体顺序添加活化糖是一个解离过程，由物理上独立的酶活性催化。
