Institute for Biochemistry and Pathobiochemistry, Department of Systems Biochemistry, Faculty of Medicine, Ruhr University of Bochum, Bochum, Germany.
Methods Mol Biol. 2023;2643:33-45. doi: 10.1007/978-1-0716-3048-8_3.
Glycosomes, belonging to the sub-class of peroxisomes, are single-membrane-bound organelles of trypanosomatid parasites. Glycosomes compartmentalize mainly glycolytic and other essential metabolic pathways such as gluconeogenesis, pentose phosphate pathway, sugar nucleotide biosynthesis, etc. Since glycosomes are parasite-specific and their biogenesis is essential for the parasite survival, they have attracted a lot of interest over the years. Understanding the glycosomal enzyme composition and machinery involved in the biogenesis of this organelle requires the knowledge of the glycosomal proteome. Here we describe a method to isolate highly purified glycosomes and further enrichment of the glycosomal membrane proteins from the pro-cyclic form of Trypanosoma brucei. The isolation method is based on the controlled rupture of the cells by silicon carbide, followed by the differential centrifugation, and density gradient centrifugation. Further, the glycosomal membrane proteins are enriched from the purified glycosomes by the successive treatments with low-salt, high-salt, and alkaline carbonate buffer extractions.
糖体属于过氧化物酶体亚类，是原生动物寄生虫的单层膜结合细胞器。糖体主要分隔糖酵解和其他必要的代谢途径，如糖异生、磷酸戊糖途径、糖核苷酸生物合成等。由于糖体是寄生虫特异性的，其发生对于寄生虫的生存是必不可少的，因此多年来引起了很多关注。了解参与该细胞器发生的糖体酶组成和机制需要了解糖体蛋白质组的知识。在这里，我们描述了一种从布氏锥虫循环前体中分离高度纯化的糖体和进一步富集糖体膜蛋白的方法。该分离方法基于碳化硅控制的细胞破裂，然后进行差速离心和密度梯度离心。此外，通过低盐、高盐和碱性碳酸盐缓冲液提取的连续处理从纯化的糖体中富集糖体膜蛋白。
