Purification, morphometric analysis, and characterization of the glycosomes (microbodies) of the protozoan hemoflagellate Trypanosoma brucei.
Purification, morphometric analysis, and characterization of the glycosomes (microbodies) of the protozoan hemoflagellate Trypanosoma brucei.
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DOI：
10.1083/jcb.98.4.1178
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发表时间：
1984-04
期刊：
影响因子：
--
通讯作者：
Misset O
中科院分区：
文献类型：
--
作者：
Opperdoes FR;Baudhuin P;Coppens I;De Roe C;Edwards SW;Weijers PJ;Misset O
布氏锥虫糖体（含有参与糖酵解的九种酶的微体）已从血流形式的锥鞭毛体中纯化至接近同质，用于形态学和生化分析。差速离心，然后在等渗Percoll和蔗糖梯度中进行两次等密度离心，分别得到12- 13倍纯化的糖体，总产率为31%。这些糖体似乎是高纯度的，并含有不到1%的线粒体污染的形态和生化分析判断。在完整细胞中，糖体显示出非常均匀的尺寸分布，平均直径为0.27微米，标准偏差为0.03微米。分离的糖体的大小分布与在完整细胞中测量的仅略有不同。一个T布氏细胞平均含有230个糖体，占总细胞体积的4.3%。所述糖体被单个膜包围，并且仅含有比例为2：1的磷脂酰胆碱和磷脂酰乙醇胺作为磷脂。纯化的糖体级分具有0.18微克/mg蛋白质的非常低的DNA含量。没有观察到不可能来自污染线粒体或核碎片的DNA分子。
Trypanosoma brucei glycosomes (microbodies containing nine enzymes involved in glycolysis) have been purified to near homogeneity from bloodstream-form trypomastigotes for the purpose of morphologic and biochemical analysis. Differential centrifugation followed by two isopycnic centrifugations in an isotonic Percoll and in a sucrose gradient, respectively, resulted in 12- to 13-fold purified glycosomes with an overall yield of 31%. These glycosomes appeared to be highly pure and contained less than 1% mitochondrial contamination as judged by morphometric and biochemical analyses. In intact cells, glycosomes displayed a remarkably homogeneous size distribution centered on an average diameter of 0.27 micron with a standard deviation of 0.03 micron. The size distribution of isolated glycosomes differed only slightly from that measured in intact cells. One T. brucei cell contained on average 230 glycosomes, representing 4.3% of the total cell volume. The glycosomes were surrounded by a single membrane and contained as phospholipids only phosphatidyl choline and phosphatidyl ethanolamine in a ratio of 2:1. The purified glycosomal fraction had a very low DNA content of 0.18 microgram/mg protein. No DNA molecules were observed that could not have been derived from contaminating mitochondrial or nuclear debris.