Purification, morphometric analysis, and characterization of the glycosomes (microbodies) of the protozoan hemoflagellate Trypanosoma brucei

Abstract

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.