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 <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 um with a standard deviation of 0.03 um . 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 hg/mg protein . No DNA molecules were observed that could not have been derived from contaminating mitochondrial or nuclear debris.

Glycolytic enzymes of Trypanosoma brucei. Simultaneous purification, intraglycosomal concentrations and physical properties

“…This predicts that a 27-fold purification of glycosomes could be obtained. These estimations, however, arc in contrast with our earlier experimental results where glycosomes were only purified 11 -12-fold and from which we concluded that they represented 7.8-8.7% of the cell's protein [4]. The latter value is significantly higher than the one we now obtain when using the purification factors for the individual enzymes.…”

“…Since the exact amount of Percoll in our glycosome preparations cannot be quantified, we can not yet correct for it. From the observation that glycosomes represent 4.3% of the total cellular volume [4] and constitute 3.7% of the total cellular protein, it can be calculated that the protein concentration inside glycosomes amounts to 150 mg/ml (assuming 175 mg protein/ml for the trypanosome [4]), a value significantly less than our previous estimate (340 mg/ml [4]). From a protein concentration of 150 mg/ml and the density of protein (approximately 1300 mglml), it can be calculated that 11.5% of the volume of glycosomes is filled with protein.…”

“…In the highly (12-fold) purified glycosomes (lane 9, obtained via subsequent differential, Percoll-gradient and sucrosegradient centrifugations [4], almost all polypeptide bands can now be attributed to glycolytic enzymes. The extra nonglycosomal polypeptides present in the other two glycosome preparations (lanes 3 and 4) are responsible for the lower purification factors obtained for these preparations (see miniprint supplement at the end of this paper).…”

“…From the glycosomal protein concentrations the concentrations of the active site and native enzyme can be calculated by dividing by the molecular mass of the subunit and native enzyme, respectively ( Table 2). The native enzyme concentrations multiplied by the volume of one glycosome (10.8 x lo-" 1 [4]) and Avogrado's number give the number of enzyme molecules present inside a glycosome ( Table 2, last two columns). A functional multienzyme complex would then consist of at least one molecule of hexokinase and triosephosphate isomerase, and if the other enzymes were present according to their relative abunddncy as indicated in the last column of Table 2, the estimated molecular mass of such a complex would be 4.2 MDa and its approximate Stokes' diameter 27 nm.…”

“…Since glycosomes contain neither DNA [4] nor ribosomes, these enzymic proteins must be synthesized elsewhere in the cytoplasm. Indeed, the genes coding for three of the enzymes involved (glyceraldehyde-phosphate dehydrogenase, triosephosphate isomerase and phosphoglycerate kinase) have now been localized on large and intermediate-sized chromosomes in Trypanosoma brucei [ll] and recent evidence indicates that the mRNAs coding for several of these enzymes are translated on free cytosolic ribosomes and that the resulting polypeptides are subsequently transported into the glycosomes [12].…”