Trypanosoma brucei: subcellular distribution and organisation of the enzymes of glycolysis
Oduro, Kwabena Konadu
The cellular distribution of eleven Trypanosoma brucei enzymes involved in glucose breakdown has been studied, using the following six methods of cell disruption: saponin, Triton X-100, digitonin, freezing and thawing, and grinding with the abrasives alumina and silicon carbide. By means of differential centrifugation of the homogenates of the bloodstream T. brucei obtained by these six different methods of cell lysis, it has been shown that the distribution pattern of the enzymes is greatly affected by the method of cell lysis as follows. Only three of the eleven enzymes, namely, phosphoglycerate mutase, enolase, and pyruvate kinase were completely solubilised by at least five of the methods adopted for cell disruption. As well as these three enzymes, saponin lysis which appeared to be the most severe method of treatment, led also to the complete solubilisation of phosphoglucose isomerase and partial solubilisation of glyceraldehyde-phosphate dehydrogenase, phosphoglycerate kinase and glycerokinase. At the other extreme, cell lysis by grinding T. brucei with alumina or silicon carbide completely solubilised phosphoglycerate mutase, enolase and pyruvate kinase, whereas hexokinase, phosphoglucose isomerase, phosphofructose kinase, aldolase, phosphoglycerate kinase and glycerophosphate dehydrogenase were found to be concentrated in the post-nuclear fraction which sediments at 14,500 g (fraction 14.5KP). The patterns of distribution of the remaining two enzymes, glyceraldehydephosphate dehydrogenase and glycerokinase were found to be polydisperse. The post-nuclear fraction was found to be capable of metabolising glucose to give glycerophosphate without auxiliary enzyme supplementation, and this multienzyme activity proved to be very sensitive to inhibition by the trypanocidal compound, suramin. By means of Biogel column chromatography and acrylamide gel electrophoresis, it was shown that the multienzyme activity is concentrated in a particle probably bigger than a globular protein with a molecular weight of 5 million. Kinetic studies of fraction 14.5KP, in the presence or absence of Triton X-100 indicated that the particles possibly possess a limiting membrane with an inner matrix to which the component enzymes are bound. Isopycnic sucrose gradient centrifugation confirmed that the multienzyme complex is associated with large particles with a median equilibrium density of 1.22. Since the only sub¬ cellular organelles in T. brucei known to band at this density are the microbodies, it has been concluded that the probable intracellular location of the multienzyme complex is the microbodies of the bloodstream long slender form T. brucei.