Subcellular Fractionation of Trypanosoma brucei Bloodstream Forms with Special Reference to Hydrolases
Open Access
- 1 March 1980
- journal article
- research article
- Published by Wiley in European Journal of Biochemistry
- Vol. 105 (1), 163-175
- https://doi.org/10.1111/j.1432-1033.1980.tb04486.x
Abstract
Bloodstream forms of Trypanosoma brucei have been screened for the presence of enzymes that could serve as markers for the plasma membrane, flagellar pocket, lysosomes, endoplasmic reticulum and Golgi apparatus in order to study the subcellular organization of the digestive system of the parasite. Acetylesterase, acid DNase, acid phosphatase, acid phosphodiesterase, acid proteinase, acid RNase, alanine aminotransferase, galactosyl transferase, α-glucosidase, inosine diphosphatase and α-mannosidase were partially characterized and their assays optimized for pH-dependent activity, linearity of reaction with respect to incubation time and enzyme concentration, and the effect of inhibitors and activators. The association of these enzymes with particulate material and the presence of structural latency were investigated. Acid proteinase and α-mannosidase are particle-bound and latent in cytoplasmic extracts; they can be activated and solubilized in part by Triton X-100. Similar results were obtained for acid phosphatase, acid phosphodiesterase and inosine diphosphatase. Neutral α-glucosidase, though partly sedimentable, does not show latency and is readily solubilized by the detergent. Galactosyl transferase is firmly membrane-bound even in the presence of 0.1 % Triton X-100. Cell fractionation by differential centrifugation and density equilibration on sucrose gradients revealed that both α-mannosidase and acid proteinase are associated with organelles that band at a density of about 1.20 g/cm3. Inosine diphosphatase, galactosyl transferase, acid phosphatase and acid phosphodiesterase sediment predominantly as microsomal constituents equilibrating at densities between 1.13 and 1.15 g/cm3. In addition, inosine diphosphatase and galactosyl transferase exhibit considerable activity at higher densities (1.18 - 1.25 g/cm3). Neutral α-glucosidase is mainly recovered in the nuclear and microsomal fraction; its particulate part equilibrates as a single band at ∂= 1.22 g/cm3. Acetylesterase and acid DNase are largely soluble, whereas acid RNase does not produce distinct sedimentation and banding profiles. In intact cells, neutral α-glucosidase and acid phosphatase appear to be highly accessible to their substrates. It is tentatively concluded that (a) acid proteinase and α-mannosidase are lysosomal enzymes, (b) acid phosphatase and acid phosphodlesterase are associated with the flagellar pocket and part of the former enzyme probably with the endoplasmic reticulum, (c) galactosyl transferase is a constituent of the Golgi apparatus, and (d) α-glucosidase may serve as a marker for the plasma membrane. Inosine diphosphatase may also be derived from the latter structure.This publication has 48 references indexed in Scilit:
- Mesure Quantitative de L'Activité de la Desoxyribonuclease Plasmatique et de Son Inhibition Par le Complexe Daunorubicine‐ADNBulletin des Sociétés Chimiques Belges, 1978
- Trypanosoma brucei: Suramin and other trypanocidal compounds' effects on sn-glycerol-3-phosphate oxidaseExperimental Parasitology, 1977
- Particle‐Bound Enzymes in the Bloodstream Form of Trypanosoma bruceiEuropean Journal of Biochemistry, 1977
- The subcellular distribution and properties of Crithidia sp. hydrolases with particular reference to pyrophosphate and orthophosphate monoester phosphohydrolasesCanadian Journal of Biochemistry, 1976
- Protein Uptake and Digestion in Bloodstream and Culture Forms of Trypanosoma brucei*The Journal of Protozoology, 1975
- STUDIES ON ACID HYDROLASES AND ON CATALASE OF THE TRYPANOSOMATID CRITHIDIA LUCILIAEPublished by Elsevier ,1972
- Fluorometric assay for the hydrolytic activity of lipase using fatty acyl esters of 4-methylumbelliferoneAnalytical Biochemistry, 1967
- Characterization and Localization of Acid Phosphatase Activity of Trypanosoma gambiense*The Journal of Protozoology, 1967
- The ingestion of protein molecules by blood forms of Trypanosoma rhodesienseExperimental Cell Research, 1965
- A Colorimetric Method for the Determination of Serum Glutamic Oxalacetic and Glutamic Pyruvic TransaminasesAmerican Journal of Clinical Pathology, 1957