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Sub-cellular fractionation of Trypanosoma brucei. Isolation and characterization of plasma membranes

Published online by Cambridge University Press:  06 April 2009

Luciana Rovis  and
Steinunn Baekkeskov
Luciana Rovis
Affiliation:
International Laboratory for Research on Animal Diseases (ILRAD)P.O. Box 30709, Nairobi, Kenya
Steinunn Baekkeskov
Affiliation:
International Laboratory for Research on Animal Diseases (ILRAD)P.O. Box 30709, Nairobi, Kenya
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Summary

A procedure is described for the isolation of sub-cellular fractions from bloodstream forms of Trypanosoma brucei. The method leaves intact most of the nuclei, mitochondria and microbodies. All the fractions have been chemically characterized and tested for 10 enzymatic markers. About 5% of total cell protein was isolated as a microsomal fraction containing mostly plasma membranes and endoplasmic reticulum vesicles. Plasma membranes were purified by high-speed centrifugation on magnesium-containing Dextran, and on linear sucrose-density gradients. The yield of membranes was approximately 0·3% of the total cell protein. The purified material had a sucrose density of 1·14 g/cm3 and consisted of smooth vesicles. Specific activity of the membrane markers Na+, K+, ouabain-sensitive ATPase and adenylate cyclase were 26-and 20-fold higher, respectively, than in total cells. Neither DNA nor RNA was detected. The sum of the cholesterol and phospholipid content was 0·99 mg/mg protein. The cholesterol/phospholipid molar ratio was 1 : 2.

Type
Research Article
Information
Parasitology , Volume 80 , Issue 3 , June 1980 , pp. 507 - 524
Copyright
Copyright © Cambridge University Press 1980

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References

Ames, G. F. (1968). Lipids of Salmonella typhimurium and Escherichia coli: structure and metabolism. Journal of Bacteriology 95, 833–43.CrossRefGoogle ScholarPubMed
Angelopoulos, E. (1970). Pellicular microtubules in the family Trypanosomatidae. Journal of Protozoology 17, 39–51.CrossRefGoogle ScholarPubMed
Avruch, J. & Wallach, D. F. H. (1971). Preparation and properties of plasma membrane and endoplasmic reticulum fragments from isolated rat fat cells. Biochimica et biophysica acta 233, 334–47.CrossRefGoogle ScholarPubMed
Barbet, A. F. & Mcguire, T. C. (1978). Crossreacting determinants in variant-specific surface antigens of African trypanosomes. Proceedings of the National Academy of Sciences, USA 75, 1989–93.CrossRefGoogle ScholarPubMed
Bartlett, G. R. (1959). Phosphorus assay in column chromatography. Journal of Biological Chemistry 234, 466–8.CrossRefGoogle ScholarPubMed
Bligh, E. C. & Dyer, W. J. (1959). A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology 37, 911–17.CrossRefGoogle ScholarPubMed
Burton, K. (1956). A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. The Biochemical Journal 62, 315–23.CrossRefGoogle ScholarPubMed
Chapman, D. (1973). Recent physical studies of phospholipids and natural membranes. In Biological Membranes, vol 2 (ed. Chapman, D. and Wallach, D. F. H.), pp. 91–144. New York and London: Academic Press.Google Scholar
Cross, G. A. M. (1975). Identification, purification and properties of clone-specific glyco-protein antigens constituting the surface coat of Trypanosoma brucei. Parasitology 71, 398–417.CrossRefGoogle Scholar
Cunningham, M. P. & Vickerman, K. (1962). Antigenic analysis in the Trypanosoma brucei group using the agglutination reaction. Transactions of the Royal Society of Tropical Medicine and Hygiene 56, 48–59.CrossRefGoogle ScholarPubMed
De Duve, C., Berthet, J. & Beaufay, H. (1959). Gradient centrifugation of cell particles: theory and applications. Progress in Biophysical Chemistry 9, 326–69.Google Scholar
Demel, R. A. & De kruyff, B. (1976). The function of sterols in membranes. Biochimica et biophysica acta 457, 109–32.CrossRefGoogle ScholarPubMed
De Raadt, P. (1974). Immunity and antigenic variation: clinical observations suggestive of immune phenomena in African trypanosomiasis. In Trypanosomiaais and Leishmaniasis with Special Reference to Chagas' Disease Ciba Fdn Symp. 20 (ed. Elliott, K., O'Connor, M. and Wolstenholme, G. E. W.), pp. 199–224. Amsterdam: Associated Scientific Publishers.CrossRefGoogle Scholar
Eibl, H. & Lands, W. E. M. (1969). A new sensitive determination of phosphate. Analytical Biochemistry 30, 51–7.CrossRefGoogle ScholarPubMed
Emmelot, P. (1977). The organisation of the plasma membrane of mammalian cells: structure in relation to function. In Mammalian Cell Membranes, vol. 2 (ed. Jamieson, G. A and Robinson, D. M.), pp. 1–54. London and Boston: Butterworths.Google Scholar
Ferber, E., Resch, K., Wallach, D. F. H. & Imm, W. (1972). Isolation and characterisation of lymphocyte plasma membranes. Biochimica et biophysica acta 266, 494–504.CrossRefGoogle ScholarPubMed
Folch, J., Lees, M. & Sloane Stanley, G. H. (1957). A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry 266, 497–509.CrossRefGoogle Scholar
Graham, J. M. (1972). Isolation and characterisation of membranes from normal and transformed tissue-culture cells. The Biochemical Journal 130, 1113–24.CrossRefGoogle ScholarPubMed
Gray, A. R. (1967). Some principles of the immunology of trypanosomiasis. Bulletin of the World Health Organization 37, 177–93.Google ScholarPubMed
Heppel, L. A. & Hilmoe, R. J. (1951). Purification and properties of 5′-nucleotidase. Journal of Biological Chemistry 188, 665–76.CrossRefGoogle ScholarPubMed
Hinz, H. J. & Sturtevant, J. M. (1972). Calorimetric investigation of the influence of cholesterol on the transition of bilayers formed from synthetic L-α-lecithins in aqueous suspension. Journal of Biological Chemistry 247, 3697–700.CrossRefGoogle Scholar
Hunt, R. C. & Ellar, D. J. (1974). Isolation of the plasma membrane of a Trypanosomatid flagellate: general characterisation and lipid composition. Biochimica et biophysica acta 339, 173–89.CrossRefGoogle ScholarPubMed
Lambin, P., Rochu, D. & Fine, J. M. (1976). A new method for determination of molecular weights of proteins by electrophoresis across a sodium dodecyl sulfate (SDS)-polyacrylamide gradient gel. Analytical Biochemistry 74, 567–75.CrossRefGoogle ScholarPubMed
Langreth, S. G. & Balber, A. E. (1975). Protein uptake and digestion in bloodstream and in culture forms of Trypanosoma brucei. Journal of Protozoology 22, 40–53.CrossRefGoogle ScholarPubMed
Lanham, S. M. & Godfrey, D. G. (1970). Isolation of salivarian trypanosomes from man and other mammals using DEAE - cellulose. Experimental Parasitology 28, 521–34.CrossRefGoogle ScholarPubMed
Le Ray, D. (1975). Structures antigéniques de Trypanosoma brucei (protozoa, kinetoplastida). Analyse immunoélectrophorétique et étude comparative. Annales de la Société Belge, de Medicine, Tropicale 55, 132–311.Google ScholarPubMed
Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). Protein measurement with the folin phenol reagent. Journal of Biological Chemistry 193, 265–75.CrossRefGoogle ScholarPubMed
Luft, J. H. (1961). Improvements in epoxy resin embedding methods. Journal of Biophysical and Biochemical Cytology 9, 409–14.CrossRefGoogle ScholarPubMed
Martin, B. R., Voorheis, H. P. & Kennedy, E. L. (1978). Adenylate cyclase in bloodstream forms of Trypanosoma (Trypanozoon) brucei sp. The Biochemical Journal 175, 207–12.CrossRefGoogle ScholarPubMed
McClare, C. W. F. (1971). An accurate and convenient organic phosphorus assay. Analytical Biochemistry 39, 527–30.CrossRefGoogle ScholarPubMed
McLaughlin, J., Injeyan, H. S. & Meerovitch, E. (1976). The subcellular distribution and properties of Crithidia, sp. hydrolases with particular references to pyrophosphate and orthophosphate monoester phosphohydrolases. Canadian Journal of Biochemistry 54, 365–81.CrossRefGoogle ScholarPubMed
Meshnick, S. R., Blobstein, S. H., Grady, R. W. & Cerami, A. (1978). An approach to the development of new drugs for African trypanosomiasis. Journal of Experimental Medicine 148, 569–79.CrossRefGoogle Scholar
Müller, M. (1975). Biochemistry of protozoan microbodies: peroxisomes, α-glycerophos-phate oxidase bodies, hydrogenosomes. Annual Review of Microbiology 29, 467–83.CrossRefGoogle ScholarPubMed
Munro, H. N. & Fleck, A. (1966). Recent developments in the measurement of nucleic acids in biological materials. Analyst 91, 78–87.CrossRefGoogle ScholarPubMed
Murray, M., Barry, J. D., Morrison, W. I., Williams, R. O., Hirumi, H. & Rovis, L. (1979). Review: Prospect for vaccination in African Trypanosomiasis. II. World minimal Review, (in the Press).Google Scholar
Neil, M. W. & Horner, M. W. (1964). Studies on acid hydrolases in adult and foetal tissues. Acid p-nitrophenyl phosphate phosphohydrolases of adult guinea-pig liver. The Biochemical Journal 92, 217–24.CrossRefGoogle Scholar
Ness, A. T., Pastewka, J. W. & Peacock, A. C. (1963). Evaluation of a recently reported stable Liebermann-Burchard reagent and its use for the direct determination of serum total cholesterol. Clinica Chimica Acta, 10, 229–37.CrossRefGoogle Scholar
Opperdoes, F. R. & Borst, P. (1977). Localization of nine glycolytic enzymes in a micro-body - like organelle in Trypanosoma brucei. The glycosome. Febs Letters 80, 360–4.CrossRefGoogle Scholar
Opperdoes, F. R., Borst, P., Bakker, S. & Leene, W. (1977). Localization of glycerol-3-phosphate oxidase in the mitochondrion and particulate NAD +-linked glycerol-3 -phosphate dehydrogenase in the microbodies of the bloodstream form of Trypanosoma brucei. European Journal of Biochemistry 76, 29–39.CrossRefGoogle ScholarPubMed
Opperdoes, F. R., Borst, P. & Spits, H. (1977). Particle bound enzymes in the bloodstream form of Trypanosoma brucei. European Journal of Biochemistry 76, 21–8.CrossRefGoogle ScholarPubMed
Ouchterlony, O. (1958). Diffusion-in-gel methods for immunological analysis. In Progress in Allergy, vol. 5 (ed. Kallós, P.), pp. 1–78. Basel and New York: Karger.Google Scholar
Rifkin, M. R. (1978). Identification of the trypanocidal factor in normal human serum: high density lipoprotein. Proceedings of the National Academy of Sciences, USA 75, 3450–4.CrossRefGoogle ScholarPubMed
Riou, G. & Pautrizel, R. (1969). Nuclear and kinetoplastic DNA from trypanosomes. Journal of Protozoology 16, 509–13.CrossRefGoogle ScholarPubMed
Risby, E. L. & Seed, J. R. (1969). Purification and properties of purified hexokinase from the African trypanosomes and Trypanosoma equiperdum. Journal of Protozoology 16, 193–7.CrossRefGoogle ScholarPubMed
Rouser, G., Fleischer, S. & Yamamoto, A. (1970). Two dimensional thin layer chroma-tographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots. Lipids 5, 494–6.CrossRefGoogle Scholar
Rovis, L., Barbet, A. F. & Williams, R. O. (1978). Characterisation of the surface coat of Trypanosoma congolense. Nature, London 271, 654–6.CrossRefGoogle ScholarPubMed
Salomon, Y., Londos, C. & Rodbell, M. (1974). A highly sensitive adenylate cyclase assay. Analytical Biochemistry 58, 541–8.CrossRefGoogle ScholarPubMed
Schrier, S. L., Giberman, E. & Katchalski, E. (1969). Variability hi ouabain-induced inhibition of human erythrocyte membrane (Na+, K+) ATPase. Biochimica et biophysica acta 183, 397–400.CrossRefGoogle Scholar
Seed, J. R., Bryam, J. & Gam, A. A. (1967). Characterisation and localization of acid phos-phatase activity in Trypanosoma gambiense. Journal of Protozoology 14, 117–25.CrossRefGoogle Scholar
Snyder, J. A. & McIntosh, J. R. (1976). Biochemistry and physiology of microtubules. Annual Review of Biochemistry 45, 699–720.CrossRefGoogle ScholarPubMed
Sokoloff, L. & Rothblatt, G. H. (1972). Regulation of sterol synthesis in L. cells: steady state and transitional responses. Biochemica et biophysica acta 280, 172–81.CrossRefGoogle Scholar
Solyom, A. & Trams, E. G. (1972). Enzyme markers in characterization of isolated plasma membranes. Enzyme 13, 329–72.CrossRefGoogle ScholarPubMed
Steck, T. L. & Wallach, D. F. H. (1970). The isolation of plasma membranes. Methods in Cancer Research 5, 93–153.Google Scholar
Vasquez, R., Cattan, A. & Herbert, W. J. (1975). Macrophage-cytophilic antibodies in trypanosome infections in mice. Transactions of the Royal Society of Tropical Medicine and Hygiene 69, 436.Google ScholarPubMed
Vickerman, K. (1974 a). Antigenic variation in African trypanosomes. In Parasites in the Immunized Host: Mechanics of Survival Ciba Fdn Symp. 25 (ed. Porter, R. and Knight, J.), pp. 63–80. Amsterdam: Associated Scientific Publishers.Google Scholar
Vickerman, K. (1974 b). The ultrastructure of pathogenic flagellates. In Trypanosomiasis and Leishmaniasis with Special Reference to Chagas' Disease Ciba Fdn Symp. 20, (ed. Elliot, K., M., O'Connor and Wolstenholme, G. E. W.), pp. 171–98. Amsterdam: Associated Scientific Publishers.CrossRefGoogle Scholar
Vickerman, K. & Luckins, A. G. (1969). Localization of variable antigens in the surface coat on Trypanosoma brucei using ferritin conjugated antibody. Nature, London 224, 1125–6.CrossRefGoogle ScholarPubMed
Voorheis, P. H., Gale, J. S., Owen, M. J. & Edwards, W. (1979). The isolation and partial characterisation of the plasma membrane from Trypanosoma brucei. The Biochemical Journal 180, 11–24.CrossRefGoogle ScholarPubMed
Wallach, D. F. H. & Kamat, V. B. (1964). Plasma and cytoplasmic membrane fragments from Ehrlich ascites carcinoma. Proceedings of the National Academy of Sciences, USA 52, 721–8.CrossRefGoogle ScholarPubMed
Wallach, D. F. H. & Kamat, V. B. (1966). Preparation of plasma-membrane fragments from mouse ascites tumour cells. In Methods in Enzymology, vol. 8 (ed. Neufield, E. F and Ginsburg, V.), pp. 164–72. New York and London: Academic Press.Google Scholar
Wybenga, D. R., Pileggi, V. J., Dirstine, P. H. & Di Giorgio, J. (1970). Direct manual determination of serum total cholesterol -with a single stable reagent. Clinical Chemistry 16, 980–3.CrossRefGoogle ScholarPubMed
Zingales, B., Carniol, C., Abrahamson, P. A. & Coli, W. (1979). Purification of an adenyl cyclase-containing plasma membrane fraction, from Trypanosoma cruzi. Biochimica et biophysica acta 550, 233–44.CrossRefGoogle Scholar