Hostname: page-component-848d4c4894-nr4z6 Total loading time: 0 Render date: 2024-06-09T12:11:04.492Z Has data issue: false hasContentIssue false
  • English
  • Français

Analysis of proteins synthesized in vitro by the erythrocytic stages of Plasmodium knowlesi

Published online by Cambridge University Press:  06 April 2009

A. A. McColm ,
P. G. Shakespeare  and
P. I. Trigg
A. A. McColm
Affiliation:
Division of Parasitology, National Institute for Medical Research, Mill Hill, London NW7 1AA
P. G. Shakespeare
Affiliation:
Division of Parasitology, National Institute for Medical Research, Mill Hill, London NW7 1AA
P. I. Trigg
Affiliation:
Division of Parasitology, National Institute for Medical Research, Mill Hill, London NW7 1AA
Get access Rights & Permissions [Opens in a new window]

Summary

Studies were performed to identify specific parasite proteins synthesized within Plasmodium knowlesi-infected rhesus erythrocytes. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of whole parasites freed from the host erythrocyte by immune lysis, of membranous and cytoplasmic parasite fractions, and of isolated merozoites, detected several parasite-specific components after Coomassie Blue staining of the separated proteins. However, significant contamination with host erythrocyte material generally occurred, particularly in the whole parasite and parasite membrane preparations. Improved identification of plasmodial proteins was subsequently afforded by a radioisotope labelling technique in which parasitized erythrocytes were cultivated in vitro with [3H] isoleucine prior to electrophoretic analysis. Of 11 principal labelled peaks ranging in molecular weight from approximately 17000 to 145000 which were detected upon electrophoresis of whole parasites harvested from culture, all were observed in the cytoplasmic fraction while at least 5 were also associated with the membranous cell fraction. Analysis of different developmental stages of the intra-erythrocytic parasite revealed no significant stage-specific qualitative variations in the electrophoretic profiles. Quantitatively, however, the middle to late trophozoites incorporated more [3H] isoleucine into protein than the other intra-erythrocytic stages. Analysis of merozoites purified from labelled schizonts showed a protein pattern similar to the other stages. This confirmed that host components did not contribute to the labelling pattern and that none of the labelled proteins were specific to the residual cytoplasm remaining after merozoite formation.

Type
Research Article
Information
Parasitology , Volume 81 , Issue 1 , August 1980 , pp. 177 - 198
Copyright
Copyright © Cambridge University Press 1980

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Bishop, C., & Surgenor, D. M., (1964). The Red Blood Cell. New York: Academic Press.Google Scholar
Bonner, W. M., & Laskey, R. A., (1974). A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. European Journal of Biochemistry 46, 83–8.CrossRefGoogle ScholarPubMed
Brown, I. N., Brown, K. N., & Hills, L. A., (1966). The separation of Plasmodium knowlesi from host cells. Transactions of the Royal Society of Tropical Medicine and Hygiene 60, 3.CrossRefGoogle Scholar
Brown, K. N., & Brown, I. N., (1965). Immunity to malaria: antigenic variation in chronic infections of Plasmodium knowlesi. Nature, London 208, 1286–8.CrossRefGoogle ScholarPubMed
Butcher, G. A., Mitchell, G. H., & Cohen, S., (1973). Mechanism of host specificity in malarial infection. Nature, London 244, 40–2.CrossRefGoogle ScholarPubMed
Chavin, S. I., (1966). Studies on the antigenic constituents of Plasmodium berghei I. Immunologic analysis of the parasite constituents. II. Fractionation of the parasite constituents. Military Medicine 131, 1124–36.CrossRefGoogle ScholarPubMed
Cohen, S., & Mitchell, G. H., (1978). Prospects for immunization against malaria. Current Topics in Microbiology and Immunology 80, 97–137.Google ScholarPubMed
Deans, J. A., Dennis, E. D., & Cohen, S., (1978). Antigenic analysis of sequential erythrocytic stages of Plasmodium knowlesi. Parasitology 77, 333–44.CrossRefGoogle ScholarPubMed
Eggitt, M. J., Tappenden, L., & Brown, K. N., (1979). Synthesis of Plasmodium knowlesi polypeptides in a cell free system. Bulletin of the World Health Organization 57, 109–13.Google Scholar
Ellman, G. L., Courtney, K. D., Andres, V., & Featherstone, R. H., (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology 7, 88–95.CrossRefGoogle ScholarPubMed
Fairbanks, G. Jr, Levinthal, C., & Reeder, R. H., (1965). Analysis of 14C-labelled proteins by disc electrophoresis. Biochemical and Biophysical Research Communications 20, 393–9.CrossRefGoogle Scholar
Finerty, J. P., & Dimopoullos, G. T., (1968). Disk electrophoretic separation of soluble preparations of Plasmodium lophurae andAnaplasma marginale. Journal of Parasitology 54, 585–7.CrossRefGoogle ScholarPubMed
Gourlay, R. N., Brocklesby, D. W., & Sellwood, S. A., (1970). Disk electrophoresis as an aid in the identification of piroplasms. Veterinary Record 86, 629–30.CrossRefGoogle ScholarPubMed
Hamburger, J., & Zuckerman, A., (1976 a). Plasmodium berghei: I. Immunochemical properties of fractions of a soluble extract. Experimental Parasitology 39, 460–78.CrossRefGoogle ScholarPubMed
Hamburger, J., & Zuckerman, A., (1976 b). Plasmodium berghei: II. Immunobiological properties of fractions of a soluble extract. Experimental Parasitology 39, 479–95.CrossRefGoogle ScholarPubMed
Ilan, J., Pierce, D. R., & Miller, F. W., (1977). Influence of 9-β-D-arabino-furanosyladenine on total protein synthesis and on differential gene expression of unique proteins in the rodent malarial parasite Plasmodium berghei. Proceedings of the National Academy of Sciences, USA 74, 3386–90.CrossRefGoogle Scholar
Jaswant Singh, , Ray, A. P., & Nair, C. P., (1953). Isolation of a new strain of Plasmodium knowlesi. Nature, London 172, 122.CrossRefGoogle Scholar
Kilejian, A., & Jensen, J. B., (1977). A histidine-rich protein from malaria and its interaction with membranes. Bulletin of the World Health Organization 55, 191–7.Google Scholar
Königk, E., & Mirtsch, S., (1977). Plasmodium chabaudi- infection of mice: specific activities of erythrocyte membrane-associated enzymes and patterns of proteins and glycoproteins of erythrocyte membrane preparations. Zeitschrift für Tropenmedizin und Parasitologie 28, 17–22.Google ScholarPubMed
Krebs, H. A., & Eggleston, L. V., (1940). The oxidation of pyruvate in pigeon breast muscle. The Biochemical Journal 34, 442–59.CrossRefGoogle ScholarPubMed
Kreier, J. P., Hamburger, J., Seed, T. M., Saul, K., & Green, T., (1976). Plasmodium berghei: characteristics of a selected population of small, free blood stage parasites. Zeitschrift für Tropenmedizin und Parasitologie 27, 82–8.Google Scholar
Laemmli, U. K., (1970). Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature, London 227, 680–5.CrossRefGoogle 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
Marchesi, V. T., & Steers, E. Jr, (1968). Selective solubilization of a protein component of the red cell membrane. Science 159, 203–4.CrossRefGoogle ScholarPubMed
Matsuda, G., Maita, T., Takei, H., Ota, H., Yamaguchi, M., Miyauchi, T., & Migita, M., (1968). The primary structure of adult haemoglobin from Macaco mulatto monkeys. Journal of Biochemistry 64, 279–82.CrossRefGoogle Scholar
McColm, A. A., Shakespeare, P. G., & Trigg, P. I., (1976 a). Proteins synthesized in vitro by the simian malaria parasite Plasmodium knowlesi. Parasitology 73, xvii.Google Scholar
McColm, A. A., Shakespeare, P. G., & Trigg, P. I., (1976 b). Protein synthesis in the erythrocytic stages of Plasmodium knowlesi. In Biochemistry of Parasites and Host-Parasite Relationships (ed. Van den Bossche, H.), pp. 59–65. Elsevier/North Holland.Google Scholar
McCormick, G. J., (1970). Amino acid transport and incorporation in red blood cells of normal and Plasmodium knowlesi-infected rhesus monkeys. Experimental Parasitology 27, 143–9.CrossRefGoogle ScholarPubMed
Morrison, D. B., & Jeskey, H. A., (1947). The pigment, lipids and proteins of the malaria parasite (P. knowlesi). Federation Proceedings 6, 279.Google ScholarPubMed
Polet, H., & Conrad, M., (1969). Malaria: extracellular amino-acid requirements for in vitro growth of erythrocytic forms of Plasmodium knowlesi. Proceedings of the Society for Experimental Biology and Medicine 127, 251–3.CrossRefGoogle Scholar
Richards, W. H. G., & Williams, S. G., (1973). Malaria studies in vitro. II. The measurement of drug activities using leucocyte-free blood dilution cultures of Plasmodium berghei and 3H-isoleucine. Annals of Tropical Medicine and Parasitology 67, 179–90.CrossRefGoogle Scholar
Schmidt-Ullrich, R., & Wallach, D. F. H., (1978). Plasmodium knowlesi-indaced antigens in membranes of parasitized rhesus monkey erythrocytes. Proceedings of the National Academy of Sciences, USA 75, 4949–53.CrossRefGoogle ScholarPubMed
Schmidt-Ullrich, R., Wallach, D. F. H., & Lightholder, J., (1979). Fractionation Of P. Knowlesi-indneed Antigens of rhesus monkey erythrocyte membranes. Bulletin of the World Health Organization 57, 115–21.Google ScholarPubMed
Shapiro, A. L., Vinuela, E., & Maizel, J. V., (1967). Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochemical and Biophysical-Research Communications 28, 815–20.CrossRefGoogle ScholarPubMed
Sherman, I. W., (1964). Antigens of Plasmodium lophurae. Journal of Protozoology 11, 409–17.CrossRefGoogle ScholarPubMed
Sherman, I. W., & Hull, R. W., (1960). The pigment (haemozoin) and proteins of the avian malaria parasite Plasmodium lophurae. Journal of Protozoology 7, 409–16.CrossRefGoogle Scholar
Sodeman, W. A. Jr., & Meuwissen, J. H. B., T. (1966). Disk electrophoresis of Plasmodium berghei. Journal of Parasitology 52, 23–5.CrossRefGoogle ScholarPubMed
Spira, D., & Zuckerman, A., (1966). Recent advances in the antigenic analysis of plasmodia. Military Medicine 131, 1117–23.CrossRefGoogle ScholarPubMed
Steck, T. L., (1974). The organization of proteins in the human red blood cell membrane. Journal of Cell Biology 62, 1–19.CrossRefGoogle ScholarPubMed
Trigg, P. I., (1968). A new continuous perfusion technique for the cultivation of malaria parasites in vitro. Transactions of the Royal Society of Tropical Medicine and Hygiene 62, 371–8.CrossRefGoogle Scholar
Trigg, P. I., (1969). Some factors affecting the cultivation in vitro of the erythrocytic stages of Plasmodium knowlesi. Parasitology 59, 915–24.CrossRefGoogle ScholarPubMed
Trigg, P. I., & Gutteridge, W. E., (1971). A minimal medium for the growth of Plasmodium knowlesi in dilution cultures. Parasitology 62, 113–23.CrossRefGoogle ScholarPubMed
Wallach, D. F. H., & Conley, M., (1977). Altered membrane proteins of monkey erythrocytes infected with simian malaria. Journal of Molecular Medicine 2, 119–36.Google Scholar
Weber, K., & Osborn, M., (1969). The reliability of molecular weight determinations by dodecyl sulphate-polyacrylamide gel electrophoresis. Journal of Biological Chemistry 244, 4406–12.CrossRefGoogle Scholar
Weidekamm, E., Wallach, D. F. H., Lin, P. S., & Hendricks, J., (1973). Erythrocyte membrane alterations due to infection with Plasmodium berghei. Biochimica et biophysica acta 323, 539–46.CrossRefGoogle ScholarPubMed
World Health Organization (1975). Developments in malaria immunology. WHO Technical Report Series No. 579.Google Scholar
Williamson, J., (1967). Antigens Of Plasmodium knowlesi. Protozoology 11, 85–104.Google Scholar
Williamson, J., & Cover, B., (1966). Separation of blood-cell free trypanosomes and malaria parasites on a sucrose gradient. Transactions of the Royal Society of Tropical Medicine and Hygiene 60, 425–7.CrossRefGoogle Scholar
Wilson, R. J. M., (1974). Soluble antigens as blocking antigens. Ciba Fdn Symp. 25, N.S., pp. 185–203. Amsterdam: Associated Scientific Publishers.Google Scholar
Wilson, R. J. M., (1977). Circulating antigens of parasites. In Immunity in Parasitic Diseases. INSERM Colloquium 72, pp. 87–102.Google Scholar