Analysis of proteins synthesized<i>in vitro</i>by the erythrocytic stages of<i>Plasmodium knowlesi</i>

McColm, A. A.; Shakespeare, P.G.; Trigg, P. I.

doi:10.1017/s0031182000055141

Cited by 8 publications

(8 citation statements)

References 36 publications

(29 reference statements)

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“…6 A-C, this rated proteins by autoradiography. However, the "new" proteins we detect are not identical to the excreted proteins described by McColm et al (12), since these are also found in the parasite cytoplasm. Our immunoelectrophoretic data with monkey antischizont serum show that membranes from parasitized erythrocytes contain at least four antigens that are not detectable in schizont preparations identical to those used to produce the antiserum.…”

Section: Resultscontrasting

confidence: 85%

Plasmodium knowlesi-induced antigens in membranes of parasitized rhesus monkey erythrocytes.

Schmidt‐Ullrich¹,

Wallach²

1978

Proc. Natl. Acad. Sci. U.S.A.

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Highly purified Plasmodium knowlesi schizonts were used to produce a hyperimmune anti-parasite serum in a rhesus monkey. Proteins of membranes from normal and P. knowlesi-infected erythrocytes, as well as purified schizonts, were solubilized in 1% Triton X-100 and analyzed by bidimensional electrophoretic techniques. Of seven parasite-specific antigens identified in membranes of parasitized erythrocytes by crossed immune electrophoresis against monkey anti-parasite serum, only three could be detected in the purified schizonts. Bidimensional focusing-dodecyl sulfate/polyacrylamide gel electrophoresis of membranes from parasitized cells revealed three proteins, in the 55,000-90,000 molecular weight region, with isoelectric points between pH 4.5 and pH 5.2,-that could not be detected in normal membranes or purified schizonts. Membranes of normal erythrocytes and uninfected erythrocytes that had been incubated with sera from monkeys with 25-50% parasitemia did not react with the monkey anti-parasite serum.Inoculation of owl or rhesus monkeys with intraerythrocytic stages of Plasmodium falciparum (1) or P. knowlesi merozoites (2), with complete Freund's adjuvant, produces effective animalarial immunity. Moreover, Collins et al. (3) have isolated a heat-stable soluble antigen from sera of P. knowlesi-infected monkeys which when injected with complete adjuvant, will suppress parasitemia and reduce mortality of animals challenged with homologous parasites. These experiments show that antimalarial immunity can be achieved experimentally by vaccination with parasite antigens. However, they do not provide information about parasite-induced host-cell antigens. These were first recognized by Brown et al. (4), who showed that the sera of rhesus monkeys chronically infected with, or immune to infection by, a given strain of P. knowlesi contained antibodies that agglutinate erythrocytes containing schizonts of that strain of P. knowles . Possibly related antigens have also been detected by immune electron microscopy of erythrocytes infected with P. falciparum (5), but it is not known whether any such antigens can elicit a protective immune response.We have approached this area by biochemical and immunochemical techniques, first developing a reliable method for the isolation of schizonts and host-cell plasma membranes from P. I6mowlessinfected rhesus erythrocytes (6) and now analyzing these fractions, by two-dimensional isoelectric focusing-dodecyl sulfate (DodSO41)/polyacrylamidp gel electrophoresis and by immunoelectrophoretic methods using high-titer monkey antisera raised against purified schizonts.MATERIALS AND METHODS Chemicals. Reagents for electrophoretic separations were as in refs. 6-8; other chemicals were as in ref. 6. Monkeys. Rhesus monkeys (Macaca mulatta), weighing 4-8 kg, obtained from the Primate Import Company (Port Washington, NY), were first infected after about a 4-week quarantine period (Tb-negative). Cure was by an initial intramuscular injection of 20 mg of chloroquine per kg, followed by two...

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Section: Resultscontrasting

confidence: 85%

Plasmodium knowlesi-induced antigens in membranes of parasitized rhesus monkey erythrocytes.

Schmidt‐Ullrich¹,

Wallach²

1978

Proc. Natl. Acad. Sci. U.S.A.

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“…A similar array of proteins was synthesized by a reticulocyte cell-free protein synthesizing system containing P. knowlesi mRNA (Eggit, Tappenden & Brown, 1979). McColm et al (1980) also found that the most intensely staining parasite protein, with a molecular weight of 63-76 kDaltons, was not labelled. Why this was so was not resolved.…”

Section: Discussionmentioning

confidence: 89%

“…Earlier studies of amino acid incorporation into plasmodial proteins showed that the degree of radioisotope incorporation was related both to the developmental stage of the parasite, as well as to the particular amino acid (Polet & Barr, 1968;McColm, Shakespeare & Trigg, 1980). In general, trophozoites showed greater incorporation than rings and late schizonts, and maximum incorporation occurred with radioisotope labelled isoleucine and leucine.…”

Section: Discussionmentioning

confidence: 99%

“…This is the first report of such a qualitative distinction in these 2 plasmodial fractions. McColm et al (1980) were unable to clearly distinguish cytoplasmic (soluble) and membrane (particulate) proteins in stained gels, largely due to cross-contamination of these fractions. Similar ambiguities have been reported when infected red cells were analysed by SDS-PAGE/Coomassie blue staining (reviewed by Sherman, 1982).…”

Section: Discussionmentioning

confidence: 99%

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Stage-specific proteins of the avian malariaPlasmodium lophurae

Sherman

Tanigoshi

1983

Parasitology

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SUMMARYThe proteins in the soluble and membrane fractions ofPlasmodium lophurae, separated by SDS-PAGE and stained with Coomassie blue, were distinct from one another and different from the soluble and membrane proteins of the host cell, the duckling erythrocyte. Pulse-labelling of malaria-infected red cells containing schizonts or trophozoites with [14C]isoleucine, followed by parasite isolation, SDS-PAGE and autoradiography, showed a pattern of incorporation into soluble proteins identical to the banding pattern of gels stained with Coomassie blue. On autoradiograms, the banding patterns of the membrane proteins of trophozoites and schizonts labelled with [14C]isoleucine were similar. No qualitative differences in the soluble proteins of trophozoites and schizonts were apparent when isoleucine was used as the radiotracer; however, the banding pattern of membrane and soluble proteins labelled with [14C]isoleucine were distinct from each other. The banding pattern of soluble proteins, after staining with Coomassie blue, was distinctly different from the array of bands seen after autoradiography of parasites derived from infected cells pulse-labelled with [14C]histidine. Only small amounts of contaminating haemoglobin in the soluble protein fraction of parasites were labelled with histidine; the membrane fraction, on the other hand, had high incorporation into a single 43 kDalton band, the histidine-rich protein. No band differences, either in the membrane or soluble fractions, were observed in autoradiograms after pulse-labelling trophozoites and schizonts with [14C]histidine. The most consistent and distinct differences in protein synthesis were seen inP. lophuraepulse-labelled with [14C]proline. Autoradiograms of both soluble and membrane fractions showed the presence of bands unique to the trophozoite and schizont–stage-specific proteins.

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“…27) and Plasmodium knowlesi (28), a simian malaria, although stage-related protein synthesis in this system was not found (29), probably owing to insensitive assay techniques. Stage-specific antigens immunoprecipitated from B. falciparum (22) were found modulated in a pattern qualitatively similar to that which we found by metabolic labeling: a predominance of intermediate-sized polypeptides synthesized by young parasites and a shift toward increased relative rates sf synthesis in both higher and lower molecular weight polypeptides with parasite maturation.…”

Section: Discussionmentioning

confidence: 97%

Developmental modulation of protein synthetic patterns by the human malarial parasite Plasmodium falciparum

Allred¹,

Sherman²

1983

Can. J. Biochem. Cell Biol.

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Under conditions of in vitro culture, Plasmodium falciparum incorporated amino acids into particulate (membrane) and soluble proteins in a pattern which changed sequentially and which was dependent upon the stage of parasite maturation. Synchronized cultures pulse labeled with a mixture of 15 14C-labeled amino acids or [14C]histidine alone displayed stage-related patterns of polypeptide biosynthesis. Certain plasmodial proteins were associated with both particulate (membrane) and soluble fractions, whereas others appeared to be specific to a given fraction. Proteolysis of intact infected cells with pronase under conditions which removed 97 +/- 2.2% of the endogenous red cell acetylcholinesterase activity did not cause the apparent removal of any radiolabeled proteins; this suggests the absence of externally exposed, parasite-synthesized proteins in the infected red cell membrane. Such a result was consistent whether the radiolabel was [14C]histidine or the 14C-labeled amino acid mixture. These results indicate that specific modulation of parasite biosynthetic patterns occurs during the asexual reproductive cycle and is probably one mechanism whereby parasite differentiation occurs. Despite the formation of surface excrescences on infected red cells containing mature parasites, results of surface digestion experiments failed to demonstrate the presence of surface-exposed plasmodial proteins.

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Analysis of proteins synthesizedin vitroby the erythrocytic stages ofPlasmodium knowlesi

Cited by 8 publications

References 36 publications

Plasmodium knowlesi-induced antigens in membranes of parasitized rhesus monkey erythrocytes.

Plasmodium knowlesi-induced antigens in membranes of parasitized rhesus monkey erythrocytes.

Stage-specific proteins of the avian malariaPlasmodium lophurae

Developmental modulation of protein synthetic patterns by the human malarial parasite Plasmodium falciparum

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