On primary structure and biosynthesis of histidine-rich polypeptide from malarial parasite Plasmodium lophurae.

Abstract

Experiments on the primary structure of a histidine-rich polypeptide isolated from the malarial parasite Plasmodium Jophurae indicate that the smaller quantities of amino acids other than histidine form an integral part of the polypeptide and do not arise from a protein contaminating a histidine homopolymer. In culture, the parasites incorporate over 50% of exogenously supplied histidine into the histidine-rich polypeptide and this incorporation is inhibited by puromycin.The intraerythrocytic stages of the avian malarial parasite Plasmodium lophurae contain numerous cytoplasmic granules (1). The main chemical constituent of isolated granules was shown to migrate as a single polypeptide on polyacrylamide gels, and analysis of hydrolysates of the polypeptide showed five major constituent amino acids: 73% histidine, 7.5% proline, 7% alanine, 6% glutamic acid, and 2% aspartic acid (2). The unusually high content of histidine raised two questions: (a) could the smaller quantities of other amino acids come from a typical protein isolated as a contaminant with the histidine homopolymer rather than being integral parts of the polypeptide, and (b) could the polypeptide possibly be synthesized by a non-ribosomal enzymatic system (3-5). Even though non-ribosomal synthesis of peptides has been shown only in prokaryotes, the possibility that it might also exist in eukaryotes cannot be excluded. The unusually high content of one amino acid, histidine, and some parallelism with the polypeptide of the cyanophycin granules of blue-green algae made the malaria polypeptide a plausible candidate for testing. The algal polypeptide was shown to be a high-molecular-weight copolymer of argine and aspartic acid, and its synthesis was not inhibited by chloramphenicol (5, 6).To answer the above questions, we undertook experiments on the primary structure of the peptide and studied the effect of puromycin on the biosynthesis of the peptide.MATERIALS AND METHODS Materials. The histidine-rich polypeptide was isolated as previously described (2). Pepsin, trypsin, and chymotrypsin were purchased from Worthington; Nagarse was from En- 900), pH 6.5. The sample was applied at tUie middle of the strip. For preparative purposes, guide strips were cut and stained with ninhydrin-cadmium reagent (7) and peptides were eluted with 30% acetic acid.Amino-Acid Analysis. Peptides were hydrolyzed in 200Ml of 6 N HC1 containing 0.2% (w/v) phenol and 0.1% (v/v) mercaptoacetic acid in evacuated sealed tubes, at 1100 for 22 hr (8). Hydrolysates were analyzed with a modified amino-acid analyzer (9). Polyacrylamide Gel Electrophoresis. Electrophoresis was according to the method of Panyim and Chalkley (10).Incorporation of [3HlHistidine In Vitro. Uninucleate trophozoites of P. lophurae were removed from their host duck erythrocytes and cultured extracellularly under conditions described previously (11). To facilitate quantitative collection of parasites at the end of the incubation period, the plasma clot lining of the culture flask was omitted. To each...

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

“…The significance of these results is therefore the much less likely that any polypeptides would be poorly following: (i) it is quite unlikely that any parasitelabeled and because a histidine-rich protein (about 70 synthesized proteins are inserted though the red cell mol%) exists in P . lophume (9) and has been hypothe-membrane in quantities sufficient to play a structural sized to be present in P.falciparurn. Subsequently, this role in reorganization of the membrane or its surface; protein was proposed to be homologous with proteins and (ii) if knob proteins are in fact histidine rich, then Can.…”

“…falciparum which causes knob formation but not by one which does not (7), was presumed to be an integral part of the knobs. The putative knob protein was also hypothesized (on the basis of differential amino acid incorporation patterns) to be homologous (8) with the 45-kd histidine-rich protein found in the dense granules of mature Plasmodium lophurue parasites when infecting erythrocytes of the Bekin duckling (9).…”

In situ enzyme-linked immunosorbent assay to quantitate in vitro development of Eimeria tenella

“…A7284, Sigma Chemical Co.) and applied to a DE-52 (Whatman, Inc.) column equilibrated in 75 mM Tris-40 mM NaH2PO4-86 mM NaCl-100 mM glucose, pH 8.2. The merozoites that flowed through this column were evaluated for purity (removal of host cell debris) by electron microscopy (12). Purified merozoites were suspended in PBS (no.…”

Ultrastructural characteristics of host‐parasite interactions in intracellular protozoan parasites

“…Indirect autoradiographic evidence suggested that a histidine-rich protein is contained in these organelles (Kilejian, 1974). More recently, indirect immunofluorescence studies (Perkins, 1981;Hall et al, 1983;Campbell et al, 1984;Sam-Yellowe et al, 1988) demonstrated that fluorescine labelled antibodies binds to rhoptries and the protein is discharged from the merozoite, and spreads around the surface of the erythrocyte.…”