Analysis of proteins synthesizedin vitroby the erythrocytic stages ofPlasmodium knowlesi

Abstract

SUMMARY: Studies were performed to identify specific parasite proteins synthesized withinPlasmodium 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 cultivatedin vitrowith [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.