Isolation and characterization of parasites and host cell ghosts from erythrocytes infected with Plasmodium chabaudi

Abstract

A new procedure has been developed which allows the concomitant isolation of viable parasites and host cell plasma membranes from erythrocytes infected with Plasmodium chabaudi trophozoites. The average final yield of parasites is 56%. Free parasites reveal a well preserved ultrastructure, incorporate [14C]isoleucine for at least 3 h, and synthesize about the same proteins as parasites within erythrocytes as monitored by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)-autoradiography. The host cell plasma membranes can be isolated in the form of ghosts with an average yield of 27%. The ghosts possess a structurally intact plasma membrane as revealed by freeze-etch electron microscopy. The ghosts are regularly associated with seven neo-proteins as identified by SDS-PAGE and isoelectric focusing (IEF)/SDS-PAGE. These neo-proteins have the following apparent molecular masses: 154 kDa, 145 kDa, 90 kDa, 72 kDa (pI 4.5), 67 kDa, 52 kDa, and 33 kDa (pI 5.7), respectively. The contamination of ghosts by parasite material and, conversely, the contamination of parasites by host cell plasma membranes is very low as demonstrated by light and electron microscopy, lactoperoxidase-mediated radioiodination and the distribution of the typical parasite marker enzymes such as choline kinase, cholinephosphotransferase and ethanolaminephosphotransferase.