Preparation of pure and intact <it>Plasmodium falciparum</it> plasma membrane vesicles and partial characterisation of the plasma membrane ATPase

• Main Authors: Smith Pete J, Elandalloussi Laurence M
• Format: Article
• Language: English
• Published: BMC 2002-04-01
• Series: Malaria Journal
• Online Access: http://www.malariajournal.com/content/1/1/6
• ISSN: 1475-2875

<p>Abstract</p> <p>Background</p> <p>In host erythrocytes, the malaria parasite must contend with ion and drug transport across three membranes; its own plasma membrane, the parasitophorous membrane and the host plasma membrane. Isolation of pure and intact <it>Plasmodium falciparum</it> plasma membrane would provide a suitable model to elucidate the possible role played by the parasite plasma membrane in ion balance and drug transport.</p> <p>Results</p> <p>This study describes a procedure for isolating parasite plasma membrane from <it>P. falciparum</it>-infected erythrocytes. With this method, the trophozoites released by saponin treatment were cleansed of erythrocyte membranes using anti-erythrocyte antibodies fixed to polystyrene beads. These trophozoites were then biotinylated and the parasite plasma membrane was disrupted by nitrogen cavitation. This process allows the membranes to reform into vesicles. The magnetic streptavidin beads bind specifically to the biotinylated parasite plasma membrane vesicles facilitating their recovery with a magnet. These vesicles can then be easily released from the magnetic beads by treatment with dithiotreithol. The parasite plasma membrane showed optimal ATPase activity at 2 mM ATP and 2 mM Mg²⁺. It was also found that Ca²⁺ could not substitute for Mg²⁺ ATPase activity in parasite plasma membranes whereas activity was completely preserved when Mn²⁺ was used instead of Mg²⁺. Other nucleoside triphosphates tested were hydrolysed as efficiently as ATP, while the nucleoside monophosphate AMP was not.</p> <p>Conclusions</p> <p>We have described the successful isolation of intact <it>P. falciparum</it> plasma membrane vesicles free of contaminating organelles and determined the experimental conditions for optimum ATPase activity.</p>