An investigation of P-type and V-type ATPase components and chloroquine sensitivity of digestive vacuoles purified from Plasmodium falciparum

Adams, Bronwen

An investigation of P-type and V-type ATPase components and chloroquine sensitivity of digestive vacuoles purified from Plasmodium falciparum

Thesis / Dissertation

1999

Publisher

University of Cape Town

Department

Department of Medicine

Faculty

Faculty of Health Sciences

Abstract

Chloroquine as a therapeutic for malaria has become limited due to the appearance of drug-resistant Plasmodium falciparum. Digestive vacuoles purified from resistant P. falciparum accumulate less chloroquine than those from sensitive strains. This study characterised the ATPase activity of intact digestive vacuoles purified from chloroquine-sensitive and -resistant P. falciparum. Marker enzyme assays, transmission electron microscopy and gel electrophoresis demonstrated that vacuolar preparations were largely free from parasite and erythrocyte contaminants. Lower [3H]-chloroquine accumulation by purified vacuoles from resistant strains was confirmed but their 60-fold less uptake compared to infected erythrocytes argued there are other nonvacuolar sites which influence sensitivity to chloroquine. P. falciparum vacuolar ATPase activity required an external pH of 7.5 and 2mM concentrations of ATP and Mg2+ for maximum levels. Proton pump (Vacuolar or V-type ATPase) and P-glycoprotein (Phosphorylated or P-type ATPase) inhibitors indicated a weakened proton pumping ATPase was not responsible for reduced chloroquine accumulation in purified digestive vacuoles from resistant P. falciparum. Similarly, there was no correlation between chloroquine sensitivity of P. falciparum and vacuolar ATPase activity associated with a P-glycoprotein homologue termed Pghl. Elevated levels of Pghl described in the resistant clone FAC8 was confirmed by the increased sensitivity of its vacuolar ATPase activity and chloroquine accumulation to the P-glycoprotein inhibitors. However, Pghl at the vacuolar level was implicated in chloroquine accumulation possibly through regulation of the intravacuolar pH as a chloride channel. A sodium/hydrogen exchanger (NHE) activity was linked to the vacuolar ATPase activity of P. falciparum although it could not be implicated in chloroquine uptake. Results can be reconciled with chloroquine binding to a haem molecule in P. falciparum driving its uptake in a process sensitive to pH which is regulated by a combination of proton pumping, Pghl or NHE activities.

Keywords

chloroquine

plasmodium falciparum

Reference:

Collections

PhD / Doctoral

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