It is proposed to evaluate the compare key properties of P. knowlesi, P,k, and P falciparum, P.f, vacuolar membranes, PVMs, (a) continuing work using P.k schizonts, isolated free of host cell membrane contamination by multiple criteria, to determine important structural and functional attributes of P.k PVM proteins, and (b) phasing our approaches into a study (already initiated) to the P.f PVM. The following approaches are planned: 1. Analysis of host cell proteins exposed at he outer surface of the PVM by vectorial surface labeling, selective peptide cleavage, and use of specific antibodies, with specific attention directed to (a) erythrocyte protein 3, (b) glycophorin, GP, and (c) hemoglobin. 2. Examination of parasite-synthesized proteins in the PVM, using metabolic labeling combined with vectorial surface labeling, selective peptide cleavage, and specific antibodies, with specific attention on the 74 kDa P.k protein which we have previously detected in the P.k PVM, and possibly related proteins of P.f. 3. Functional studies (a) measuring anion transport through the PVM, (b) using anion channel probes to evaluate the role of PVM in this process, (c) evaluating the ability of exogenous ATP to support protein synthesis of isolated schizonts, (d) using photoaffinity probes to localize the route of atractyloside- sensitive ATP entry. 4. Develop methods for (a) selective solubilization of the PVM, and (b) the insertion of PVM proteins into phospholipid bilayers, to purify integral PVM proteins, allow more extensive future studies on the PVM, and as means for eventual exploration of membrane traffic in intraerythrocytic Plasmodium.
