VACUOLAR & MEMBRANES OF ISOLATED PLASMODIUM

提出了一种评价P的比较关键性质的方法。 knowlesi，P，k，和恶性疟原虫，P.f，空泡膜，PVM， (a)继续使用分离的无宿主细胞的P.k酵母进行研究 膜污染的多重标准，以确定 P.k PVM重要结构和功能属性 蛋白质，以及（B）将我们的方法逐步纳入研究（已经 启动）到P.f PVM。 计划采取以下办法： 1. 暴露在细胞外表面的宿主细胞蛋白的分析 通过载体表面标记、选择性肽切割， 和使用特异性抗体，特别注意 (a)红细胞蛋白3，（B）血型糖蛋白，GP，和（c）血红蛋白。 2. PVM中寄生虫合成蛋白的检查，使用 代谢标记结合矢量表面标记， 选择性肽切割和特异性抗体， 注意74 kDa的P.k蛋白，我们以前有 在P.kPVM中检测到的蛋白质，以及可能与P.f. 3. 功能研究（a）测量阴离子通过 PVM，（B）使用阴离子通道探针评估PVM的作用 在这个过程中，（c）评估外源性ATP的能力， 支持分离重组子的蛋白质合成，（d）使用 光亲和探针定位的途径， 敏感的ATP输入。 4. 开发方法，用于（a）PVM的选择性溶解， 和（B）将PVM蛋白插入磷脂双层中， 纯化PVM蛋白，为今后更广泛的研究奠定基础 在PVM上，作为最终探索膜的手段， 红细胞内疟原虫的运输。

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.