Sam-Yellowe T Y, Fujioka H, Aikawa M, Messineo D G
Department of Biology, Cleveland State University, OH 44115-2403, USA.
J Eukaryot Microbiol. 1995 May-Jun;42(3):224-31. doi: 10.1111/j.1550-7408.1995.tb01570.x.
To investigate in more detail the structure of the high molecular weight rhoptry protein complex of Plasmodium falciparum, Rhop-H (140/130/110 kd), the complex was affinity purified from parasite extracts using rhoptry protein specific antisera prepared against Rhop-H proteins bound to and eluted from Balb/c mouse erythrocytes, using 0.5 M NaCl. The individual proteins (140 kd/Rhop-1, 130 kd/Rhop-2, and 110 kd/Rhop-3) were separated, electroeluted, and monospecific polyclonal antisera prepared against the individual proteins, and against the affinity purified complex. Immunofluorescence assays and immunoelectron microscopic studies were performed to verify the subcellular localization of the Rhop-H epitopes. Immunoblotting and immunoprecipitation assays were also performed. We report novel findings regarding the localization of the rhoptry proteins to an electron lucent compartment in the neck of the rhoptries. Analysis of the amino acid composition of the individually purified Rhop-H proteins demonstrated a predominance of negatively charged (E, D) as well as hydrophobic residues (L, A, P, S) in the three proteins. The percentage of negatively charged residues was high for all three proteins. Similarities in amino acid composition for the three proteins supports the previous data demonstrating shared properties such as erythrocyte and liposome binding, for the three proteins. Results of antibody characterizations using rhoptry protein specific antisera demonstrate the immunodominance of the Rhop-H complex.
为了更详细地研究恶性疟原虫高分子量棒状体蛋白复合物Rhop-H（140/130/110kd）的结构，使用针对与Balb/c小鼠红细胞结合并经0.5M NaCl洗脱的Rhop-H蛋白制备的棒状体蛋白特异性抗血清，从寄生虫提取物中亲和纯化该复合物。分离出各个蛋白质（140kd/Rhop-1、130kd/Rhop-2和110kd/Rhop-3），进行电洗脱，并针对各个蛋白质以及亲和纯化的复合物制备单特异性多克隆抗血清。进行免疫荧光测定和免疫电子显微镜研究以验证Rhop-H表位的亚细胞定位。还进行了免疫印迹和免疫沉淀测定。我们报告了关于棒状体蛋白定位于棒状体颈部电子透明区室的新发现。对单独纯化的Rhop-H蛋白的氨基酸组成分析表明，这三种蛋白质中带负电荷的（E、D）以及疏水残基（L、A、P、S）占主导。所有三种蛋白质中带负电荷残基的百分比都很高。这三种蛋白质在氨基酸组成上的相似性支持了先前的数据，即这三种蛋白质具有共同的特性，如与红细胞和脂质体结合。使用棒状体蛋白特异性抗血清进行抗体表征的结果表明Rhop-H复合物具有免疫优势。
