Isolation and characterization of rhoptries of Plasmodium falciparum.
Rhoptries have been isolated from Plasmodium falciparum schizont-infected erythrocytes by isopycnic density centrifugation. Gradient fractions were analyzed by immunoblotting with antibodies against two polypeptides of 140 and 110 kDa, known to be components of the rhoptry. The proteins were present primarily in fractions with a density of 1.16 g ml-1. Electron microscopy of these fractions indicated they were enriched in rhoptries. For the most part, the isolated organelle retained in situ morphology, although some rhoptries were distorted, indicating the structure of some of the organelles is not rigid. Electrophoretic analysis of the rhoptry fractions indicated the presence of a number of proteins, many of which have not been identified to date. Properties of proteins in the isolated rhoptry were examined using the 140 and 110 kDa proteins as representative markers. Both proteins are present in a complex with a 130-kDa protein, as all three co-immunoprecipitate. At the late schizont stage, the rhoptry proteins are present in two distinct forms; a soluble form with an Mr of 480 000 which would correspond to a single copy of the 140/130/110 kDa complex and a form that can be sedimented at 130 000 x g. Properties of the sedimentable form suggest that the proteins are included in structures that resemble membranes. Ionic detergents were required to solubilize the proteins while high concentrations of NaCl and Na2CO3 resulted in only partial solubilization. Furthermore, treatment of disrupted rhoptries with phospholipase A and C resulted in the release of proteins into the soluble form.
Etzion
Z
Z
Laboratory of Biochemical Parasitology, Rockefeller University, New York, NY 10021.
Murray
M C
MC
Perkins
M E
ME
D000818:Animals; D000953:Antigens, Protozoan / Q000737:chemistry* / Q000302:isolation & purification; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D004912:Erythrocytes / Q000469:parasitology*; D006801:Humans; D008970:Molecular Weight; D015388:Organelles / Q000737:chemistry / Q000469:parasitology* / Q000648:ultrastructure; D010963:Plasmodium falciparum / Q000648:ultrastructure*; D015800:Protozoan Proteins / Q000737:chemistry* / Q000302:isolation & purification; D012995:Solubility; D013347:Subcellular Fractions / Q000737:chemistry / Q000469:parasitology / Q000648:ultrastructure
D000953:Antigens, Protozoan; D015800:Protozoan Proteins; C065352:rhoptry associated protein, Plasmodium