Phase separation in Triton X-114 of antigens of transmission blocking immunity in Plasmodium gallinaceum.
The distribution of proteins of mosquito midgut forms of Plasmodium gallinaceum in the detergent-free (aqueous) and detergent-enriched phases was studied using a phase separation technique in Triton X-114. Of the three surface proteins on gametes and newly fertilized zygotes (240, 56, and 54 kDa) immunoprecipitated by transmission blocking monoclonal antibodies, 240 kDa protein was recovered in the aqueous phase, whereas 56 and 54 kDa proteins were found preferentially in the detergent phase. The hydrophobic properties of the 56 and 54 kDa proteins were also shown by their strong tendency to interact with the lipid bilayers and a hydrophobic matrix phenyl-Sepharose. Monoclonal antibody IID3B3 immunoprecipitated all the three proteins from the whole Triton extract but in the phase-separated extracts reacted only with the 240 kDa protein in the aqueous phase and not with the 56 and 54 kDa doublet in the detergent phase. In Western blot analysis also monoclonal antibody IID3B3 reacted only with the 240 kDa protein. The 240 kDa protein in the aqueous phase was retained by monoclonal antibody IID3B3 linked to Sepharose 4B beads and could be eluted either with 0.1 M acetic acid or 50 mM diethylamine. The 56 and 54 kDa doublet in the detergent phase could be bound to and eluted from Sepharose 4B beads-linked monoclonal antibody IID4 or rabbit anti-male P. gallinaceum gamete serum. Two stage-specific glycoproteins of 26 and 28 kDa on the surface of ookinetes of P. gallinaceum were also separated in the detergent phase following Triton X-114 extraction. Phase separation in Triton X-114 offers a simple approach to the separation of a select group of proteins from the bulk of the cellular proteins.
Kumar
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D000818:Animals; D000911:Antibodies, Monoclonal; D000953:Antigens, Protozoan / Q000276:immunology / Q000302:isolation & purification*; D055598:Chemical Phenomena; D011232:Chemical Precipitation; D002627:Chemistry, Physical; D009033:Culicidae; D005306:Fertilization; D005718:Gametogenesis; D006023:Glycoproteins / Q000276:immunology* / Q000302:isolation & purification; D008289:Malaria, Avian / Q000276:immunology / Q000635:transmission; D008565:Membrane Proteins / Q000276:immunology* / Q000302:isolation & purification; D017830:Octoxynol; D010961:Plasmodium / Q000276:immunology*; D010964:Plasmodium gallinaceum / Q000254:growth & development / Q000276:immunology*; D011092:Polyethylene Glycols; D011817:Rabbits; D015053:Zygote / Q000032:analysis / Q000276:immunology
D000911:Antibodies, Monoclonal; D000953:Antigens, Protozoan; D006023:Glycoproteins; D008565:Membrane Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40