Radioiodination of new protein antigens on the surface of Plasmodium knowlesi schizont-infected erythrocytes |
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Authors: | Russell J. Howard John W. Barnwell Vivien Kao Wendell A. Daniel Stephen B. Alley |
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Affiliation: | Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20205, U.S.A. |
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Abstract: | Schizont-infected red blood cells (SI-RBCs) from Plasmodium knowlesi-infected rhesus monkeys (Macaca mulatta) have been radioiodinated and the 125I-proteins and 125I-antigens compared with those of uninfected RBCs. New 125I-proteins of Mr 230 000, 180 000, 165 000, 155 000, 135 000, 107 000, 72 000 and 65 000 were shown to be parasite-dependent components of SI-RBCs, the Mr 230 000 and 72 000 bands being quantitatively the major new components. New 125I-antigens of SI-RBCs that were absent from uninfected cells and were only immunoprecipitated by sera from infected monkeys had Mr 230 000, 200 000, 180 000, 165 000, 155 000, 135 000, 130 000, 107 000, 72 000, 65 000 and 47 000. The following approaches were used to test which new radioiodinated components are on the SI-RBC outer membrane: analysis of radioactivity in haemoglobin; electron microscopic analysis of the integrity and purity of the SI-RBCs; treatment of intact 125I-SI-RBCs with trypsin to ascertain the sensitivity of new proteins to exogenous protease; immunoprecipitation of antigen-antibody complexes after addition of antibody to intact 125I-SI-RBCs. Although each test has inherent disadvantages, the accumulated evidence suggests that many, if not all of the new antigens identified for the first time in this report are on the SI-RBC outer membrane. The SICA variant antigen on P. knowlesi SI-RBCs was not identified by this approach. |
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Keywords: | Schizont-infected erythrocytes Surface membrane antigens Radioiodination Lactoperoxidase IODOGEN 1,3,4,6-tetrachloro-3α,6α-diphenylglycoluril D-RBC ‘dense’ red blood cells from infected blood consisting of uninfected cells plus cells infected with immature parasites EDTA ethylenediaminetetraacetic acid GO glucose oxidase IODOGEN 1,3,4,6-tetrachloro-3α,6α-diphenylglycoluril LPO lactoperoxidase apparent molecular weight by SDS-polyacrylamide gradient gel electrophoresis NET-T 0.15 M NaCl, 0.005 M EDTA, 0.05 M Tris, 0.002 M PMSF, 0.02% sodium azide (pH 7.2) plus 1% w/v Triton X-100 PBS phosphate-buffered saline PMSF phenylmethylsulfonyl fluoride SDS sodium dodecyl sulfate SICA schizont-infected cell agglutination SI-RBC schizont infected red blood cell STI soybean trypsin inhibitor TCA trichloroacetic acid TLCK TPCK L-1-tosylamide-2-phenylethylchloromethyl ketone HCl |
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