Radioiodination and identification of externally disposed membrane components of Leishmania tropica |
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Authors: | P R Gardiner D M Dwyer |
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Affiliation: | Cell Biology and Immunology Section, 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: | Two methods, one involving a lactoperoxidase-glucose oxidase coupled reaction and the other employing the insoluble catalyst 1,3,4,6-tetrachloro-3 alpha, 6-alpha-diphenyl-glycoluril (Iodo-Gen), were used to label the surface membrane of promastigotes of Leishmania tropica major. Both methods labelled approximately 20 proteins or glycoproteins (apparent size range 10 to 110 kDa) in a qualitatively similar manner, however the lactoperoxidase method labelled one additional constituent (260 kDa). By omission of both enzymes, or of Iodo-Gen; by comparison of radioactivity incorporated by particulate and soluble cell fractions; and through the action of proteases on live, labelled promastigotes, the surface-labelling specificity of both procedures was confirmed. Immunoprecipitation of Triton X-100 extracts of labelled cells with rabbit antisera revealed a minimum of twelve (seven major) protein antigens in the homologous system and different but cross-reactive protein species from two other isolates of L. tropica. Lectin precipitation of radiolabelled surface components was possible with concanavalin A (but not with other lectins tested) identifying a minimum of 12 glycoproteins. Two of these glycoproteins (210 and 88 kDa) were not recognized by rabbit antiserum. |
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Keywords: | Cell surface iodination Immunoprecipitation Lectin precipitation Iodo-Gen is Pierce's Registered Trade name for 1,3,4,6-tetrachloro-3α,6α-diphenyl-glycoluril kDa kilodaltons D-PBS Dulbecco's phosphate-buffered saline D-PBSG D-PBS containing 20 mM glucose SDS sodium dodecyl sulphate SB slab gel sample buffer TCA trichloroacetic acid Staph A Con A concanavalin A SBA soy bean agglutinin UEA WGA wheat germ agglutinin RCA SDS-PAGE SDS-polyacrylamide gel electrophoresis |
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