Date of Award:
5-1986
Document Type:
Dissertation
Degree Name:
Doctor of Philosophy (PhD)
Department:
Biology
Department name when degree awarded
Biology (Microbiology)
Committee Chair(s)
Frederick Post
Committee
Frederick Post
Committee
Gary Resnick
Abstract
Protective antigen component of B. anthracis toxin was produced and purified to the >99% level. Toxin was purified from culture supernatant utilizing concentration and liquid chromatography techniques. Purity was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
The purified protective antigen retained biological and antigenic activity as evidenced respectively by lethality in Fischer 344 rats when injected in combination with lethal factor, and by positive results on the Ouchterlony double diffussion assay.
Radioiodinated protective antigen was used both in the in vivo and the in vitro experiments.
In vivo distribution of labelled protective antigen was determined in Fischer 344 rats. Assay of organ tissues for labelled protective antigen aided in the decision to use Maden-Darby bovine kidney cells for the cell cultures in the protective antigen binding studies.
Protective antigen binding studies, all performed at 37°C, evaluated criteria for receptor existence. Labelled protective antigen was found to bind specifically and reversibly to Maden-Darby bovine kidney cells. Receptors proved to be saturable. Scatchard analysis showed a relatively high dissociation constant (KD= 17 X 10-9 M) compared to other toxins in similar studies. This indicated moderately low affinity for protective antigen.
The receptor was also partially characterized. It was shown that cholera toxin subunit B blocked the binding of labelled protective antigen to Maden-Darby bovine kidney cells and that the protective antigen receptor was insensitive to trypsin treatment. Both of these observations suggest a ganglioside as the receptor for protective antigen.
Checksum
0d795a1d8a4359087b1f52e73026d39c
Recommended Citation
Martin, Daniel Dalton, "Purification of Anthrax Toxin Protective Antigen Component and Characterization of its Binding Interaction with Bovine Kidney Cells" (1986). All Graduate Theses and Dissertations, Spring 1920 to Summer 2023. 4641.
https://digitalcommons.usu.edu/etd/4641
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