The long term goals of this project are to define the mechanism for calmodulin (CaM) regulation of Bordetella pertussis adenylate cyclase and to elucidate the mechanism for entry of the enzyme into animal cells. B. pertussis is the pathogen responsible for whooping cough and the organism releases several toxins into its growth media including a CaM sensitive adenylate cyclase. Recent evidence from our laboratory using a highly purified B. pertussis adenylate cyclase demonstrated that the enzyme preparation can cause significant increases in intracellular cAMP of animal cells. We hypothesize that the enzyme enters animal cells, is activated by CaM, and elevates intracellular cAMP levels. The specific objectives of this proposal include purification of the enzyme to homogeneity, elucidation of its subunit composition and quaternary structure and identification of its catalytic subunit and CaM binding subunits by affinity labeling with 8-azido[3H]ATP and azido[125I]CaM. Furthermore, we propose to demonstrate unambiguously that the enzyme enters animal cells and to define the mechanism for cellular entry. The availability of the highly purified toxin may aid in the development of a safer vaccine for whooping cough and the study should contribute to a better understanding of the molecular basis of B. pertussis pathology.
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