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Purification and characterization of a calmodulin-sensitive adenylate cyclase from Bordetella pertussis
Abstract:
Bordetella pertussis, the bacterium responsible for whooping cough, releases a soluble, calmodulin-sensitive adenylate cyclase into its culture medium. B. pertussis mutants deficient in this enzyme are avirulent, indicating that the adenylate cyclase contributes to the pathogenesis of the disease. It has been proposed that B. pertussis adenylate cyclase may enter animal cells and increase intracellular adenosine cyclic 3',5'-phosphate (cAMP) levels. We have purified the enzyme extensively from culture medium using anion-exchange chromatography in the presence and absence of calmodulin and gel filtration chromatography. The enzyme was purified 1600-fold to a specific activity of 608 mumol of cAMP min-1 mg-1 and was free of islet activating protein. The molecular weight of the enzyme was 43 400 in the absence of calmodulin and 54 200 in the presence of calmodulin. The Km of the bacterial enzyme for adenosine 5'-triphosphate was 2.0 mM, whereas the Km of the calmodulin-sensitive adenylate cyclase from bovine brain was 0.07 mM. Although the enzyme was not purified to homogeneity, its turnover number of 27 000 min-1 is the highest documented for any adenylate cyclase preparation.
Insights
Bordetella pertussis adenylate cyclase is crucial for whooping cough pathogenesis. Extensive purification revealed its calmodulin sensitivity and high turnover rate, suggesting a key role in disease development.
Area of Science:
- Microbiology
- Biochemistry
Background:
- Bordetella pertussis causes whooping cough and releases adenylate cyclase.
- This enzyme is essential for B. pertussis virulence.
- The enzyme's role in increasing intracellular cAMP levels in host cells is proposed.
Purpose of the Study:
- To purify and characterize the Bordetella pertussis adenylate cyclase.
- To investigate the enzyme's properties, including calmodulin sensitivity and molecular weight.
Main Methods:
- Extensive purification of the enzyme from bacterial culture medium.
- Anion-exchange chromatography and gel filtration chromatography were employed.
- Enzyme activity, molecular weight, and kinetic parameters were determined.
Main Results:
- The adenylate cyclase was purified 1600-fold with a specific activity of 608 μmol cAMP min⁻¹ mg⁻¹.
- The enzyme's molecular weight was 43,400 Da without calmodulin and 54,200 Da with calmodulin.
- A high turnover number of 27,000 min⁻¹ was observed, the highest documented for any adenylate cyclase.
Conclusions:
- Bordetella pertussis adenylate cyclase is a potent enzyme contributing to whooping cough pathogenesis.
- Calmodulin binding significantly alters the enzyme's molecular weight.
- The enzyme's high catalytic efficiency supports its proposed role in disease.