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Calcium-independent stimulation of Bordetella pertussis adenylate cyclase by calmodulin
Abstract:
Bordetella pertussis produces an extracellular adenylate cyclase activity that is present in the culture medium of exponentially growing cells. We have determined that calmodulin (CaM) stimulates this enzyme both in the presence and in the absence of free Ca2+. In the presence of 90 micron Ca2+ half-maximal stimulation of the enzyme occurred at 95 pM calmodulin. Comparable levels of calmodulin stimulation were observed when free Ca2+ levels were minimized by using EGTA-containing buffers. However, the concentration of CaM required for half-maximal stimulation of B. pertussis adenylate cyclase in the presence of 1 nM free Ca2+ was 24 nM. The apparent affinity of the enzyme for calmodulin was also significantly enhanced by Mn2+. In addition, troponin I inhibited calmodulin stimulation of the bacterial adenylate cyclase. Photoaffinity cross-linking experiments using azido[125I]calmodulin and B. pertussis adenylate cyclase revealed only one major cross-linked product having a molecular weight of 97000. It is proposed that the catalytic subunit of the calmodulin-sensitive adenylate cyclase is 77000.
Insights
Bordetella pertussis adenylate cyclase is stimulated by calmodulin (CaM), a calcium-binding protein. This bacterial enzyme
Area of Science:
- Microbiology
- Biochemistry
- Enzymology
Background:
- Bordetella pertussis secretes an extracellular adenylate cyclase.
- This enzyme's activity is regulated by cellular factors.
Purpose of the Study:
- To investigate the role of calmodulin (CaM) in modulating Bordetella pertussis adenylate cyclase activity.
- To characterize the interaction between bacterial adenylate cyclase and CaM.
Main Methods:
- Enzyme activity assays with varying Ca2+ and CaM concentrations.
- Use of EGTA to minimize free Ca2+ levels.
- Photoaffinity cross-linking with azido[125I]calmodulin.
- Molecular weight determination of cross-linked products.
Main Results:
- Calmodulin (CaM) stimulates Bordetella pertussis adenylate cyclase activity independently of Ca2+ levels.
- Mn2+ enhances the enzyme's affinity for CaM.
- Troponin I inhibits CaM-stimulated cyclase activity.
- Photoaffinity labeling identified a 97,000 molecular weight cross-linked product.
Conclusions:
- Bordetella pertussis adenylate cyclase is a calmodulin-sensitive enzyme.
- The catalytic subunit of the calmodulin-sensitive adenylate cyclase is estimated to be 77,000 molecular weight.