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Characterization of the bacterial cell associated calmodulin-sensitive adenylate cyclase from Bordetella pertussis
1Department of Pharmacology, School of Medicine, University of Washington, Seattle 98195.
Abstract:
Bordetella pertussis produces a calmodulin-sensitive adenylate cyclase that is associated with the whole bacteria and released into its culture media. Preparations of this enzyme invade animal cells, causing elevations in intracellular cAMP levels. Cell-associated adenylate cyclase accounted for 28% of the total adenylate cyclase activity while 72% was released into the culture supernatant. Over 90% of the cell-associated adenylate cyclase activity was sensitive to trypsin treatment of whole cells, indicating that the catalytic domain of the enzyme is localized on the outer surface of the bacterial cells. Enzyme activity was released from whole cells by treatment with SDS. This activity was resolved as a large form (Mr 215,000) by SDS-polyacrylamide gel electrophoresis. In contrast, the culture supernatant contained only the 45,000-dalton catalytic subunit. Enzyme activity released from spheroplasts by sonication was resolved into a large form (Mr 215,000) and a small form (Mr 45,000). The appearance of the small form with spheroplast formation was probably the result of proteolytic degradation. Antibodies generated against the catalytic subunit purified from culture supernatants cross-reacted with and immunoprecipitated both the large and small forms of adenylate cyclase isolated from bacterial cells. Furthermore, incubation of the cell-associated enzyme with a crude bacterial extract resulted in a time-dependent disappearance of the 215,000-dalton form and a concomitant increase in the amount of the smaller 45,000-dalton form. There was also a parallel increase in the ability of the cell-associated preparation to elevate intracellular cAMP levels in N1E-115 mouse neuroblastoma cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Bordetella pertussis adenylate cyclase is released from bacteria and invades cells, increasing cAMP. The enzyme
Area of Science:
- Microbiology
- Biochemistry
- Molecular Biology
Background:
- Bordetella pertussis secretes adenylate cyclase, an enzyme that elevates intracellular cAMP levels in host cells.
- This calmodulin-sensitive adenylate cyclase exists in both cell-associated and released forms.
Purpose of the Study:
- To investigate the localization and forms of Bordetella pertussis adenylate cyclase.
- To understand the relationship between the different molecular weight forms of the enzyme.
Main Methods:
- SDS-polyacrylamide gel electrophoresis to resolve enzyme forms.
- Antibody cross-reactivity and immunoprecipitation assays.
- Enzyme activity assays measuring cAMP levels.
Main Results:
- Cell-associated adenylate cyclase (28%) is mostly external, sensitive to trypsin.
- SDS treatment releases a large form (215,000 Da) from cells; culture supernatant contains a smaller catalytic subunit (45,000 Da).
- Proteolytic degradation likely produces the smaller form; antibodies recognize both forms.
Conclusions:
- Bordetella pertussis adenylate cyclase has a large cell-associated form and a smaller catalytic subunit released extracellularly.
- The enzyme's catalytic domain is on the bacterial surface, and it can be processed into smaller forms.
- These forms contribute to the enzyme's ability to increase intracellular cAMP.