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Immunological distinction between calmodulin-sensitive and calmodulin-insensitive adenylate cyclases
The Journal of Biological Chemistry
|June 5, 1987
Summary
Researchers developed an antibody targeting the calmodulin-sensitive adenylate cyclase. This antibody distinguished between calmodulin-sensitive and insensitive forms, revealing distinct isozymes in rat tissues.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Previous research suggested the presence of both calmodulin-sensitive and -insensitive adenylate cyclase activities in bovine brain.
- Calmodulin-Sepharose affinity chromatography was used to initially differentiate these enzyme forms.
Purpose of the Study:
- To generate a specific antibody against the calmodulin-sensitive adenylate cyclase.
- To investigate the distribution of calmodulin-sensitive adenylate cyclase in various rat tissues.
- To confirm the immunological distinction between calmodulin-sensitive and -insensitive adenylate cyclase isozymes.
Main Methods:
- Immunization of mice with purified calmodulin-sensitive adenylate cyclase from bovine brain to produce a polyclonal antiserum.
- Characterization of the antiserum's specificity using calmodulin-sensitive and -insensitive adenylate cyclase preparations.
- Immunoprecipitation assays to quantify calmodulin-sensitive adenylate cyclase activity in detergent-solubilized membranes from different rat tissues (brain, kidney, liver, testes, heart, lung).
Main Results:
- A specific polyclonal antiserum was successfully generated against the calmodulin-sensitive adenylate cyclase.
- The antiserum specifically precipitated the 135,000 Mr catalytic subunit of the calmodulin-sensitive adenylate cyclase.
- Calmodulin-sensitive adenylate cyclase activity was detected in rat brain (40-60%), kidney (40-60%), heart (approx. 15%), and lung (approx. 15%).
- No detectable calmodulin-sensitive adenylate cyclase activity was found in rat liver and testes.
Conclusions:
- The generated antiserum is specific to the catalytic subunit of the calmodulin-sensitive adenylate cyclase isozyme.
- Calmodulin-sensitive and insensitive adenylate cyclases are immunologically distinct.
- The findings support the existence of multiple adenylate cyclase isozymes in the brain and suggest differential tissue distribution.