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A rat monoclonal antibody which interacts with mammalian ornithine decarboxylase at an epitope involved in
Abstract:
Ornithine decarboxylase was purified from androgen-treated mouse kidney to homogeneity and high specific activity. The purified enzyme was utilized for production and screening of rat monoclonal and polyclonal antibodies. A rat monoclonal antibody was isolated which was capable of immunoprecipitation of native mouse kidney ornithine decarboxylase activity or the [3H]difluoromethylornithine-inactivated enzyme. Phosphorylation of mouse ornithine decarboxylase by casein kinase-II prior to immunoprecipitation led to complete loss of the epitope recognized by the monoclonal antibody but did not alter recognition by polyclonal antibody. Mammalian ornithine decarboxylase activity obtained from several species, in crude or partially purified extracts, was subjected to quantitative immunoprecipitation with monoclonal and polyclonal antibody. Polyclonal antibody immunoprecipitated all of the ornithine decarboxylase activity from every extract tested, while monoclonal antibody was capable of only limited immunoprecipitation (60-80%). Due to the inability of the monoclonal antibody to recognize ornithine decarboxylase phosphorylated in vitro by casein kinase-II and the partial immunoprecipitation of ornithine decarboxylase activity from cell extracts, a portion of the ornithine decarboxylase molecule population must exist in a phosphorylated state. This immunological evidence further confirms existing data that the enzyme exists in at least two distinct forms.
Insights
Researchers developed antibodies to study ornithine decarboxylase (ODC). A monoclonal antibody distinguished between phosphorylated and non-phosphorylated ODC forms, confirming the enzyme exists in multiple states.
Area of Science:
- Biochemistry
- Immunology
- Enzymology
Background:
- Ornithine decarboxylase (ODC) is a key enzyme in polyamine synthesis.
- Understanding ODC's regulation is crucial for cellular processes.
- Previous studies suggested ODC may exist in different forms.
Purpose of the Study:
- To purify mouse kidney ornithine decarboxylase (ODC).
- To generate and characterize monoclonal and polyclonal antibodies against ODC.
- To investigate the different forms of ODC using immunological methods.
Main Methods:
- Purification of ODC from androgen-treated mouse kidney.
- Production and screening of rat monoclonal and polyclonal antibodies.
- Immunoprecipitation assays using native and modified ODC.
- Quantitative immunoprecipitation of ODC from various mammalian extracts.
Main Results:
- A specific monoclonal antibody was generated, capable of immunoprecipitating ODC.
- Phosphorylation of ODC by casein kinase-II abolished recognition by the monoclonal antibody.
- Polyclonal antibodies recognized all ODC activity, while monoclonal antibodies recognized only 60-80% in cell extracts.
- These findings indicate the presence of phosphorylated ODC in vivo.
Conclusions:
- The study successfully produced antibodies for ODC research.
- Immunological evidence supports the existence of at least two distinct forms of ODC.
- A significant portion of ODC in cell extracts exists in a phosphorylated state, affecting its recognition by specific antibodies.