Localization of the catalytic subunit of cyclic AMP-dependent. Protein kinase in cultured cells using a specific antibody
| Autor: | Alton L. Steiner, Peter J.A. Davies, Michael P. Murtaugh |
|---|---|
| Rok vydání: | 1982 |
| Předmět: |
medicine.diagnostic_test
Macromolecular Substances Immunoprecipitation Protein subunit Chinese hamster ovary cell Articles Cell Biology Biology Immunofluorescence Molecular biology Cell Compartmentation Antigen-Antibody Reactions Biochemistry Antigen Affinity chromatography Immunologic Techniques medicine biology.protein Animals Cattle Antibody Protein kinase A Protein Kinases Cells Cultured |
| Zdroj: | The Journal of Cell Biology |
| ISSN: | 1540-8140 0021-9525 |
| DOI: | 10.1083/jcb.95.1.64 |
| Popis: | We developed a specific antibody to the catalytic subunit (C-subunit) of cyclic AMP-dependent protein kinase and used it to localize C-subunit in cultured cells. C-subunit antigen was purified from bovine cardiac muscle and cross-linked to hemocyanin with glutaraldehyde. Immunized goat serum showed a low titer of antibody after boosting; it was enriched 100-fold by affinity chromatography on catalytic subunit-Sepharose. The antibody immunoprecipitated C-subunit from type I and type II holoenzyme and depleted enzymatic activity from solution. At 12.5 nM antigen, 1 microgram antibody immunoprecipitated 10 ng of C-subunit. Immunoprecipitation of 35S-labeled cell extracts and 125I-antibody detection on nitrocellulose paper revealed that the antibody specifically reacts with C-subunit in Chinese hamster ovary (CHO) whole cell extracts. Using indirect immunofluorescence to localize C-subunit, we found a pattern of diffuse staining in the cytoplasm of CHO cells with little or no nuclear staining. A similar distribution of the enzyme was observed in Swiss 3T3 cells, bovine endothelial tracheal cells, human lung fibroblasts and NRK cells. Treatment of CHO cells with 8-bromo-cyclic AMP produced no change in the pattern or intensity of immunofluorescence. We conclude that the majority of C-subunit is localized in cytoplasm and that in cultured fibroblasts exposure to cyclic AMP analogues causes no apparent redistribution of catalytic subunit. |
| Databáze: | OpenAIRE |
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