Localization of the catalytic subunit of cyclic AMPdependent protein kinase in cultured cells using a specific antibody

Michael P. Murtaugh, Alton L. Steiner, Peter J.A. Davies

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27 Scopus citations

Abstract

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, I µg antibody immunoprecipitated 10 ng of Csubunit. Immunoprecipitation of 35S-labeled cell extracts and 125l-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 Csubunit, 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.

Original languageEnglish (US)
Pages (from-to)64-72
Number of pages9
JournalJournal of Cell Biology
Volume95
Issue number1
DOIs
StatePublished - Oct 1 1982

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