A study of the interaction between bovine cardiac-muscle cyclic AMP-dependent protein kinase and cyclic AMP using fluorescence-polarization spectroscopy.
The C-subunit of type II cyclic AMP-dependent protein kinase from bovine heart was labelled with the fluorophore fluorescamine (FAM). The association of the dye-labelled subunit (CFAM) with the R-subunit isolated from the same source was monitored by fluorescence polarization spectroscopy. The stoichiometry of C to R in the final complex was close to 1:1. The affinity of the two subunits could be described by a dissociation constant in the nanomolar range. Holoenzyme (formed from CFAM and R) was titrated with cyclic AMP, and the changes in fluorescence anisotropy, due to dissociation of the holoenzyme, recorded. The titration curves were analysed in terms of a model which required computer simulation. Cyclic AMP-induced dissociation proceeds via one or more ternary complexes, and all four cyclic AMP-binding sites on the R-dimer are accessible in the holoenzyme. The dissociation constants describing the release of the C-subunits from the two ternary complexes containing four cyclic AMP molecules were both approx. 9 microM. The binding of two cyclic AMP molecules to protein kinase is necessary and sufficient to cause the dissociation of both C-subunits. The state of association at 'in vivo' concentrations of protein and cyclic AMP is discussed.
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D000242:Cyclic AMP / Q000378:metabolism*; D005454:Fluorescence Polarization; D007700:Kinetics; D046911:Macromolecular Substances; D008956:Models, Chemical; D009206:Myocardium / Q000201:enzymology*; D011494:Protein Kinases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D046911:Macromolecular Substances; D000242:Cyclic AMP; D011494:Protein Kinases