Dissociation and reassociation of the phosphorylated and nonphosphorylated forms of adenosine 3':5' -monophosphate-dependent protein kinase from bovine cardiac muscle.
Adenosine 3':5' -monophosphate (cyclic AMP) -dependent protein kinase from bovine heart muscle catalyzes the phosphorylation of its regulatory, cyclic AMP-binding subunit. Phosphorylation enhances net dissociation of the enzyme by cyclic AMP. Chromatography on omega-aminohexyl-agarose was used to study the effects of phosphorylation on cyclic AMP binding and subunit dissociation and reassociation. This method permitted rapid separation of the catalytic subunit from the cyclic AMP -binding protein and holoenzyme. Phospho- and dephosphoprotein kinases were found to dissociate to the same extent at any given concentration of cyclic AMP and completely at saturation. At equilibrium, the amount of cyclic AMP bound was the same for both forms of enzyme and was directly proportional to the degree of dissociation of the holoenzyme. In the absence of cyclic AMP, phospho- and dephospho-cyclic AMP-binding proteins reassociated completely with the catalytic subunit. However, the rate of reassociation of the dephospho-cyclic AMP-binding protein was at least 5 times greater than the phospho-cyclic AMP-binding protein. Retardation of reassociation was directly proportional to the extent of phosphorylation. We conclude that the degree to which the cyclic AMP-binding protein is phosphorylated markedly affects its intrinsic ability to combine with the catalytic subunit to regenerate the inactive cyclic nucleotide-dependent kinase and that the state of phosphorylation of this subunit may be important in detemining the proportion of dissociated (active) and reassociated (inactive) protein kinase at any given time.
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002846:Chromatography, Affinity; D000242:Cyclic AMP / Q000494:pharmacology*; D006152:Cyclic GMP; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D046911:Macromolecular Substances; D009206:Myocardium / Q000201:enzymology*; D010750:Phosphoproteins / Q000378:metabolism; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism; D012685:Sepharose
D046911:Macromolecular Substances; D010750:Phosphoproteins; D012685:Sepharose; D000242:Cyclic AMP; D011494:Protein Kinases; D006152:Cyclic GMP