Conversion of bovine cardiac adenosine cyclic 3',5'-phosphate dependent protein kinase to a heterodimer by removal of 45 residues at the N-terminus of the regulatory subunit.
The type II adenosine cyclic 3',5'-phosphate (cAMP) dependent protein kinase from bovine heart, consisting of a dimeric regulatory subunit and two catalytic subunits, was converted to a heterodimer by limited tryptic digestion. Loss of the tetrameric structure was accompanied by proteolysis of the regulatory subunit to a form with an apparent molecular weight of 45 000 vs. 52 000 for the native subunit. The proteolyzed subunit behaved as a monomer, in contrast to the dimeric native subunit. Amino acid sequence analysis established that proteolysis removed 45 residues at the N-terminus, indicating that these 45 residues constitute the dimerizing domain of this protein. The kinetic properties of this heterodimer were indistinguishable from those of the native tetramer: half-maximal kinase activation occurred at 48 nM cAMP with a Hill coefficient of 1.45, the regulatory subunit bound 1.5 equiv of cAMP with half-maximal binding occurring at 33 nM, and kinetics for dissociation of bound cAMP were biphasic, indicating the presence of two different binding sites. These observations suggest that residues 1-45 function only in the formation of dimers and that dimerization has little influence on other functional properties of the regulatory subunit. More extensive proteolysis cleaved the monomeric fragment at Lys-311. The fragments resulting from this second cleavage did not dissociate, and the complex inhibited the catalytic subunit in a cAMP-dependent manner.
D000818:Animals; D002417:Cattle; D000242:Cyclic AMP / Q000378:metabolism / Q000494:pharmacology; D004789:Enzyme Activation; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism*
D046911:Macromolecular Substances; D000242:Cyclic AMP; D011494:Protein Kinases; D014357:Trypsin