title: The regulatory subunit monomer of cAMP-dependent protein kinase retains the salient kinetic properties of the native dimeric subunit.
abstract: Monomeric regulatory subunit (R) fragments of type II cAMP-dependent protein kinase were compared with the parent dimeric R. The monomeric fragments were generated by either endogenous proteolysis of rabbit muscle R or by trypsin treatment of bovine heart R in the holoenzyme form. During isolation of pure R from rabbit muscle, carboxyl-terminal fragments of Mr = 42,000 (42 K) and Mr = 37,000 by denaturing gels are generated by endogenous proteolysis. Although the autophosphorylation site is retained, the 42 K is not dimeric (as is its native 56 K precursor) but, in contrast to the monomeric 37 K product, actively reassociates with purified catalytic subunit (C). Several lines of evidence indicate a type II R origin of the 42 K. N-terminal sequence analysis of the 42 K shows some homology with known bovine RI, RII, and cGMP-dependent protein kinase sequences. Both cyclic nucleotide-binding sites (two/42 K or 37 K) and the site selectivity of cAMP analogs are retained in the monomeric fragments. When purified bovine heart holoenzyme, which contains a dimeric Mr = 56,000 R (denaturing gel analysis) and two C subunits, is treated with trypsin followed by separation procedures, the product is a fully recovered active enzyme with an unaltered ratio of cAMP binding to catalytic activity. From Mr considerations, the product is a dimer containing one intact C and a proteolyzed R of Mr = 48,000 on denaturing gels. This dimeric enzyme is not significantly different from the parent tetramer in cAMP concentration dependence (Hill constant = 1.63), [3H]cAMP dissociation behavior (both intrasubunit cAMP-binding sites are present), stimulation of [3H]cIMP binding by site-selective cAMP analogs, and synergism between two analogs in kinase activation. The data indicate that 1) proteolytic cleavage of the native R dimer can cause monomerization without appreciably affecting the inhibition of C and 2) essentially all of the cAMP binding cooperativity is an intrasubunit interaction.
authors.forename: S R
authors.initials: SR
authors.lastname: Rannels
authors.forename: C E
authors.initials: CE
authors.lastname: Cobb
authors.forename: L R
authors.initials: LR
authors.lastname: Landiss
authors.forename: J D
authors.initials: JD
authors.lastname: Corbin
mesh_terms: D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism; D004789:Enzyme Activation; D007700:Kinetics; D008168:Lung / Q000201:enzymology; D046911:Macromolecular Substances; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D011494:Protein Kinases / Q000378:metabolism*; D011817:Rabbits; D014357:Trypsin / Q000378:metabolism
chemical_list: D046911:Macromolecular Substances; D000242:Cyclic AMP; D011494:Protein Kinases; D014357:Trypsin