Activation of cyclic AMP-dependent protein kinase isoenzymes: studies using specific antisera.
A novel method for rapidly determining the amount and degree of association-dissociation of the Type I and Type II cAMP-dependent protein kinases has been developed and validated. Antibodies directed against the regulatory subunits of Type I and Type II cAMP-dependent protein kinases were used. The antibodies formed complexes with holoenzymes and regulatory subunits which were precipitated by goat anti-rabbit IgG (immunoglobulin G). These complexes bound [3H]cAMP with an apparent Kb of 20 nM for protein kinase I and 80 nM for protein kinase II. Immunoprecipitated protein kinases I and II were catalytically active when incubated with cAMP, [gamma-32P]ATP, and histone H2B. When mixtures of the two kinase isoenzymes or cytosol were incubated with various amounts of [3H]cAMP and the isoenzymes were separated by precipitation with antisera specific for each isoenzyme, the amount of [3H]cAMP associated with immunoprecipitates was proportional to the concentration of [3H]cAMP. In contrast, the catalytic activity that was immunoprecipitated varied inversely with the concentration of [3H]cAMP, showing that the activation of protein kinase could be assessed by the disappearance of catalytic activity from the immunoprecipitates. In the absence of MgATP protein kinase I was activated by a 10-fold lower concentration of cAMP than protein kinase II. However, when MgATP was added to the incubation, there was no significant difference in the binding of [3H]cAMP or dissociation of catalytic subunits of the two isoenzymes. The anti-R antibodies were also used to rapidly quantitate the concentration of regulatory subunits and the relative ratio of protein kinases I and II in tissue cytosols.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001666:Binding Sites, Antibody / Q000187:drug effects; D002384:Catalysis; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D007106:Immune Sera; D007527:Isoenzymes / Q000276:immunology / Q000378:metabolism*; D009132:Muscles / Q000201:enzymology; D011494:Protein Kinases / Q000276:immunology / Q000378:metabolism*
D007106:Immune Sera; D007527:Isoenzymes; D000255:Adenosine Triphosphate; D011494:Protein Kinases