Isolation and elucidation of some functional properties of the "mute" catalytic subunit of cAMP-dependent protein kinase.
A mute isoenzyme of type II cAMP-dependent protein kinase from rat muscle has been reported that is released from the regulatory subunit by cAMP but remains inactive until combination with heat- and acid-stable modulator has occurred. This enzyme has now been obtained in isolation free of the normal catalytic subunit using affinity chromatography with both an ATP analog (Blue Dextran/Sepharose) and a protein substrate analog (Kemptide/CH-Sepharose). Separation can be effected in both cases before activation of the mute enzyme. Affinity of the mute enzyme for Blue Dextran--a ligand specific for the dinucleotide fold in this kinase--is somewhat higher than that of the normal enzyme. Conversely, before reaction with the modulatory protein the mute enzyme will not bind at all to Kemptide/CH-Sepharose, where the normal enzyme elutes at 50 mM KCl. When pretreated with the modulatory protein and so activated, mute enzyme binds to Kemptide with a very high affinity and can only be eluted using a natural substrate (phosphorylase kinase), up to 500 mM salt being ineffective. The modulator thus appears to act through alteration of the protein substrate binding site on the enzyme.
D000818:Animals; D002384:Catalysis; D002417:Cattle; D002846:Chromatography, Affinity; D000242:Cyclic AMP / Q000494:pharmacology*; D004789:Enzyme Activation; D007527:Isoenzymes / Q000302:isolation & purification; D009132:Muscles / Q000201:enzymology; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism / Q000502:physiology; D051381:Rats; D013379:Substrate Specificity
D007527:Isoenzymes; D000242:Cyclic AMP; D011494:Protein Kinases