Coelution of the type II holoenzyme form of cAMP-dependent protein kinase with regulatory subunits of the type I form of cAMP-dependent protein kinase.
The types and subunit composition of cAMP-dependent protein kinases in soluble rat ovarian extracts were investigated. Results demonstrated that three peaks of cAMP-dependent kinase activity could be resolved using DEAE-cellulose chromatography. Based on the sedimentation of cAMP-dependent protein kinase and regulatory subunits using sucrose density gradient centrifugation, identification of 8-N3[32P]cAMP labeled RI and RII in DEAE-cellulose column and sucrose gradient fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Scatchard analysis of the cAMP-stimulated activation of the eluted peaks of kinase activity, the following conclusions were drawn regarding the composition of the three peaks of cAMP-dependent protein kinase activity: peak 1, eluting with less than or equal to 0.05 M potassium phosphate, consisted of the type I form of cAMP-dependent protein kinase; peak 2, eluting with 0.065-0.11 M potassium phosphate, consisted of free RI and a type II tetrameric holoenzyme; peak 3, eluting with 0.125 M potassium phosphate, consisted of an apparent RIIC trimer, followed by the elution with 0.15 M potassium phosphate of free RII. The regulatory subunits were confirmed as authentic RI and RII based upon their molecular weights and autophosphorylation characteristics. The more basic elution of the type II holoenzyme with free RI was not attributable to the ionic properties of the regulatory subunits, based upon the isoelectric points of photolabeled RI and RII and upon the elution location from DEAE-cellulose of RI and RII on dissociation from their respective holoenzymes by cAMP. This is the first report of a type II holoenzyme eluting in low salt fractions with free RI, and of the presence of an apparent RIIC trimer in a soluble tissue extract.
D000818:Animals; D002499:Centrifugation, Density Gradient; D002848:Chromatography, DEAE-Cellulose; D000242:Cyclic AMP / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007526:Isoelectric Point; D046911:Macromolecular Substances; D008970:Molecular Weight; D009994:Osmolar Concentration; D010053:Ovary / Q000201:enzymology*; D010710:Phosphates; D010766:Phosphorylation; D011188:Potassium; D017680:Potassium Compounds; D011247:Pregnancy; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D012741:Sexual Maturation
D046911:Macromolecular Substances; D010710:Phosphates; D017680:Potassium Compounds; C013216:potassium phosphate; D000242:Cyclic AMP; D011494:Protein Kinases; D011188:Potassium