Co-elution of type I and type II cAMP-dependent protein kinase holoenzymes in high salt fractions from DEAE-cellulose.
Post-mitochondrial supernatants from adult and neonatal mouse lung were each separated using DEAE-cellulose chromatography, and fractions were assayed for cAMP-dependent kinase activity and photoincorporation of the analog, 8-azido-cyclic adenosine 3':5'-monophosphate. Although there is no significant developmental differences in the amount of kinase activity which elutes in these high salt or DEAE peak II fractions, such fractions derived from neonatal lung contain only half as much photodetectable regulatory subunit of the type II protein kinase isozyme (RII) as do corresponding fractions from the adult. Much of the kinase activity associated with these neonatal DEAE peak II fractions may actually be due to the type I holoenzyme. Significant amounts of photodetectable regulatory subunit of the type I protein kinase isozyme (RI) co-elute with this peak of type II kinase activity, and several lines of evidence suggest that much of this RI exists in a holoenzyme state. Incubation of these peak II fractions with cAMP prior to a second elution from DEAE-cellulose causes much of the RI to elute at lower salt concentrations characteristic of the free RI subunit. After sucrose gradient sedimentation of peak II fractions, RI cosediments with the peak of cAMP-dependent kinase activity. Finally, kinase activation by cAMP is biphasic, suggesting that both the type I and II holoenzymes contribute to the kinase activity in the type II peak. Two-dimensional gel electrophoresis of photolabeled proteins does not suggest any structural difference between RI subunits present in various DEAE fractions. The elution of the type I holoenzyme over a range of salt concentrations may be due to structural variations in the C subunit of the type I isozyme or possibly to the stable association of the type I holoenzyme with other molecules in the cell.
D000375:Aging; D000818:Animals; D000831:Animals, Newborn; D001665:Binding Sites; D002848:Chromatography, DEAE-Cellulose; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008168:Lung / Q000201:enzymology* / Q000254:growth & development; D051379:Mice; D008805:Mice, Inbred A; D009994:Osmolar Concentration; D011485:Protein Binding; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism
D007527:Isoenzymes; D011494:Protein Kinases