The abilities of cyclic
adenosine 3':5'-monophosphate (cAMP) and cyclic
8-azidoadenosine 3':5'-[32P]monophosphate (8-N3-[32P]cAMP) to bind to the regulatory subunit (RII) of the type II
cAMP-dependent protein kinase isozyme and to cause subsequent dissociation of the
holoenzyme were compared in extracts from adult and neonatal mouse lung and lung
adenoma. RII in extracts from adult lung exhibits equal numbers of high- (Kd 15 nM) and low- (Kd 230 nM) affinity 8-N3-[32P]cAMP binding sites. In the neonate, the proportion of high-affinity sites is reduced to 20% while, in lung
adenoma, only low-affinity RII binding is observed. Low-affinity RII binding is correlated with an inability of cAMP to dissociate the type II
holoenzyme completely.
Sucrose gradient sedimentation of adult lung cytosol in the presence of cAMP shows complete dissociation of the type I
isozyme, while only some of the type II
holoenzyme is dissociated. This is in contrast to the case with lung
tumor cytosol, in which only low-affinity binding is observed and no apparent dissociation of the type II
isozyme occurs. cAMP does promote RII dephosphorylation within the
holoenzyme, however, suggesting that cAMP can bind to RII without dissociating the tetramer. Consistent with this interpretation, photoincorporation of 8-N3-[32P]cAMP prior to
sucrose gradient sedimentation results in the formation of a photolabeled RII complex which sediments at the same rate as does the
holoenzyme. Two-dimensional gel electrophoresis of RII photolabeled at low and high concentrations of 8-N3-[32P]cAMP suggests that these altered binding and dissociation characteristics of the type II
isozyme are not due to the presence of a structurally altered RII molecule. After
DEAE-cellulose chromatography of lung cytosol, only high-affinity RII binding is observed, and all of the RII can now be dissociated with cAMP. Low-affinity binding may thus reflect either an altered conformational state of RII or the interaction of the type II
kinase with other cytosolic molecules which can affect RII binding and dissociation without altering the functional properties of the type I
isozyme.