Mechanism of Activation of Protein Kinase I from Rabbit Skeletal Muscle. Investigation with Agarose-Immobilized cAMP Derivatives

Six different affinity gels were prepared by coupling adenosine 3′: 5′-monophosphate (cAMP) to agarose via spacers of different lengths attached to adenine at the postitions N-6, C-8 and C-2. The interaction of protein kinase I from rabbit skeletal muscle with these immobilized cAMP ligands was investigated, in order to elucidate the mechanism of activation of this enzyme. It was confirmed that detachment of the dimeric regulatory subunit from cAMP-Sepharose by free cAMP is not possible when cAMP is coupled with long spacers (hexyl) to either N-6 or C-8 of adenine. Detachment is possible with a short spacer (ethyl); however, the efficiency depends on the coupling position (C-2>N-6>C-8). These results are in accord with the steric and electronic features of the cAMP site on the kinase, elaborated in the preceding paper. It was further shown that the tight binding of the dimeric regulatory subunit towards the N6 -(6-aminohexyl) derivative of cAMP-Sepharose involves only one of its two cAMP sites, the other site is able to bind free cAMP. However, both catalytic subunits are set free when the tetrameric holoenzyme interacts with this cAMP-Sepharose, indicating the cooperative feature of this process. On the other hand, in the presence of ATP a considerable fraction of the catalytic subunit is retained by the affinity gel. Furthermore, most of the regulatory subunit can be detached (hexyl spacer at N-6) through incubation with the catalytic subunit and ATP (without free cAMP). These findings can be explained assuming that the two high-affinity cAMP sites at the free dimeric regulatory subunit are transformed to low-affinity sites after formation of the holoenzyme. ATP is known to favor association of the protein subunits of type I enzyme.