Interaction with Phosphodiesterase of Free and Kinasecomplexed Cyclic Adenosine 3',5'-Monophosphate

• Published in: The Journal of biological chemistry Vol. 246; no. 20; pp. 6183 - 6190
• Main Authors: Robert F. O'Dea, Mari K. Haddox, Nelson D. Goldberg
• Format: Journal Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology 25-10-1971
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)61773-9

Some characteristics of cyclic adenosine 3',5'-monophosphate (cyclic AMP) interaction with a cyclic AMP-stimulatable protein
kinase from bovine skeletal muscle and reactivity of the unbound and complexed cyclic nucleotide with cyclic nucleotide phosphodiesterase
from bovine heart were examined. The velocity of the phosphodiesterase-catalyzed hydrolysis of cyclic AMP was substantially
lowered at 0° and 30° with the addition of purified protein kinase to the system. The apparent inhibition of cyclic AMP hydrolysis
under these conditions was determined to be due primarily to the nonreactivity with phosphodiesterase of protein-complexed
cyclic AMP and not a direct inhibitory effect of the kinase or cyclic AMP-complexed protein. This conclusion derived from
the following observations. At 0° where the dissociation of cyclic AMP from the protein complex was undetectable over a 30-
to 60-min period by Millipore filtration analysis, the first order rates of 5'-[ 3 H]AMP generation from cyclic [ 3 H]AMP were not appreciably different in the presence or absence of previously formed cyclic AMP complex. The decrement in
5'-[ 3 H]AMP generated by phosphodiesterase at 0° was proportional to the amount of kinase added to the reaction mixture. From a
study of the temperature dependence of cyclic AMP dissociation from complex with kinase, it was found that the rate increased
with temperature to where a half-time of less than 3 min was achieved at 40°. At 20° where the half-time of cyclic AMP dissociation
from complex was approximately 20 min, the production of 5'-[ 3 H]AMP paralleled the rate of cyclic AMP dissociation after the unbound species of cyclic AMP in the system was rapidly degraded
by phosphodiesterase. The rate or extent of cyclic [ 3 H]guanosine monophosphate hydrolysis by this preparation of phosphodiesterase which catalyzed the degradation of both cyclic
AMP and cyclic guanosine monophosphate was unaffected by addition of the kinase alone or kinase complexed with cyclic AMP.
These findings are consistent with the hypothesis that phosphodiesterase hydrolyzes only the unbound species of cyclic AMP
intracellularly and that the proportion of activated kinase ( i.e. catalytic subunit dissociated from the cyclic AMP-bound regulatory subunit) could be adjusted through the enzymic degradation
of the unbound species alone because of the rapid equilibrium achievable at higher temperatures between free and protein-complexed
cyclic nucleotide. The results also indicate that the absolute amounts of free and complexed cyclic AMP may be estimated in
a reaction mixture containing both species by the amount of 5'-nucleotide generated from the unbound cyclic AMP at 0° with
phosphodiesterase.