A mechanistic and kinetic analysis of the interactions of the diastereoisomers of adenosine 3',5'-(cyclic)phosphorothioate with purified cyclic AMP-dependent protein kinase.
The binding affinities of the diastereoisomers of adenosine 3',5'-(cyclic)phosphorothioate, Sp-cAMP[S] and Rp-cAMP[S], for the cyclic AMP- (cAMP-)binding sites on purified and reconstituted pig heart type II cAMP-dependent protein kinase holoenzyme were determined by measuring the ability of these compounds to displace [3H]cAMP from this enzyme. Sp-cAMP[S], a cAMP agonist, displaced 50% of the [3H]cAMP bound to the holoenzyme at a concentration 10-fold higher than that of cAMP; Rp-cAMP[S], a cAMP antagonist, required a 100-fold higher concentration relative to cAMP. Activation of the isolated holoenzyme, determined as phosphotransferase activity, was measured in the presence of the agonist and in the absence and in the presence of increasing concentrations of the antagonist. The results of fitting the activation data to sigmoid curves with a non-linear-regression program and to Hill plots by using a linear-regression program showed that Rp-cAMP[S] had no effect on Vmax, increased the EC50 values for agonist activation and had no effect on the co-operativity of activation (h). A Ki value of 11 microM was determined for Rp-cAMP[S] inhibition of cAMP-induced activation of purified type II cAMP-dependent protein kinase. Electrophoresis of the holoenzyme on polyacrylamide gels under non-denaturing conditions in the presence of saturating concentrations of the diastereoisomers resulted in 100% dissociation of the subunits with Sp-cAMP[S] and 0% dissociation with Rp-cAMP[S]. Sp-cAMP[S], the isomer with an axial exocyclic sulphur atom, binds to the holoenzyme, releases the catalytic subunit and activates the phosphotransferase activity. Rp-cAMP[S], the isomer with an equatorial exocyclic sulphur atom, binds to the holoenzyme but does not result in dissociation, and thus acts as a competitive inhibitor of phosphotransferase activity.
Rothermel
J D
JD
Sandoz Research Institute, Sandoz Pharmaceuticals Corporation, East Hanover, NJ 07936.
Parker Botelho
L H
LH
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D010770:Phosphotransferases / Q000037:antagonists & inhibitors; D011485:Protein Binding; D047428:Protein Kinase Inhibitors; D013045:Species Specificity; D013237:Stereoisomerism; D013552:Swine; D013873:Thionucleotides / Q000494:pharmacology*
D007527:Isoenzymes; D047428:Protein Kinase Inhibitors; D013873:Thionucleotides; C016957:adenosine-3',5'-cyclic phosphorothioate; D000242:Cyclic AMP; D010770:Phosphotransferases