Complete Amino Acid Sequence of the Catalytic Subunit of Bovine Cardiac Muscle Cyclic AMP-Dependent Protein Kinase (original) (raw)
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European Journal of Biochemistry, 1989
cAMP analogs, all 96 of which were modified in the adenine moiety, were examined quantitatively for their ability to inhibit the binding of [3H]cAMP to each of the two classes (A and B) of cAMP-binding sites of type I (rabbit skeletal muscle) and type II (bovine heart) cAMP-dependent protein kinase. The study showed that analogs can be constructed that have a higher affinity than cAMP for a binding site. N6-phenyl-cAMP had 18-fold increased affinity for site A of RI (AI) and 40-fold increased affinity for site AII. 2-chloro-8-methylamino-cAMP had a 7-fold increased affinity for BI, and 8-(4-chlorophenylthio)-cAMP had 17-fold increased affinity for BII. Analogs could discriminate between the two classes of binding sites by more than two orders of magnitude in binding affinity: 2-chloro-8-methylamino-cAMP had 170-fold higher affinity for BI than for AI, and 2-n-butyl-8-thiobenzyl-cAMP had 700-fold higher affinity for BII than for AII. Analogs could also discriminate between the homologous binding sites of the isozymes: 2-n-butyl-8-bromo-cAMP had 260-fold higher affinity for AI than for AII (22-fold higher for BII than BI), and 8-piperidino-cAMP had 50-fold higher affinity for BII than for BI (and 50-fold higher for AI than for AII). The data suggest the following conclusions. (a) Stacking interactions are important for the binding of cAMP to all the binding sites. (b) Subtle differences exist between the sites as to the optimal electron distribution in the adenine ring since modifications that withdraw electrons at C2 and donate at C8 favour binding to BI, and disfavour binding to AI and AII. (c) There are no hydrogen bonds between the adenine ring of cAMP and any of the binding sites. (d) All sites bind cAMP in the syn conformation. (e) The subsites adjacent to the N6 and C8 positions may have nonpolar neighbouring regions since hydrophobic substituents at N6 could increase the affinity for AI and AII and similar substituents at C8 could increase the affinity for BII. Finally, (f) the sites differed in their ability to accomodate bulky substituents at C2 and C8. For all compounds tested, their potency as activators of protein kinases I and II was found to correlate, in a predictable fashion, to their mean affinity for the two classes of binding sites, rather than to the affinity for only one of the sites.
The Journal of biological chemistry, 1983
Eighty different adenine-modified cAMP analogs were tested as activators of rabbit muscle protein kinase I (cAKI) in an in vitro phosphotransferase assay. All the analogs tested were able to activate completely the kinase. The affinities of the cAMP derivatives for the two types (A and B) of binding sites associated with the regulatory moiety of cAKI were determined under conditions similar to those used in the phosphotransferase assay. The potency of the cAMP analogs as cAKI activators was found to correlate with the mean affinity for sites A and B, rather than to the affinity for only one of the sites. This was true whether cAKI was assayed at low or near physiological ionic strength, whether the concentration of cAKI binding sites was 0.2 or 400 nM, and whether the kinase substrate was mixed histones or homogeneous phenylalanine-4-monooxygenase. Furthermore, site A-selective and site B-selective cAMP analogs activated cAKI synergistically. Finally, it was shown that the degree of...