EJB 85 0044 104 CAMP analogs, most of them modified in the adenine moiety, were tested as activators of CAMP-dependent protein kinase I (from rabbit or rat skeletal muscle) and kinase 11 (from bovine heart or rat skeletal muscle). When tested singly, only 2-phenyl-1 ,M-etheno-cAMP showed a considerably (sevenfold) higher potency as an activator of kinase 11 than of kinase I. Analogs containing an %amino modification preferentially activated kinase I, some being more than 10-fold more potent as activators of kinase I than kinase 11. When two analogs were combined, the concentration of one (complementary) analog required to halfmaximally activate each isozyme was determined in the presence of a fixed concentration of another (priming) analog. Analogs tested in combination had been analyzed for their affinity for the intrasubunit binding sites (A, B) of isozyme I and 11. The degree to which complementary analogs preferentially activated one isozyme was plotted against the mean site selectivity, i.e. (affinity A/B isozyme I. affinity A/B isozyme 11)' / 2. This plot produced a straight line, the slope of which reflected the ability of the priming analog to discriminate homologous sites on the isozymes. This means that the isozyme discriminating power of an analog pair can be quantitatively predicted from the affinity of the analogs for site A and B of the two enzymes. It also means that a systematic analysis of those features of analogs imparting a high mean site selectivity or the ability to discriminate between homologous isozyme sites will facilitate the synthesis of new even more isozyme-selective analogs. The pivotal role of CAMP-dependent protein kinase (cAK) as a mediator of CAMP action is well established [l-51. However, the biological significance of the presence of two distinct isozyme forms, cAKI and cAKI1 [6], is still largely unknown. Both isozymes contain one regulatory subunit (R) dimer and two catalytic (C) moieties [2-31. Each regulatory subunit has two types of binding sites for CAMP, termed A and B according to the rate with which they exchange bound labelled nucleotide [7-91. Activation of cAK by cAMP occurs according to the overall equation [lo]: RzCz + 4 CAMP + Rz CAMP)^ + 2 C. The free catalytic subunits transfer the terminal phosphate group of ATP to acceptor proteins, whose function thus may Correspondence to S. 0. Deskeland, Anatomisk Institutt, Universitetet i Bergen, Arstadveien 19, N-5000 Bergen, Norway Abbreviations. cAK1, cAKT1, CAMP-dependent protein kinase type I, 11; R', R", the regulatory subunit of cAKI, cAKII; C, the catalytic subunit of cAKI or cAKII; K,, apparent activation constant, i.e. the concentration of CAMP or an analog required for half-maximal kinase activation; KL, relative activation constant, i.e. K, CAMP/ K, analog; Kcom,,, the concentration of one (complementary) analog required to activate further, half-maximally, the kinase in the presence of a fixed level of another (priming) analog; Ksynr Ka/Kcomb. AI, AII, CAMP binding site type A of R' and R", respectively; BI...

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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 I1 (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 AIL 2-ch!oro-S-methylarninocAMP had a 7-fold increased affinity for BI, and 8-(4-~hloropheny!thio)-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 AIL Analogs could also discriminate between the homologous binding sites of the isozymes: 2-n-butyl-8-bromo-CAMP had 260-fold higher affinity for A1 than for A11 (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 A1 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 BE, and disfavour binding to A1 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 A1 and A11 and similar substituents at C8 could increase the affinity for €311. Finally, (0 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 I1 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 major receptor for cAMP in mammalian cells is the regulatory subunit of CAMP-dependent protein kinase [1 -31. Two general types (cAKI and cAKII) of CAMP-dependent protein kinases have been distinguished based on their orderCorrespondence to R. Ekanger,