Studies of two different intrachain cGMP-binding sites of cGMP-dependent protein kinase

The binding of [3H]cGMP to purified beef lung cGMP-dependent protein kinase (cG kinase) was examined using two methods of membrane filtration which avoided loss of bound [3H]cGMP. The enzyme bound 1.6-2.0 mol of [3H]cGMP/mol of monomer. If the kinase was saturated with [3H]cGMP and then excess unlabeled cGMP was added, [3H]cGMP dissociated from the enzyme as two approximately equal components (Sites 1 and 2). When 8-bromo-cGMP or cIMP was added to the [3H]cGMP-binding reaction at a concentration sufficient to competitively inhibit binding by greater than 50%, the relative amount of the slower or faster component, respectively, of [3H]cGMP dissociation decreased during the cGMP chase. The data indicated that the cG kinase, like its cAMP-dependent protein kinase homologue, possesses two highly conserved intrachain cyclic nucleotide-binding sites which have different dissociation rates and analog specificity. The Ka of the kinase for cGMP was about 20-fold lower using histone instead of heptapeptide as substrate. Aging of the enzyme caused conversion to a higher Ka form of the kinase and an apparent increase in the Site 1 cGMP dissociation rate. Using fresh enzyme and heptapeptide as substrate, Site 1 occupation occurred at lower concentrations of cGMP than did Site 2 occupation, and was associated with an increase in protein kinase activity. However, kinase activity appeared to correlate better with total cGMP binding than with binding to either of the two sites, and the activation by cGMP exhibited positive cooperativity (n = 1.57). It is suggested that both intrachain sites are involved in protein kinase activation. E2 + 4 cGMP in equilibrium E2 . cGMP4 The cG kinase could be photoaffinity-labeled using 8-azido-[32P]cAMP. When the labeled cG kinase was trypsin-treated followed by sodium dodecyl sulfate-slab gel electrophoresis, a single major peptide of approximate Mr = 12,000 was resolved.     

Two different intrachain cAMP binding sites of cAMP-dependent protein kinases

The regulatory subunits of both isozymes of cAMP-dependent protein kinase bind 2 mol of cAMP/mol of monomer. cAMP dissociation studies indicate similar cAMP binding behavior for each isozyme. Each has two different intrachain cAMP binding components present in approximately equal amounts and the rate of cAMP dissociation is 5- to 10-fold slower from one site (Site 1) than from the other (Site 2). Equilibrium [3H]cAMP binding is inhibited by several competing cyclic nucleotides. Following equilibrium binding using saturating [3H]cAMP in the presence of competing nucleotide, the pattern of release of [3H]cAMP, monitored in the presence of an excess of nonradioactive cAMP, suggests site-specific selectivity of some of the cyclic nucleotides. As compared with cAMP, cIMP prefers Site 2 for both regulatory subunits, whereas N6, O2-dibutyryl-cAMP shows a similar preference only with isozyme II regulatory subunit. 8-Bromo-cAMP, 8-bromo-cGMP, and 8-azido-cAMP prefer Site 1 of both proteins. The results indicate that for each isozyme the two intrachain binding sites have different analogue specificities and cAMP dissociation rates. Site 1 or Site 2 of one isozyme has a similar but not identical cyclic nucleotide specificity and cAMP dissociation rate to the corresponding site of the other isozyme.     

Two types of cyclic GMP binding site associated with the cyclic AMP-dependent protein kinase from lymphocytes.

Low- and high-affinity binding sites for cyclic GMP were found to be associated with the cyclic AMP-dependent protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) from human tonsillar lymphocytes, but neither of them was identical with the cyclic AMP binding site. The enzyme activated by cyclic GMP phosphorylated the same site of calf thymus H2b histone as the cyclic AMP activated enzyme; however, more complex kinetics of activation were found with cyclic GMP. Two classes of cyclic GMP binding site were demonstrated by kinetic analysis of cyclic [3H]GMP binding in the enzyme preparations eluted by 0.1 M potassium phosphate (pH 7.0) from DEAE cellulose. The high-affinity cyclic GMP binding site (Kd about 4 . 10(-8) M) belonged to some complex form of the protein kinase, as evidenced by the mutual inhibition of cyclic AMP binding and high affinity cyclic GMP binding. However, the high-affinity cyclic GMP binding site disappeared on Sephadex G-100 gel chromatography of the enzyme preparation, whereas the cyclic AMP binding activity was recovered quantitively as separate fractions. The low-affinity cyclic GMP binding site (Kd 2--5 . 10(-6) M) was demonstrated by the inhibitory effect of 10(-5) M cyclic GMP on cyclic AMP binding in each cyclic AMP binding fraction obtained by gel chromatography. However, cyclic AMP did not inhibit the binding of cyclic GMP to the low-affinity binding site.     

Characterization of histamine H1-receptor on rat hepatocytes

The binding sites for [3H]pyrilamine in isolated rat hepatocytes were characterized. Scatchard analysis revealed two kinds of binding sites in hepatocytes, a high-affinity site and a low-affinity one. The rates of binding of the radioligand with the high-affinity binding site and its dissociation were rapid. The specificity of the sites for various histamine antagonists indicated that the high-affinity [3H]pyrilamine binding site is representative of the histamine H1 receptor. Treatment of hepatocytes with protease or phospholipase A2 significantly decreased the maximum binding capacity of the high-affinity site without affecting its dissociation constant, suggesting that the binding site is proteinaceous and is sensitive to a change in the lipid moiety of the membrane. Hepatocytic cyclic AMP and cyclic GMP were not significantly modulated by incubating hepatocytes with histamine. Thus, the action of histamine on hepatocytes might not be mediated by the cyclic nucleotides.     

Two types of cyclic GMP binding site associated with the cyclic AMP-dependent protein kinase from lymphocytes

Low- and high-affinity binding sites for cyclic GMP were found to be associated with the cyclic AMP-dependent protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) from human tonsillar lymphocytes, but neither of them was identical with the cyclic AMP binding site.The enzyme activated by cyclic GMP phosphorylated the same site of calf thymus H2b histone as the cyclic AMP activated enzyme; however, more complex kinetics of activation were found with cyclic GMP.Two classes of cyclic GMP binding site were demonstrated by kinetic analysis of cyclic [³H]GMP binding in the enzyme preparations eluted by 0.1 M potassium phosphate (pH 7.0) from DEAE cellulose. The high-affinity cyclic GMP binding site (K_d about 44 · 10^?8 M belonged to some complex form of the protein kinase, as evidenced by the mutual inhibition of cyclic AMP binding and high affinity cyclic GMP binding. However, the high-affinity cyclic GMP binding site disappeared on Sephadex G-100 gel chromatography of the enzyme preparation, whereas the cyclic AMP binding activity was recovered quantitively as separate fractions. The low-affinity cyclic GMP binding site (K_d 2–5 · 10^?6 M) was demonstrated by the inhibitory effect of 10^?5 M cyclic GMP on cyclic AMP binding in each cyclic AMP binding fraction obtained by gel chromatography. However, cyclic AMP did not inhibit the binding of cyclic GMP to the low-affinity binding site.     

Activation of type I cyclic AMP-dependent protein kinases with defective cyclic AMP-binding sites

Two S49 mouse lymphoma cell variants hemizygous for expression of mutant regulatory (R) subunits of type I cyclic AMP-dependent protein kinase were used to investigate functional consequences of lesions in the putative cAMP-binding sites of R subunit. Kinase activation properties of wild-type and mutant enzymes were compared using cAMP and six site-selective analogs of cAMP. Kinases from both mutant sublines were relatively resistant to cyclic nucleotide-dependent activation, but they were fully activable by at least some effectors. Relative resistances of the mutant kinases varied from about 5-fold for analogs selective for their nonmutated sites to as much as 700-fold for analogs selective for their mutated sites; resistance to cAMP was intermediate. Apparent affinities of wild-type and mutant R subunits for [3H]cAMP were not appreciably different, but competition experiments with site-selective analogs of cAMP suggested that binding of cAMP to mutant R subunits was primarily to their nonmutated sites. Analyses of cooperativity in cyclic nucleotide-dependent activation of mutant kinases, synergism between site I- and site II-selective analogs in activating the mutant enzymes, and dissociation of bound cAMP from mutant R subunits provided additional evidence that the mutations in these strains selectively inactivated single classes of cAMP-binding sites: phenomena attributable in wild-type enzyme to intrachain interactions between sites I and II were always absent or severely diminished in experiments with the mutant enzymes. These results confirm that R subunit sequences implicated in cAMP binding by homology with other cyclic nucleotide-binding proteins actually correspond to functional cAMP-binding sites. Furthermore, occupation of either cAMP-binding site I or II is apparently sufficient for activation of cAMP-dependent protein kinase. The presence of four functional cAMP-binding sites in wild-type kinase enhances the cooperativity and sensitivity of cAMP-mediated activation.     

A cyclic GMP-dependent histone kinase bound to liver nucleoli

A method of steady-state electrophoresis in polyacrylamide gels was used to analyze the presence of cyclic nucleotide binding components in cell extracts. Multiple cyclic AMP and cyclic GMP binding components were detected in soluble cytoplasmic and nuclear extracts derived from avian liver, but only a single cyclic GMP binding protein was found in the 0.3 M NaCl extract of liver nucleoli. In the presence of cyclic GMP, this protein phosphorylated efficiently a calf thymus histone mixture and an endogenous nucleolar protein which migrated identically with histone H4 in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isoelectric point of the cyclic GMP-binding protein was 4.8. Addition of cyclic GMP did not influence the activity of the endogenous nucleolar RNA polymerase.     

pH induced increase in cyclic GMP reactivity with cyclic AMP-dependent protein kinases

Cyclic AMP-dependent protein kinases have been found which exhibit an enhanced capacity to bind cyclic GMP at acidic values of pH. The binding of cyclic GMP to a protein kinase from skeletal muscle, eluted as a single peak from DEAE cellulose columns, is inversely proportional to pH between the values of 7 to 4; the enzyme exhibits a 5 fold greater ability to bind cyclic [³H]-GMP (10^?8M) at pH 4.0 than 7.0. Protein kinases prepared from skeletal or uterine muscle, eluted as the first of two peaks from DEAE cellulose, exhibited similar pH dependent changes in specificity for cyclic GMP as determined by inhibition of cyclic [³H]-AMP binding. Acidic pH did not appreciably enhance the binding of cyclic [³H]-AMP to kinases prepared from aged skeletal muscle or kinase eluted as the second peak from DEAE cellulose.     

Intracellular cyclic GMP-binding proteins in cellular slime molds.

The complexity of cyclic GMP-binding activity in the 48,000 X g supernatant of three species of the cellular slime molds (Dictyostelium discoideum, Dictyostelium rosarium, and Polysphondylium violaceum) was studied by gel filtration chromatography on AcA 34 Ultrogel. All these species have in common a cyclic GMP-binding protein of molecular weight of about 2.5 X 10(5) which specifically binds this nucleotide. In addition, Scatchard plots of assays carried out with the 48,000 X g supernatant of these species exhibit cyclic GMP-binding activity with an apparent dissociation constant of about 1 nM. None of the cyclic GMP-binding proteins separated by chromatography on AcA 34 Ultrogel was associated to protein kinase activity stimulation. In view of the cyclic GMP function during chemotactic transduction in the cellular slime molds, the possible molecular function for this 2.5 X 10(5)-dalton cyclic GMP-binding protein is discussed.     

Guanosine 3':5'-monophosphate-dependent protein kinase from silkworm, properties of a catalytic fragment obtained by limited proteolysis.

Although guanosine 3':5'-monophosphate (cyclic GMP)-dependent protein kinase (protein kinase G) which was partially purified from silkworm pupae was not dissociated by cyclic GMP into catalytic and regulatory subunits as described for adenosine 3':5'-monophosphate-dependent protein kinase (protein kinase A) (Takai, Y., Nakaya, S., Inoue, M., Kishimoto, A., Nishiyama, K., Yamamura, H., and Nishizuka, Y. (1976) J. Biol. Chem. 251, 1481-1487), limited proteolysis with trypsin resulted in the formation of catalytic and cyclic GMP-binding fragments which showed molecular weights of approximately 3.4 X 10(4) and 3.6 X 10(4), respectively (the molecular weight of native protein kinase G was 1.4 X 10(5)). The catalytic fragment did not bind cyclic GMP and was fully active in the absence of the cyclic nucleotide. The fragment did not show an absolute requirement for a sulfhydryl compound and high concentrations of Mg2+ (50 to 100 mM), both of which were necessary for the maximal activation of native protein kinase G. The catalytic fragment was not inhibited by the cyclic GMP-binding fragment nor by the regulatory subunit of protein kinase A. Inversely, the cyclic GMP-binding fragment was unable to inhibit the catalytic subunit of protein kinase A. Protein inhibitor, which was described for protein kinase A, was inert for the catalytic fragment.     

Effect of cyclic nucleotide analogs on intrachain site I of protein kinase isozymes

The effects of numerous cAMP analogs present in the [3H]cAMP binding reaction on subsequent dissociation of [3H]cAMP from the regulatory subunit of cAMP-dependent protein kinase I and II were analyzed. Certain analogs with modification at either C-8 or C-2 showed relative selectivity for one (site 1) of two intrachain cAMP binding sites of both isozymes. Modification at C-6 caused selectivity for the second site (site 2). The combination of a site-1-directed and site-2-directed analog inhibited [3H]cAMP binding much more than did either analog alone. In general, there was a correlation between the site 1 selectivity and the ability of the analog to stimulate the binding of [3H]cIMP, which selects site 2. The site-1-directed analogs stimulated the initial rate of [3H]cIMP binding. The stimulatory effect was enhanced in the presence of a polycationic protein such as histone and was inhibited by high ionic strength. The type I and II isozymes exhibited large differences in analog specificity for this effect. For type I, of the analogs tested the most efficacious for stimulating [3H]cIMP binding were those containing a nitrogen atom attached to C-8, 8-aminobutylamino-cAMP being the most effective. Type II responded best to analogs containing a sulfur atom attached to C-8, 8-SH-cAMP being the most effective of those tested. The stimulatory effect was accentuated in the presence of MgATP when using type I, but this nucleotide had no effect when using type II. It is proposed that in intact tissues cAMP binding to site 1 of either isozyme stimulates the binding to site 2.     

Site-directed mutagenesis of the cAMP-binding sites of the recombinant type I regulatory subunit of cAMP-dependent protein kinase

The type I regulatory subunit (R-I) of rat brain cAMP-dependent protein kinase was expressed in E. coli and site-directed mutagenesis was used to substitute amino acids in the putative cAMP-binding sites. The wild-type recombinant R-I bound 2 mol of cAMP/mol subunit, while two mutant R-Is with a single amino acid substitution in one of the two intrachain cAMP-binding sites (clone N153:a glutamate for Gly-200, and clone C254:an aspartate for Gly-324) bound 1 mol of cAMP/mol subunit. When these two substitutions were made in one mutant, cAMP did not bind to this mutant, indicating that binding of cAMP to N153 or C254 was to their nonmutated sites. Competition experiments with site-selective analogs and dissociation of bound cAMP from mutant R-Is provided evidence for strong intrachain interactions between the two classes of cAMP-binding sites in R-I.     

Phosphorylation of cGMP-dependent protein kinase increases the affinity for cyclic AMP

Cyclic-GMP-dependent protein kinase contains two binding sites for cGMP, which have different affinities for cGMP. Autophosphorylation of the enzyme affects mainly the binding of cGMP to the 'high'-affinity site (site 1). The enzyme binds cAMP and cAMP stimulates the phosphotransferase activity of the native enzyme half-maximally at 44 microM. Autophosphorylation of the enzyme decreases the apparent Ka value to 7 microM. Autophosphorylation does not affect the catalytic rate of the enzyme if measured at a saturating concentration of ATP. Tritiated cAMP apparently binds at 4 degrees C to one site with a Kd value of 3 microM. Binding to the second site is not measurable. Autophosphorylation of the enzyme increases the affinity of the high-affinity site for cAMP sixfold (Kd 0.46 microM) and allows the detection of a second site. In accordance with these data the dissociation rate of [3H]cAMP from the high-affinity site is decreased from 4.5 min-1 to 1.2 min-1 by autophosphorylation. Experiments in which unlabeled cAMP competes with [3H] cGMP for the two binding sites confirmed these results. Recalculation of the competition curves by a computer program for two binding sites indicated that autophosphorylation decreases the Kd value for binding of cAMP to the high-affinity site from 1.9 microM to 0.17 microM. Autophosphorylation does not affect significantly the affinity for the second site. Kd values for site 2 varied from 17 microM to 40 microM. These results suggest that autophosphorylation of cGMP-dependent protein kinase increases the affinity of the enzyme for cAMP by affecting mainly the properties of binding site 1.     

Molecular model of the cyclic GMP-binding domain of the cyclic GMP-gated ion channel.

The structure of the cyclic GMP-binding domain of the cyclic GMP-gated ion channel from bovine retinal rod photoreceptors has been modeled by analogy to the crystal structure of the homologous cyclic AMP-binding domain of catabolite gene activator protein (CAP). The modeled cyclic GMP-binding domain has a three-residue deletion and a five-residue insertion between beta strands compared to CAP. The major interactions of the ion channel with cyclic GMP are similar to those observed for cyclic AMP bound to CAP and predicted for cGMP bound to the cGMP-dependent protein kinase: Gly 543 and Glu 544 make hydrogen-bond interactions with the ribose 2'-OH, Arg 559 forms an ion pair with the charged phosphate oxygen, and Thr 560 forms hydrogen-bond interactions with an exocyclic phosphate oxygen and with the 2-amino group of cGMP. Three additional potential interactions were predicted from the model structure. Ile 545 O and Ser 546 OH form hydrogen-bond interactions with an exocyclic phosphate oxygen, and Phe 533 may interact with the aromatic ring of cGMP. This model is in agreement with both the analogue binding experiments and the mutational analysis of Thr 560.     

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.     

Characterization of the cyclic AMP-dependent protein kinase from rat pancreas, further purification of the catalytic subunit, substrate specificity, effect of the pancreatic heat stable inhibitor.

In order to investigate the sequence of events triggered by cyclic AMP and cyclic GMP in exocrine pancreatic cells, the identification of the various protein kinases possibly present in this tissue is of major interest. Further analysis of the two cyclic AMP-dependent protein kinases previously reported [11] suggests that KI is a degraded form of KII. It is therefore likely that a single holoenzyme is present in exocrine cells. In addition no protein kinase, specifically stimulated by cyclic GMP, has been detected in any fraction obtained in the course of purification of the cyclic AMP-dependent protein kinase. A faster and more efficient method than the one previously described [11] allows the purification (5000 times) of the protein kinase catalytic subunit. Analysis of the subunit by sodium dodecyl sulphate polyacrylamide gel electrophoresis indicates a molecular weight of 40 000 +/- 1 000. The enzyme phosphorylates specifically histone H2B (Vm = 236 min(-1), Km = 1.15 10(-5) M) and to a lesser extent H2A, H5 and H1 (Vm = 55--77 min(-1), Km 5--25 10(-5) M). Histones H3 and H4 are not phosphorylated. The effect of the heat stable inhibitor, extracted from rat pancreas, on the phosphorylation of H2B has been investigated. The inhibition is of the non competitive type with respect to ATP. The inhibition at various histone concentrations cannot be described by the Michaelis-Menten equation.     

Site-specific cyclic nucleotide binding and dissociation of the holoenzyme of cAMP-dependent protein kinase

The photoaffinity reagent 8-azidoadenosine 3':5'-monophosphate (8-N3cAMP) was previously shown to modify a single tyrosine residue on the type II regulatory subunit of cAMP-dependent protein kinase (Kerlavage, A.R., and Taylor, S.S. (1980) J. Biol. Chem, 255, 8483-8488). In the present studies, the binding stoichiometries of type II holoenzyme for cAMP and 8-N3cAMP were determined using Millipore filtration assays in the absence (Assay A) and presence (Assay B) of 2 M NaCl and histone. The binding stoichiometry of holoenzyme for cAMP was 2 mol/mol with Assay A, and 4 mol/mol with assay B. The binding stoichiometry for 8-N3cAMP was 2 mol/mol with Assay B or with Assay A following photolysis of the holoenzyme:8-N3cAMP mixture. In the absence of photolysis, the binding stoichiometry for 8-N3cAMP was 0.4 mol/mol with Assay A. Both 8-N3cAMP and cAMP fully dissociated the holoenzyme. Holoenzyme, labeled with 8-N3[3H]cAMP on a preparative scale, incorporated 1 mol of 8-N3[3H]cAMP/mol of regulatory subunit (RII) monomer. The labeled RII was separated from catalytic subunit, cleaved with cyanogen bromide, and the resultant peptides were separated by high performance liquid chromatography. A single radioactive peptide was observed which had the same NH2 terminal residue and amino acid composition as the peptide obtained when dissociated RII was labeled with 8-N3cAMP.     

Presence of the sites for interacting with cyclic AMP and with catalytic subunit on small fragments of protein kinase regulatory subunit.

During the purification of cyclic AMP binding proteins from rat liver, some smaller active fragments were obtained, possibly as the result of proteolysis. The binding proteins detected had approximate molecular weights of 50,000, 36,000, and 10,000. Each of these components bound cyclic [3H]AMP with high affinity (apparent dissociation constants ranging from 2 to 10 nM) and had a similar ability to inhibit the purified catalytic subunit of rat liver protein kinase. Cyclic AMP prevented this inhibition in each instance. These results suggest that the binding site for cyclic AMP and the site for interacting with catalytic subunit occur relatively close to one another on the regulatory subunit and can remain functional when a substantial fraction of the subunit is lost.     

The preparation of cyclic nucleotide-depleted regulatory subunits from type II adenosine 3':5'-monophosphate-dependent protein kinase by a nondenaturing method

A nondenaturing method for the preparation of R subunits from type II cyclic AMP-dependent protein kinase is described. The procedure is based on the exchange of cyclic AMP, which is tightly bound to the R subunit, for more weakly bound cyclic GMP, which can be removed by washing and dialysis. Less than 5% of the available cyclic nucleotide-binding sites of R subunit prepared by this method contained cyclic AMP and less than 3% contained cyclic GMP. The C-subunit contamination (mol of C/mol of R monomer) was approximately 0.2%. These levels of contamination did not affect the properties of the R subunit as judged by (a) the ability of the R subunit to inhibit the activity of the C subunit and (b) the rate of exchange of cAMP into R2 . etheno-cAMP. The advantages of our method are that the protein is not subjected to denaturing conditions and that large amounts of material can be processed relatively rapidly.     

Characterization of the interchain and intrachain interactions between the binding sites of the free regulatory moiety of protein kinase I

The interaction between the four binding sites (two A sites and two B sites) of the regulatory subunit dimer of protein kinase I (RI2) was studied. The rate of association of c[3H]AMP to site B was slower when site A had already been occupied. Occupation of site A also retarded the rate of dissociation of c[3H]AMP from site B. This site A-B interaction was intrachain since it was observed also for a monomeric fragment of RI2. Thus, each monomer of RI2 must have one A site and one B site. Quantitative analysis of the rate constants for cAMP binding to variously liganded RI2 suggested little or no thermodynamic coupling between site A and B. This conclusion was supported by equilibrium binding data. Occupation of one A site retarded the dissociation of c[3H]AMP from the A site of the other subunit (interchain interaction). The rate kinetic constants as well as equilibrium binding data indicated a positively cooperative site A-A interaction. The interaction between cAMP and either site was enthalphy-driven (25 degrees C), the process being accompanied by a loss of entropy. The thermodynamic parameters did not support the occurrence of an abrupt conformational change at a certain level of ligandation of RI2. Half-maximal saturation of either site occurred at 1-2 nM cAMP (37 degrees C, pH 7.0, 0.15 M KCl). The concentration of RI2 did not detectably influence any binding parameters. Aging of RI2 produced a form with minimally, if at all, altered Mr, but which showed a more rapid release of c[3H]AMP bound to site B.