Microheterogeneity of adenosine cyclic monophosphate-dependent protein kinases from mouse brain and heart.

1. DEAE-cellulose chromatography of mouse brain cytosol indicated the presence of only the type II isoenzyme of cyclic AMP-dependent protein kinase. Mouse heart cytosol contained approximately equal amounts of the type I and type II isoenzymes. 2. Both brain and heart type II isoenzymes reassociated after a transient exposure to cyclic AMP, but the heart type I isoenzyme remained dissociated. 3. Elution of brain cytosol continuously exposed to cyclic AMP resolved multiple peaks of protein kinase and cyclic AMP-binding activities. A single peak of kinase and multiple peaks of cyclic AMP-binding activities were found under the same conditions with heart cytosol. Various control experiments suggested that the heterogeneity within the brain type II isoenzymic class had not been caused by proteolysis. 4. Kinetic experiments with unfractionated brain cytosol showed that the binding of cyclic AMP, the dissociation of cyclic AMP from protein and the rate of heat denaturation of the cyclic AMP-binding activity gave results consistent with the presence of multiple binding species. 5. It concluded that the type II isoenzymic peak obtained by DEAE-cellulose chromatography of mouse brain cytosol represents a class of enzymes containing multiple regulatory and catalytic subunits. The two heart cytosol isoenzymes contain a common catalytic subunit. The degree of protein kinase 'microheterogeneity", defined as the presence of multiple regulatory and/or catalytic subunits within a single isoenzymic class, appears to be tissue-specific.     

Adenosine 3'':5''-cyclic monophosphate-dependence of protein kinase isoenzymes from mouse liver.

Conditions influencing the cyclic AMP-dependence of protein kinase (ATP-protein phosphotransferase, EC 2.7.1.37) during the phosphorylation of histone were studied. Protein kinase from mouse liver cytosol and the two isoenzymes [PK (protein kinase) I and PK II] isolated from the cytosol by DEAE-cellulose chromatography were tested. A relation between concentration of enzyme and cyclic AMP-dependence was observed for both isoenzymes. Moderate dilution of isoenzyme PK II decreased the stimulation of the enzyme by cyclic AMP. Isoenzyme PK I could be diluted 200 times more than isoenzyme PK II before the same decrease in cyclic AMP-dependence appeared. Long-term incubation with high concentrations of histone increased the activity in the absence of cyclic AMP relative to the activity in the presence of the nucleotide. This was more pronounced for isoenzyme PK II than for isoenzyme PK I. The cyclic AMP concentration needed to give half-maximal binding of the nucleotide was the same as the cyclic AMP concentration (Ka) at which the protein kinase had 50% of its maximal activity. The close correlation between binding and activation is also found in the presence of KCl, which increased the apparent activation constant (Ka) for cyclic AMP. With increasing [KCl], a progressively higher proportion of the histone phosphorylation observed in cytosol was due to cyclic AMP-independent (casein) kinases, leading to an overestimation of the degree of activation of the cyclic AMP-dependent protein kinases present. The relative contributions of cyclic AMP-dependent and -independent kinases to histone phosphorylation at different ionic strengths was determined by use of heat-stable inhibitor and phospho-cellulose chromatography.     

Properties of the cyclic AMP-dependent protein kinases in mouse mastocytoma cells

The properties of type I and type II protein kinases in PY815 mouse mastocytoma cells were shown to change following growth inhibition by prostaglandin E1 and 3-isobutyl-1-methylxanthine. These changes included a large reduction in type I protein kinase consistent with a role for this isoenzyme as a positive effector of growth, a decrease in free cyclic AMP binding protein and an increase in type II protein kinase. Some properties of the fully activated isoenzymes are presented that may be important in determining their activity in vivo.     

Low molecular weight cyclic AMP binding protein isolated from the extract of human tonsillar lymphocytes

A protein fraction of molecular weight 33,000-36,000 accounted for about 40% of the cyclic AMP binding capacity of the cytoplasmic extract of human tonsillar lymphocytes. This cyclic AMP binding fraction (designated as R' protein [10]) proved to be a proteolytic fragment of the regulatory subunit of the cyclic AMP-dependent protein kinase. The Scatchard plot of cyclic AMP binding by the isolated R' fraction indicated positive cooperativity. 50% saturation of the cyclic AMP binding sites was achieved at about 4 . 10(-9) M cyclic AMP. An upward concave curve was obtained in the Scatchard plot of cyclic GMP binding by the R' protein. These results strongly suggest that more than one molecule of cyclic nucleotide can be bound by one molecule of the R' protein. The R' protein could not be detected in the physiological salt extract of isolated nuclei in which type I cyclic AMP-dependent protein kinase was the dominating isoenzyme (according to the terminology used by Corbin, S.D., Keely, S.L. and Park, C.R. (1975) J. Biol. Chem. 250, 218-225). The cytoplasm of cells contained a higher amount of type II than type I regulatory subunit. In the cytoplasm the predominant part of RII was present in the dissociated state in all preparations, while when the RII was found in the nucleus it was mainly in the holoenzyme form. The R' protein presumably from the dissociated type II regulatory subunit.     

Epinephrine effects on cyclic AMP-dependent protein kinases from rat diaphragms

Diaphragm extracts were subjected to electrophoresis on polyacrylamide gels to separate the different molecular species of th cyclic AMP-dependent protein kinase. Using cyclic [3H]AMP, three peaks of binding activity were observed. The peak closest to the origin (peak I) was associated with cyclic AMP-dependent protein kinase activity and was abolished by incubation of the extracts with cyclic AMP prior to electrophoresis. The peak farthest from the origin (peak III) was devoid of kinase activity and was increased by incubation of extracts with cyclic AMP before electrophoresis; furthermore, when extracts were incubated with cyclic [3H]AMP before electrophoresis, essentially all the radioactivity appeared in peak III. Peak II, in an intermediate position, was also abolished by preincubation of the extracts with cyclic AMP and both its binding capacity and cyclic AMP-dependent protein kinase activity were lower than in Peak I. A peak of cyclic AMP-independent protein kinase (peak 0) that migrated more slowly than peak II was also detected. From these and other data it is concluded that peaks I and II are cyclic AMP-dependent protein kinase and that peak III is the dissociated regulatory subunit, respectively. Peak 0 is cyclic AMP-independent protein kinase together with free catalytic subunits from cyclic AMP-dependent protein kinase. Incubation of rat diaphragms with epinephrine resulted in dose- and time-dependent decrease in peak I and increase in peak III. These changes correlated with the decrease of cyclic AMP-dependent protein kinase associated with peak I. No changes in Peak II were observed with epinephrine, but an increased peak 0 was noted. Changes in peak I and peak III correlated with the modification of glycogen synthase and glycogen phosphorylase activities. No regulatory subunits (peak III) were detected as phosphorylated forms in diaphragms previously equilibrated with 32P. Treatment with epinephrine produce no noticeable phosphorylation of these regulatory subunits.     

Characteristics of selective activation of cyclic AMP-dependent protein kinase isoenzymes by calcitonin and prostaglandin E2 in human breast cancer cells.

The characteristics of the cyclic AMP-dependent protein kinase isoenzyme response to calcitonin stimulation have been studied in two human breast cancer cell lines, T47D and MCF 7. Both cell lines possess calcitonin receptors, a calcitonin-responsive adenylate cyclase and the two isoenzymes of the cyclic AMP-dependent protein kinase, types I and II. The adenylate cyclase also responds to prostaglandin E2. Acute activation of the cyclic AMP-dependent protein kinase isoenzymes was determined by using a modification of a multiple small anion exchange column method [Livesey, Kemp, Re, Partridge & Martin (1982) J. Biol. Chem. 257, 14983-14987]. Control experiments showed that post-extraction activation did not influence the data. Calcitonin caused a rapid, selective activation of isoenzyme II in the T 47D cells with half-maximal response at 10(-10)M, and persisting for at least 24h. In MCF 7 cells calcitonin also caused a highly selective activation of isoenzyme II with half-maximal response at 5 X 10(-11) M, but the response was transient with a return to basal isoenzyme activity by 4-6 h. At this time further addition of calcitonin did not restimulate the cyclic AMP-dependent kinase activity. In neither cell line did calcitonin treatment result in activation of isoenzyme I. Prostaglandin E2, on the other hand, the only significant alternative agonist of adenylate cyclase in T 47D cells, activated isoenzymes I and II to an equal extent in these cells, illustrating that two hormones activating adenylate cyclase in the one cell type might exert different effects by their selective actions upon protein kinase isoenzymes.     

Properties of soluble cyclic AMP-dependent protein kinase activity of renal inner medulla

Studies of the chromatographic distribution of soluble protein kinase in rat kidney demonstrated that the type I isoenzyme predominates in cortex, whereas activity in outer and inner medulla is almost exclusively the type II form. The type II isoenzyme also predominates (95% or greater) in human, canine, bovine, porcine and rabbit inner medulla. Compared to soluble type I activities from rat renal cortex or medulla, type II activity of inner medulla demonstrates a marked resistance to activation by NaCl and/or urea in subcellular preparations. However, with respect to solute activation, the resistance of the type II enzyme of inner medulla does not differ from that of type II activities from other tissues. In contrast to the effects on basal activity, NaCl and urea potentiated inner medullary type II activation by cyclic AMP and also delayed the rate of subunit reassociation after chromatographic removal of cyclic AMP. Incubation of inner medullary slices in high osmolality buffer (NaCl and urea) did not alone activate soluble protein kinase, an observation which implied that the enzyme was also resistant to solute activation in the intact cell system. Moreover, at 1650 mosM, vasopressin activation of soluble protein kinase was enhanced compared to responses at 750 mosM despite comparabel levels of cyclic AMP accumulation at the two osmolalities. However, a cyclic AMP-independent action of high osmolality to reduce the rate of inactivation of arginine vasopressin-stimulated protein kinase was not demonstrable in inner medullary slices.The present data suggest the possibility that the resistance of inner medullary protein kinase to solute activation could be related to the isomeric form of enzyme (type II) present in this tissue. The high concentrations of NaCl and urea routinely found in inner medulla during hydropenia also influenced protein kinase responses to arginine vasopressin, and may do so in part by directly potentiating the action of cyclic AMP on subunit dissociation.     

Myosin isoenzymes in single muscle fibres of Xenopus laevis: analysis of five different functional types

An analysis has been performed of the native myosin isoenzyme composition of isolated skeletal muscle fibres from Xenopus laevis with well-defined isotonic contraction properties. Fast twitch 'white' (type 1) fibres contained three isomyosins; fast twitch 'red' (type 2) fibres showed two major myosin bands with migration velocities very similar to those of the two slower bands in type 1. Slow twitch (type 3) fibres yielded a single, slowly migrating band as did slow tonic (type 5) fibres, whereas the myosin from type 4 (very slow twitch, 'intermediate') fibres migrated with a somewhat higher mobility. The results suggest that amphibian skeletal muscle may possess the principal fibre types found in mammals and birds.     

Species-dependent isoenzyme subtypes of membrane-bound cyclic AMP-dependent protein kinase in highly purified cardiac sarcolemma.

Highly purified sarcolemma from dog and pig cardiac muscle has been shown to contain significant activities of a membrane-bound cyclic AMP-dependent protein kinase. In addition, these membranes undergo endogenous phosphorylation when incubated with Mg2+ and [gamma-32P]ATP. By comparing 32P-labelled patterns obtained with [gamma-32P]ATP and the photoaffinity label 8-azidoadenosine 3':5'-[32P]monophosphate (8-azido-cyclic [32P]AMP), we have demonstrated that, whereas the major kinase isoenzyme in dog sarcolemma was Type II, that in the pig membrane was the Type I isoenzyme.     

Studies on the role of adenosine 3':5'-monophosphate in the activation of liver phosphorylase.

Crude extracts of rabbit liver, preincubated to promote the dephosphorylation of enzymes or other regulatory proteins, were used to study the role of cyclic AMP in the activation of glycogen phosphorylase. Inasmuch as endogenous liver phosphorylase was irreversibly altered by the preincubation procedure, crystalline skeletal muscle phosphorylase was used as the substrate in these studies. In the presence of magnesium ions and ATP, phosphorylase b was converted to phosphorylase a, and in an apparent biphasic process the phosphorylase a formed was subsequently converted to phosphorylase b. In the presence of adenosine 3':5'-monophosphate the rate of phosphorylase a formation and the maximal amount of phosphorylase a formed were increased. The cyclic AMP effect was enhanced by glucose-6-P and required the presence of glycogen. The catalytic subunit of cyclic AMP-dependent protein kinase could replace cyclic AMP in the stimulation of phosphorylase a formation. The effects of cyclic AMP or the catalytic subunit were shown to be due to stimulation of phosphorylase kinase rather than to inhibition of phosphorylase phosphatase. Preliminary fractionation experiments showed that it is possible to separate phosphorylase kinase catalytic activity from a factor or factors required for stimulation of its activation by the catalytic subunit.     

Promoter-like mutants with increased expression of the Escherichia coli uridine phosphorylase structural gene.

From an Escherichia coli K-12 strain lacking adenylate cyclase (cya) and cyclic AMP receptor protein (crp), two mutants were isolated that synthesize uridine phosphorylase constitutively. The mutations differ from one another and also from a wild type in the maximum rate of uridine phosphorylase synthesis. They have constitutive expression of the uridine phosphorylase gene (udp) in the presence of repressor protein coded by the cytR regulatory gene and decrease the sensitivity of the udp gene simultaneously with catabolite repression. Both mutations cause a high level of udp expression whether they are in a cya crp or in a cya+ crp+ background. Another mutation (udpP1) isolated previously alters the response of udp gene to the ctyR repressor and produces a higher constitutive level of uridine phosphorylase in a cytR+ than in a cytR background when bacteria are grown in glucose. The synthesis of uridine phosphorylase in this mutant is dependent on an intact cyclic AMP-cyclic AMP receptor protein complex. All mutations studied are cis-acting and extremely closely linked to the udp structural gene, and appear to affect the uridine phosphorylase promoter-operator region. The data obtained are in accordance with a suggestion that the cytR repressor protein normally asserts its function by preventing the positive action of cyclic AMP-cyclic AMP receptor protein complex.     

Location and properties of two isoenzymes of cardiac adenylate kinase.

Adenylate kinase catalyses the equilibrium 2ADP = ATP + AMP. There are two isoenzymes of adenylate kinase in bovine ventricular tissue, one cytoplasmic, the other mitochondrial. Mitochondrial subfractionation locates this isoenzyme between the mitochondrial membranes with fatty acid-CoA ligase. The cytoplasmic and mitochondrial isoenzymes are distributed in ratio 3:2, and both forms were purified to homogeneity. They differ principally by charge, Km values for ATP, ADP and AMP, pH-stability and -activity profiles, and susceptibility to the inhibitor adenosine pentaphosphoadenosine. The forward and reverse reactions show similar energies of activation for the cytoplasmic enzyme, but differ for the mitochondrial enzyme. The molecular weights are indistinguishable. An integrated mechanism is formulated whereby one isoenzyme suppresses the activation of fatty acid and the other enhances carbohydrate utilization in hypoxic myocytes.     

Beta-adrenergic receptor subtypes and subcellular compartmentation of cyclic AMP and cyclic AMP-dependent protein kinase in rabbit cardiomyocytes

In purified ventricular myocytes from adult rabbit, beta-adrenergic stimulation causes cyclic AMP accumulation and cyclic AMP-protein kinase activation in both particulate and soluble fractions of the cell, whereas prostaglandin E1 elevates cyclic AMP and cyclic AMP-protein kinase activity in the soluble fraction exclusively. Only activation of particulate cyclic AMP-protein kinase activity results in phosphorylase b----a conversion. Using radioligand binding technics, we have determined whether beta 1- and beta 2-receptor subtypes mediate beta-adrenergic effects in particulate and soluble subcellular compartments, respectively. The non-selective antagonist [125I]iodocyanopindolol binds to intact ventricular myocytes with KD of 25 pM and a Bmax of 2.6 X 10(5) receptors/myocyte. Competition for [125I]iodocyanopindolol binding to intact myocytes by the beta-receptor subtype-specific antagonists practolol (beta 1) and zinterol (beta 2) results in monophasic curves with antagonist KD values of 1 microM and 1.5 microM, respectively. We conclude that adult rabbit cardiac myocytes do not possess detectable beta 2 receptors. Further, the ability of isoproterenol to cause elevation of cyclic AMP in two functionally distinct regions within the myocyte must pertain to the actions of a single subtype of beta-receptor, the beta 1-receptor.     

Differential sensitivity of neural and non-neural protein kinase isozymes to cyclic AMP

Cyclic AMP-dependent protein kinase, which plays a major role in metabolic and genetic regulation, consists of two classes of isozymes denoted as type I and type II. The type II isozyme, moreover, consists of two subclasses denoted as neural and non-neural based upon immunochemical differences between the enzyme isolated from bovine brain and heart, respectively. Whereas the catalytic (C) subunits of these three isozymes are quite similar, all three isozymes differ with respect to their regulatory (R) subunits. In the present report, we have compared the sensitivities to cyclic AMP of the type I and type II isozymes in several tissues from a single species (rat). The sensitivities of the three isozymes to cyclic AMP were type I much greater than non-neural type II greater than neural type II. We suggest that the differences in sensitivity to cyclic AMP of isozymes present in the same cell provides the cell with a dynamic range of responses to the widely varying alterations in cellular cyclic AMP levels produced by regulatory first messengers.     

Comparison of cyclic nucleotide specificity of guanosine 3',5'-monophosphate-dependent protein kinase and adenosine 3',5'-monophosphate-dependent protein kinase.

Guanosine 3',5'-monophosphate-dependent protein kinase (cyclic GMP-dependent protein kinase) and adenosine 3',5'-monophosphate-dependent protein kinase (cyclic AMP-dependent protein kinase) exhibited a high degree of cyclic nucleotide specificity when hormone-sensitive triacylglycerol lipase, phosphorylase kinase, and cardiac troponin were used as substrates. The concentration of cyclic GMP required to activate half-maximally cyclic dependent protein kinase was 1000- to 100-fold less than that of cyclic AMP with these substrates. The opposite was true with cyclic AMP-dependent protein kinase where 1000- to 100-fold less cyclic AMP than cyclic GMP was required for half-maximal enzyme activation. This contrasts with the lower degree of cyclic nucleotide specificity of cyclic GMP-dependent protein kinase of 25-fold when histone H2b was used as a substrate for phosphorylation. Cyclic IMP resembled cyclic AMP in effectiveness in stimulating cyclic GMP-dependent protein kinase but was intermediate between cyclic AMP and cyclic GMP in stimulating cyclic AMP-dependent protein kinase. The effect of cyclic IMP on cyclic GMP-dependent protein kinase was confirmed in studies of autophosphorylation of cyclic GMP-dependent protein kinase where both cyclic AMP and cyclic IMP enhanced autophosphorylation. The high degree of cyclic nucleotide specificity observed suggests that cyclic AMP and cyclic GMP activate only their specific kinase and that crossover to the opposite kinase is unlikely to occur at reported cellular concentrations of cyclic nucleotides.     

Energy metabolism of fast- and slow-twitch skeletal muscle in the rat: Thyroid hormone induced changes

Summary Male Wistar rats were made hypothyroid or hyperthyroid over a period of six weeks, by administration of carbimazole or triiodothyronine (T₃). Serial frozen sections of soleus and extensor digitorum longus (EDL) muscle were stained histochemically for myosin ATPase, succinic dehydrogenase and phosphorylase. Muscle fibres were classified as either slow twitch oxidative (SO), fast twitch oxidative glycolytic (FOG) or fast twitch glycolytic (FG). In addition the activities of phosphorylase, phosphofructokinase (PFK), fructose-1,6-diphosphatase (FDP), lactate dehydrogenase (LDH), hexokinase, citrate synthetase, cytochrome oxidase, 3-hydroxyacyl-CoA dehydrogenase (HAD) and 5-AMP aminohydrolase were measured in both muscles.Increasing plasma levels of T₃ are associated with marked alterations in the fibre type populations in both muscles. In the soleus there is conversion of SO to FOG fibres while in the EDL, FG fibres are converted to FOG fibres. The quantitative changes in metabolic enzyme activity however, are in the main restricted to the soleus. Increased T₃ levels result in an increased capacity for the aerobic metabolism of both fat and carbohydrate and an increase in anaerobic glycolytic activity in the soleus muscle which parallels the change in fibre types. However, the extent of these increases cannot be explained solely on this basis and there is also an overall increase in aerobic activity in all fibres including slow oxidative ones. It is concluded that the effects of thyroid hormone on muscle phenotype and respiratory capacity involve both primary and secondary sites of action and the possible mechanisms are discussed.Abbreviations EDL extensor digitorum longus - FDP fructose-1,6-diphosphatase - FG fast twitch glycolytic - FOG fast twitch oxidative glycolytic - HAD 3-hydroxyacyl-CoA-dehydrogenase - LDH lactate dehydrogenase - PFK phosphofructokinase - SO slow twitch oxidative - T ₃ triiodothyronine - T ₄ thyroxine     

Comparison of cyclic nucleotide specificity of guanosine 3′,5′-monophosphate-dependent protein kinase and adenosine 3′,5′-monophosphate-dependent protein kinase

Guanosine 3′,5′-monophosphate-dependent protein kinase (cyclic GMP-dependent protein kinase) and adenosine 3′,5′-monophosphate-dependent protein kinase (cyclic AMP-dependent protein kinase) exhibited a high degree of cyclic nucleotide specificity when hormone-sensitive triacylglycerol lipase, phosphorylase kinase, and cardiac troponin were used as substrates. The concentration of cyclic GMP required to activate half-maximally cyclic dependent protein kinase was 1000- to 100-folds less than that of cylic AMP with these substrates. The opposite was true with cyclic AMP-dependent protein kinase where 1000- to 100-fold less cyclic GMP was required for half-maximal enzyme activation. This contrasts with the lower degree of cyclic nucleotide specificity of cyclic GMP-dependent protein kinase of 25-fold when histone H2b was used as a substrate for phosphorylation. Cyclic IMP resembled cyclic AMP in effectiveness in stimulating cyclic GMP-dependent protein kinase but was intermediate between cyclic AMP and cyclic GMP in stimulating cyclic. AMP-dependent protein kinase. The effect of cyclic IMP on cyclic GMP-dependent protein kinase was confirmed in studies of autophosphorylation of cyclic GMP-dependent protein kinase where both cyclic AMP and cyclic IMP enhanced autophophorylation. The high degree of cyclic nucleotide specificity observed suggests that cyclic AMP and cyclic GMP activate only their specific kinase and that crossover to the opposite kinase is unlikely to occur at reported cellular concentrations of cyclic nucleotides.     

Thiophosphate-activated phosphorylase kinase as a probe in the regulation of phosphorylase phosphatase.

Rabbit muscle nonactivated phosphorylase kinase (EC 2.7.1.38) is converted to thiophosphate-activated phosphorylase kinase by cyclic AMP dependent protein kinase, Mg2+ and ATP-gamma-S/adenosine-5'-O-(s-thiotriphosphate)/. The formation of thiophosphate-activated phosphorylase kinase wal also observed in the protein-glycogen complex from skeletal muscle. This new form of kinase is resistant to the action of phosphatase and behaves as a competitive inhibitor in the dephosphorylation of phosphorylase alpha by phosphorylase phosphatase (Ki = 0.04 mg per ml). The fact that the inhibitory effect of thiophosphate-activated phosphorylase kinase is 3 times higher than in the case of nonactivated kinase, may explain the transient inhibition of phosphorylase phosphatase in the protein-glycogen complex. The use of activated (phosphorylated) phosphorylase kinase supports this assumption since it causes a delay in the dephosphorylation of phosphorylase alpha, i.e. the conversion of phosphorylase alpha into beta could start only after the dephosphorylation of activated phosphorylase kinase.     

Phosphorylation of the inhibitory subunit of troponin in perfused hearts of mice deficient in phosphorylase kinase. Evidence for the phosphorylation of troponin by adenosine 3'':5''-phosphate-dependent protein kinase in vivo.

When hearts from control and phosphorylase kinase-deficient (I strain) mice were perfused with 0.1 micrometer-DL-isoprenaline, there was a parallel increase in contraction, cyclic AMP concentration and troponin I phosphorylation. However, there was no increase in phosphorylase a in the I-strain hearts, whereas the control hearts showed a large increase. Assays of I-strain heart extracts showed a normal cyclic AMP-dependent protein kinase activity but no phosphorylase kinase activity. It is concluded that troponin I is phosphorylated in intact hearts by protein kinase and not phosphorylase kinase.     

Binding proteins for adenosine 3′:5′-cyclic monophosphate in bovine adrenal cortex

Five peaks of cyclic AMP-binding activity could be resolved by DEAE-cellulose chromatography of bovine adrenal-cortex cytosol. Two of the binding peaks co-chromatographed with the catalytic activities of cyclic AMP-dependent protein kinases (ATP-protein phosphotransferase, EC 2.7.1.37) of type I or type II respectively. A third binding protein was eluted between the two kinases, and appeared to be the free regulatory moiety of protein kinase I. Two of the binding proteins for cyclic AMP, sedimenting at 9S in sucrose gradients, could also bind adenosine. They bound cyclic AMP with an apparent equilibrium dissociation constant (K(d)) of about 0.1mum, and showed an increased binding capacity for cyclic AMP after preincubation in the presence of K(+), Mg(2+) and ATP. The two binding proteins differed in their apparent affinities for adenosine. The isolated regulatory moiety of protein kinase I had a very high affinity for cyclic AMP (K(d)<0.1nm). At low ionic strength or in the presence of MgATP, the high-affinity binding of cyclic AMP to the regulatory subunit of protein kinase I was decreased by the catalytic subunit. At high ionic strength and in the absence of MgATP the high-affinity binding to the regulatory subunit was not affected by the presence of catalytic subunit. Under all experimental conditions tested, dissociation of protein kinase I was accompanied by an increased affinity for cyclic AMP. To gain some insight into the mechanism by which cyclic AMP activates protein kinase, the interaction between basic proteins, salt and the cyclic nucleotide in activating the kinase was studied.