Cyclic AMP Binding Capacity Research Articles

Effects of glucagon and insulin on the cyclic AMP binding capacity of hepatocyte cyclic AMP-dependent protein kinase.

Extracts obtained from rat hepatocytes incubated with saline, glucagon or insulin were electrophoresed on polyacrylamide gels and then assayed for cyclic (3H)AMP binding capacity. Analysis of the binding patterns demonstrated that glucagon dissociated a holoenzyme of cyclic AMP-dependent protein kinase in a dose-dependent manner. The increase in free regulatory subunits and, hence, in free catalytic subunits explains the activation of this enzyme by glucagon in the liver. Insulin decreased both the amount of cyclic (3H)AMP bound to the holoenzyme and the capacity of the enzyme to be dissociated when the extracts were incubated with increasing concentrations of this cyclic nucleotide. We propose that these insulin-induced effects are determined by an inhibition of the cyclic AMP binding capacity of this protein kinase. This mechanism could account for the inactivation of cyclic AMP-dependent protein kinase that insulin causes in the liver.

Protein kinases in human leukemic cells.

Protein kinase activities and cyclic AMP binding capacity were investigated in human peripheral blood cells from leukemic patients and normal controls. Using [gamma 32P] ATP as phosphoryldonor, the phosphorylating activities were not found to be significantly different in either normal or leukemic cells when measured on both artificial basic and acidic substrates. In contrast, the GTP-dependent casein kinase activity, CK2, which is almost undetectable in normal granulocytes, was markedly increased in highly proliferating myeloblastic cells from patients with acute myelogenous leukemia (AML) or with chronic myelogenous leukemia in blastic crisis (BC-CML). Levels of endogenous phosphotyrosine were not higher in leukemic cells than in normal peripheral lymphocytes or granulocytes. Finally, cAMP binding capacity was found to be increased in several types of proliferating leukemic cells, due to a higher amount of the R1-type regulatory subunit of the cAMP-dependent protein kinases. Specific patterns of cAMP binding proteins observed in the different types of normal blood cells were rather blurred in leukemic cells. In conclusion, modifications observed in human leukemic cells seem to be more related to proliferation or blockage in normal differentiation than to their cellular origin.

Relationship of cyclic AMP binding capacity and estrogen receptor to hormone sensitivity in human breast cancer.

Estrogen receptor (ER) and total binding capacity for cyclic AMP (cAR) were measured in cytosols from human mammary tumors. Patients with advanced, evaluable breast cancer were biopsied before the start of endocrine treatment, and ER and cAR measurements were performed. All patients included in this study were ER positive. Sixteen of 30 patients (53%) had an objective response to endocrine treatment. When ER and cAR were expressed as a ratio and this ratio was related to treatment response, it was found that all objective responders had ratio values above 2.5 X 10(-3). Nine of 14 nonresponders had ER/cAR ratios below this value. In our limited series of patients a threshold limit of 2.5 X 10(-3) would have correctly predicted the response to endocrine treatment in 25 of 30 patients (83%). In patients with ER values below 100 fmol/mg protein and PgR negative or unknown, cAR might strengthen the predictive value of steroid receptor measurements.

Functional Pool of Cyclic Adenosine 3',5'-Monophosphate in Rabbit Platelets

Accumulation of the newly formed 14C-cyclic adenosine 3',5'-monophosphate (cyclic AMP) was found in the P1 (1.0) fraction, i.e. a platelet plasma membrane fraction which was obtained from 14C-adenine-labeled platelets. On the other hand, total cyclic AMP as determined simultaneously was located mainly in the platelet soluble fraction. Furthermore, the highest value of the cyclic AMP-binding capacity was found in the P1 (1.0) fraction. The cyclic AMP-binding activity of platelet membranes was attributed to two proteins with molecular weights of approximately 48,000 and 68,000. The treatment of 14C-adenine-prelabeled platelets with thrombin (1 unit per ml) led to about 40% decrease in the newly formed 14C-cyclic AMP level and 18% reduction of 14C-adenosine triphosphate level in whole platelets within 10 sec. On the other hand, the 14C-cyclic AMP level in the P1 fraction decreased by about 80% of the control value while the total cyclic AMP in this fraction was almost unchanged. This rapid and striking fall in the membrane 14C-cyclic AMP level could be correlated with the more than 2fold stimulation of the membrane-bound cyclic AMP phosphodiesterase, together with the more than 20% inhibition of both the cyclic AMP-binding capacity and the adenyl cyclase in platelet membranes by thrombin treatment. These observations suggest the possibility that functional pool of cyclic AMP related to thrombin-induced aggregation is located in rabbit platelet plasma membrane.

Cyclic AMP-binding and cyclic AMP-dependent protein kinase activities in the cytosol of differentiating bone marrow erythroblasts.

Cytosolic cyclic AMP-binding capacity and cyclic AMP-dependent protein kinase activity have been studied in relation to differentiation and maturation of rabbit bone marrow erythroblasts. Using cells fractionated by velocity sedimentation at unit gravity, it was found that both activities decreased in dividing cells when calculated in terms of cell number but remained constant per cell volume. After the final cell division, cyclic AMP-dependent protein kinase activity did not change further, whereas cyclic AMP-binding capacity declined. There were no qualitative, but only quantitative, changes in the cyclic AMP-binding proteins that are present in the cytosol of developing erythroblasts. In the immature cells, the apparent KD for the interaction of binding proteins with cyclic AMP was 4 X 10(-8) M. The data suggest that changes in cyclic AMP-binding activity during differentiation of erythroid cells are due both to changes in the amount of binding proteins and in their affinity for cyclic AMP. Plasma membranes of erythroblasts were also able to bind cyclic AMP but only in dividing cells.

Adenosine 3′: 5′ Monophosphate Receptor Sites in Xenopus laevis Oocytes

Adenosine 3':5' cyclic monophosphate (cyclic AMP) binds to proteins present in the 100,000 g supernatant fractions of full-grown oocytes of Xenopus laevis. Optimal pH for the binding is 4.0. Two receptor binding sites have been characterized by density gradient centrifugation as peaks with sedimentation constants of 4.6 S and 5.9 S. The apparent dissociation constants for the two cyclic AMP binding sites are 7 nM and 40 nM. The total cyclic AMP binding capacity of oocyte cytosol is 15.8 +/- 2.2 fmoles/oocyte and remains constant during meiotic maturation induced by progesterone.

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, 218–255). The cytoplasm of cells contained a higher amount of type II than type I regulatory subunit. In the cytoplasm the predominant part of R II was present in the dissociated state in all preparations, while when the R II was found in the nucleus it was mainly in the holoenzyme form. The R′ protein derived presumably from the dissociated type II regulatory subunit.

Effect of vasopressin on cyclic AMP-dependent protein kinase in toad urinary bladder

The effect of vasopressin on the toad urinary bladder has been shown to be mediated by cyclic AMP. It has been assumed that, as demonstrated for other systems, this involves activation of cyclic AMP-dependent protein kinase. In order to test this hypothesis we investigated the effect of vasopressin on cyclic AMP-dependent protein kinases in epithelial cells of toad bladders. About 80% of protein kinase activity and cyclic AMP-binding capacity was found to be in the cytosol. DEAE-cellulose chromatography showed a pattern of 15–20% type I and 80–85% type II cyclic AMP-dependent protein kinase. Cytosolic kinase was activated 3–4-fold by cyclic AMP with half-maximal activation at 5 · 10 − M. Similarly, half-maximal binding of cyclic AMP occurred at 7 · 10 −8 M. Incubation of toad bladders in Ringer's solution containing 0.1 mM 3-isobutyl-1-methylxanthine, prior to homogenization assay, showed stable cyclic AMP-binding capacity and protein kinase ratio —cylic AMP/+cyclic AMP. Exposure of bladders to 10 mU/ml of vasopressin for 10 min caused intracellular activation of protein kinase and decrease in cyclic AMP-binding capacity that were maintained for at least 30 min. Incubation of bladders with increasing concentrations of vasopressin (0.5–100 mU/ml) resulted in a discrepancy between a progressive increase in cyclic AMP levels and levelling off at 10 mU/ml of vasopressin for the changes in protein kinase ratio and cyclic AMP-binding capacity. The increase in kinase ratio was due to higher activity in the absence of exogenous cyclic AMP and was fully inhibitable by a specific protein kinase inhibitor. Using Sephadex G-25-CM50 column chromatography for separation of holonzyme and free catalytic subunit we demonstrated that the activation of protein kinase in the vasopressin-treated bladders is due to intracellular dissociation of the kinase. These results show that the effect of vasopressin on the toad bladder involves of a cytosolic cyclic AMP-dependent protein kinase. The time course and the dose-response curve of the kinase activation closely parallel vasopressin's effect on osmotic water flow.

Changing pattern of cyclic AMP-binding proteins during germination of Mucor racemosus sporangiospores

Interation of cyclic AMP with a profoundly changing pattern of specific binding proteins was shown during aerobic germination of sporangiospores from the fungus Mucor racemosus. 32P-labeled 8-azido-cycli AMP, an analogue of cyclic AMP that forms a covalent linkage with the binding proteins under u.v. light, was used as the ligand. Binding proteins carrying this photoaffinity label were separated by polyacrylamide-gel electrophoresis and identified by radioautography. Equibiltrium dissociation constants (Kd) and binding-response curves in the presence of competing nucleotides were identical for both 8-azido-cyclic [32P]AMP and cyclic [3H]AMP. A quantitative binding assay with both 8-azido-cyclic [32P]AMP and cyclic [3H]AMP over the time course of sporangiospore germination indicated a parallel relationship between cyclic AMP-binding capacity and the intracellular concentrations of cyclic AMP reported in a previous study [Paznokas & Sypherd (1975) J. Bacteriol. 124, 134--139]. Both of these parameters attained transient high values at a time of development when addition of exogenous cyclic AMP prevents hyphal-germ-tube emergence. The measured Kd values did not change during sport germination.

Distribution and properties of protein kinase and protein phosphatase activities in synaptosomal plasma membranes and synaptic junctions

Some characteristics of the protein kinase activity associated with a synaptosomal plasma membrane (synaptic membrane) fraction and a synaptic junction fraction have been compared. Autoradiography of the phosphorylated fractions separated on sodium dodecyl sulfate polyacrylamine gels showed that cyclic AMP stimulates the phosphorylation of five polypeptides in synaptic membranes, whereas no cyclic AMP dependency could be detected in synaptic junctions. Kinetic studies demonstrated that synaptic junctions contain at high K m and a low K m protein kinase activity while only the high K m activity could be detected in synaptic membranes. The intrinsic ATPase activity of synaptic membranes was shown to strongly interfere with measurements of protein kinase activity. Cyclic AMP binding experiments revealed a 2.6-fold enrichment of cyclic AMP binding capacity in synaptic junctions as compared to synaptic membranes. Protein phosphatase activity was not detected in synaptic junctions but was associated with synaptic membranes, where cyclic AMP was shown to either stimulate or inhibit the dephosphorylation of different polypeptides.

Adenosine 3′:5′-cyclic monophosphate-dependent protein kinase(s) of rat ovarian cells. Gonadotropin regulation of adenosine 3′:5′-cyclic monophosphate-receptor activity

Regulation of cyclic AMP-dependent protein kinase, cyclic AMP-receptor activity and intracellular cyclic AMP concentrations by choriogonadotropin was studied in ovarian cells prepared from 26-day-old rats. A close correlation was observed between phospho-transferase activity and cyclic AMP-receptor activity in 12000g supernatant fractions from rat ovarian homogenate. The apparent activation constant (K(a)) and I(50) (concentration required to produce 50% inhibition) of different cyclic nucleotides for phosphotransferase and cyclic AMP receptor activities respectively were also determined. Cyclic AMP and 8-bromo cyclic AMP were most effective, giving K(a) values of 0.08 and 0.09mum and I(50) of 0.12 and 0.16mum respectively. Other nucleotides were also effective, but required higher concentrations to give a comparable effect. An increased concentration of cyclic AMP produced by choriogonadotropin (1mug/ml) treatment was accompanied by decreased cyclic AMP binding as early as 5min after hormone addition. Choriogonadotropin also stimulated the protein kinase activity ratio (-cyclic AMP/+cyclic AMP) under identical experimental conditions. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine potentiated the action of choriogonadotropin on the three parameters measured in a dose- and time-dependent manner. The maximal cyclic AMP-binding capacity, as determined by cyclic AMP-exchange assay, remained unchanged before and after hormone addition. The endogenously bound cyclic AMP was determined from the difference between the maximal binding capacity and the exogenously bound cyclic AMP. With different choriogonadotropin concentrations, a quantitative correlation was established between maximal binding capacity, exogenous binding and endogenous binding activities. Approx. 60% of total binding sites were endogenously occupied in untreated cells, and choriogonadotropin (1mug/ml) treatment fully saturated available binding sites with a parallel 10-fold increase in cellular cyclic AMP. The present results provide evidence for a probable intracellular compartmentalization of cyclic AMP in the ovarian cell, and suggest that in the unstimulated state all cyclic AMP present in the ovarian cell may not be available for protein kinase activation.

An Adenosine 3′:5′‐Monophosphate/Adenosine Binding Protein from Mouse Liver

Synthetic and naturally occurring adenosine derivatives were tested for their ability to inhibit the binding of cyclic [3H]AMP and [3H]adenosine to the activated cyclic AMP/adenosine binding protein from mouse liver [Ueland, P. M. and Døskeland, S. O. (1977) J. Biol. Chem. 252, 677–689]. The structural requirements for inhibition of the binding of the two ligands were different, as demonstrated by the existence of adenosine derivatives that inhibited the binding of [3H]adenosine but not the binding of cyclic [3H]AMP and vice versa. The interaction of the binding protein with AMP, ADP and adenine was investigated in more detail. The binding of radioactive AMP and ADP to the protein was determined under equilibrium conditions. Competition studies indicate that AMP binds to the cyclic AMP binding site with an apparent dissociation constant of 2 × 10−6 M and to a low‐affinity site. The double‐reciprocal plot for the binding of ADP deviated slightly from linearity suggesting the possibility of heterogeneity also for the ADP binding sites. Competition studies indicate that this nucleotide binds mainly to the cyclic AMP binding site.Cyclic AMP has previously been shown to activate its own sites under certain conditions (30°C, in the presence of KCl and Mg2+). This phenomenon has been termed homologous activation of binding sites [Ueland, P. M. and Døskeland, S. O. (1978) J. Biol. Chem., in the press]. Similar results were obtained with AMP and ADP.Displacement of [3H]adenosine binding from the sites specific for this adenine derivative was observed with unlabelled adenosine, adenine, AMP and ADP with decreasing efficiency in the order mentioned. This indicates that adenine, AMP and possibly ADP interact with the adenosine binding sites. Adenine and AMP inhibited the activation of the cyclic AMP binding capacity by ATP as previously shown for adenosine [Ueland, P. M. and Døskeland, S. O. (1978) Arch. Biochem. Biophys. 185, 195–203]. The inhibitory power closely parallelled the displacement of [3H]adenosine binding. Adenine was also demonstrated to inhibit the homologous activation of the cyclic AMP binding site.

Some properties of the cyclic AMP dependent protein kinase of epithelial cells and cortical fibers of bovine eye lens

Cyclic AMP dependent protein kinase activity was demonstrated in extracts of epithelial cells and cortical fibers of bovine eye lens. The histone fractions can be phosphorylated by these enzymes. Highest phosphorylation was obtained with f2b and f1 histone fractions. The K m values of the epithelial and cortical enzymes were very similar with all histone fractions. Protein kinase in the two extracts had a similar affinity to ATP and showed a pH optimum at 6·0–6·2. The enzymes were activated by cyclic AMP and the concentration of cyclic AMP necessary to half maximal activation was similar for both enzymes: 5×10 −8 m for the epithelial enzyme and 9×10 −8 m for the cortical enzyme. The magnitude of activation, however, was very different. The activity of the epithelial enzyme was increased 5–8-fold, while that of the cortical enzyme up to 25-fold. The high cyclic AMP sensitivity of the cortical enzyme was accompanied by a relatively high cyclic AMP binding capacity.

Factors affecting the binding of [3H]adenosine 3′:5′-cyclic monophosphate to protein kinase from bovine adrenal cortex

Inorganic salts, several proteins and traces of protein precipitants were tested to find out by what mechanisms they modulate the binding of cyclic [3H]AMP to protein kinase (ATP-protein phosphotransferase; EC 2.7.1.37). The separation of free and bound cyclic AMP by (NH4)2SO4 precipitation was unaffected by the above agents and was more reliable than the Millipore filtration technique. Several binding sites for cyclic AMP were revealed in adrenal-cortex extract. When this extract was used as binding reagent in an assay for cyclic AMP, the standard curve was distorted in the presence of KCl because the salt affected the different binding sites to a varying extent. At high ionic strenth the protein kinase isoenzyme I dissociated and showed an extraordinarily high affinity for cyclic AMP. Trichloroacetate and perchlorate at very low concentrations were able to dissociate the protein kinase and modulate its binding characteristics as well. A progressive decrease in the cyclic AMP-binding capacity occurred on prolonged incubations. The binding protein was protected against inactivation by 2-mercaptoethanol, EDTA and several proteins. It was more resistant to denaturation when complexed to cyclic AMP. The enhancement of cyclic AMP binding by bovine serum albumin was investigated in some detail and appeared to be a pure stabilizing effect. It is proposed that the competitive-binding assays for cyclic AMP based on protein kinase be conducted at high ionic strength and in the presence of stabilizers (protein, EDTA, 2-mercaptoethanol). The interference from agents that may dissociate the protein kinase or influence its stability will thus be decreased.

Possible involvement of cyclic amp-dependent protein kinase in p, p′-ddt-induced changes in hepatic metabolism

Administration of an acute dose of p, p-1,1,1-trichloro-2,2-bis ( p-chlorophenyl) ethane ( p, p′-DDT) (600 mg/kg, p.o.) resulted in a significant increase in hepatic adenylate cyclase activity (40 per cent) and endogenous cyclic AMP levels (31 per cent). Although the cytosol cyclic AMP-independent protein kinase activity was only slightly affected (18 per cent), the activity of the nucleotide-dependent enzyme was significantly decreased to 74 per cent of the control values. Whereas the protein kinase activity ratio (−cyclic AMP/+cyclic AMP) of this soluble enzyme was increased, the ability of the enzyme to bind [ 3H]cyclic AMP in vitro was significantly decreased after pesticide administration. As observed with the soluble enzyme, maximal stimulation of the crude nuclear protein kinase (1500 g pellet) was observed 1 hr after p, p′-DDT treatment, as indicated by changes in the phosphorylation of endogenous nuclear substrates and cyclic AMP-binding capacity. Studies with lower dosages of DDT revealed that a dose as low as 100 mg/kg was sufficient to produce statistically significant alterations in the activities of both the soluble and paniculate enzymes. In most cases, doubling the dosage (200 mg/kg) resulted in greater changes in these biochemical parameters. The present study supports the concept that the DDT-induced alterations in hepatic carbohydrate metabolism may be related to an initial stimulation of the hepatic cyclic AMP-adenylate cyclase-protein kinase system.

Effects of isoproterenol on cyclic-AMP metabolism in rat ventral prostate.

Beta-Adrenergic stimulation of the ventral prostate cyclic-AMP system was investigated by examining the influence of isoproterenol on endogenous cyclic-AMP levels as well as on the activities of adenylate cyclase CEC 4.6.1.1) and cyclic-AMP-dependent and independent protein kinases (EC 2.7.1.37). Administration of isoproterenol (1 mg/kg, ip) resulted in rapid elevation of adenylate cyclase activity (119%) and cyclic-AMP levels (593%). The observed isoproterenol-stimulated changes in cyclic-AMP metabolism of the ventral prostate were time-dependent and maximal stimulation was seen 5 min after treatment with this beta-adrenergic agonist. The increases in prostatic adenylate cyclase and cyclic-AMP also were related to the dose of isoproterenol administered and maximal enhancement of these parameters was seen with 1 mg/kg dose of the agonist. Whereas pretreatment of rats with propranolol (3mg/kg, ip) partially reversed these alterations, administration of an alpha-adrenergic antagonist, phentolamine, even at a dose of 5 mg/kg, failed to elicit any appreciable effect. Stimulation of prostatic soluble protein kinase by isoproterenol was associated with a decrease (33%) in the activity of the cyclic-AMP-dependent protein kinase with a concomitant increase (25%) in that of the independent enzyme. Whereas the ability of the enzyme to bind cyclic-(3H) AMP in vitro was decreased (54%) following isoproterenol treatment, the protein kinase activity ratio (-cyclic-AMP/+cyclic AMP) was significantly elevated from 0.51+/0.05 to 0.95+/0.08. Although propranolol alone had little or no effect on these parameters, it inhibited partially the isoproterenol-induced alterations in cyclic-AMP-dependent protein kinase and the cyclic-AMP binding capacity. Treatment with propranolol also blocked the increases in the kinase activity ratio and in the activity of cyclic-AMP-independent enzyme seen with isoproterenol. Data suggest that the concentration of ventral prostate cyclic-AMP as well as the activities of adenylate cyclase and cyclic-AMP-dependent and independent form of protein kinases are subject to modulation by beta-adrenergic stimulation.

Cyclic AMP and cyclic GMP content and binding in malignancy

Summary 3′,5′-cyclic AMP and 3′,5′-cyclic GMP concentrations in several strains of Morris hepatomas as well as the ability of the hepatoma cytosol to bind the cyclic nucleotides were measured. The tumors were found to have levels of cyclic AMP either equal to or depressed below those of the host livers. Hepatomas with cyclic AMP levels equal to those of livers disclosed at least one of the following characteristics: low cyclic AMP binding capacity, high cyclic GMP concentration, high cyclic GMP binding capacity. The tumors with high cyclic GMP levels were particularly fast-growing. We suggest that malignancy is associated with a low effective level of cyclic AMP and that the effective level of cyclic AMP is a function not only of its concentration but also of the level of the various proteins which react with cyclic AMP and also the activity of its antagonists, such as cyclic GMP.

Histone kinases and cyclic AMP-binding capacity of nuclei of human tonsillar lymphocytes

Isotonic salt extract of nuclei of human tonsillar lymphocytes contained about 60–65% of the total histone kinase activity (EC 2.7.1.37) of the cells. The nuclear histone kinase activity was apparently cyclic AMP independent, although 20–25% of the total cyclic AMP-binding capacity of the cells was present in the nuclear extract. A cyclic AMP-dependent kinase (I), and an independent kinase (II), similar to those found previously in the hypotonic extract of the cells, were demonstrated in the nuclear extract by DEAE-cellulose chromatography. In addition a second type of cyclic AMP independent histone kinase (III) was found. On the basis of its catalytic properties, (kinetic parameters and the phosphorylation site of F2b histone), kinase III differed from kinase II, but it was similar to kinase I. Kinase III may be identical with the catalytic subunit of the cyclic AMP dependent enzyme. A cyclic AMP-binding fraction accompanied by a negligible amount of kinase activity was also demonstrated in the nuclear extract by DEAE-cellulose chromatography.

Distribution subcellulaire des protéine-kinases stimulables par l'AMP cyclique et des protéines réceptrices de l'AMP cyclique dans la thyroïde de porc

The distribution of cyclic AMP-dependent protein kinase activity in porcine thyroid glands has been studied. Enzyme activity catalyzing phosphorylation of exogenous substrate (protamine) from ATP, and cyclic AMP binding were determined in parallel in subcellular fractions purified by differential centrifugation and flotation on sucrose density layers. Both activities were found in all the studied fractions; they were quantitatively the highest in the cytosol but particles showed the highest specific activities. Latent protein-kinase activity was unmasked by action of detergents on microsomes (× 5–10 fold) and solubilized (85 to 99 p. cent of the initial total activity). Cyclic AMP binding capacity was also recovered in detergent-treated microsomal extracts in spite of reduced cyclic AMP binding in the presence of detergent. Protein kinase activity and cyclic AMP-binding proteins were less represented in purified nuclei than in microsomes. Again both activities were unmasked by detergent. Preparations highly enriched in Golgi membranes were compared to rough microsomal preparations. Higher protein kinase activity was detected in rough microsomes as compared to Golgi membranes, whereas the reverse was true for cyclic AMP binding. Both activities were equalized after detergent treatment. Since unmasking of protein kinase activity was the highest in Golgi membranes, this fraction contains more enzyme activity and cyclic AMP binding capacity than rough microsomes. The localization of endogeneous protein substrates of cyclic AMP-dependent protein kinases was investigated using purified soluble protein kinase subcellular fractions. The better endogeneous substrates seemed to be localized in the rough microsomal and in the nuclear fractions. L'activité de protéine-kinase stimulable par l'AMP cyclique a été étudiée dans la thyroïde de porc. L'isolement de fractions subcellulaires obtenues par centrifugations différentielles et par flottation sur des barrières de densité a permis de localiser l'activité enzymatique catalysant la phosphorylation d'un substrat exogène (protamine) par l'ATP et l'activité liante de protéines fixatrices de l'AMP cyclique. La répartition cellulaire de ces deux activités est parallèle. La majeure partie se situe dans le cytosol, mais les fractions particulaires présentent des activités spécifiques plus élevées. Les microsomes possèdent une activité enzymatique latente qui peut être démasquée par les détergents (× 5 à 10 fois) et solubilisée (85 à 90 p. cent de l'activité totale initiale). L'activité liante est aussi retrouvée dans les extraits microsomaux malgré une inhibition partielle de la liaison de l'AMP cyclique due aux détergents. Les activités enzymatiques et les protéines liant l'AMP cyclique sont moins représentées dans les noyaux que dans les microsomes. Elles sont aussi démasquées en présence de détergent. Des préparations enrichies en fragments de l'appareil de Golgi ont été étudiées comparativement à des préparations de microsomes granulaires. Dans ces fractions les activités enzymatiques et fixatrices de l'AMP cyclique semblent réparties différemment. L'activité kinasique paraît plus élevée dans les microsomes granulaires alors que la capacité de fixer l'AMP cyclique est plus importante dans la fraction de Golgi. Le parallélisme entre les activités est rétabli lorsqu'un détergent libère les activités latentes; le démasquage de l'activité enzymatique étant beaucoup plus important dans la fraction enrichie en membranes golgiennes, cette dernière se révèle être le site prépondérant des deux activités. Une localisation des protéines cellulaires susceptibles d'être phosphorylées par le système ATP-protéine kinase-AMP cyclique, a été effectuée en présence d'une protéine-kinase cytoplasmique purifiée. Elle indique que les meilleurs substrats endogènes sont localisés dans la fraction microsomale granulaire et dans la fraction nucléaire.