The role of cAMP on neoplastic cell growth and regression. III. Altered cAMP-binding in DBcAMP-unresponsive Walker 256 mammary carcinoma.

An exogenous supply of N⁶,O^2′-dibutyryl cyclic adenosine 3′,5′-monophosphate (DBcAMP) $\text{in vivo}$ produces regression of one type of Walker 256 mammary carcinoma cell population (DBcAMP-responsive); a second type of cell population continues to grow despite DBcAMP treatment (DBcAMP-unresponsive). A correlation was found between altered cAMP-binding of the tumor cytosol and DBcAMP-unresponsiveness. It was found that there was: a) a higher apparent dissociation constant (Kd) for cAMP-binding in unresponsive tumor cytosol $\text{in vitro}$, and b) unsaturability of cAMP-binding by unresponsive tumor cytosol in response to elevated cAMP levels $\text{in vivo}$. Cycloheximide abolished the saturation of cAMP binding $\text{in vivo}$ as well as tumor regression produced by DBcAMP.     

Modulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity with cAMP and wth protein fractions of rat liver cytosol

3-Hydroxy-3-methylglutaryl coenzyme A reductase activity is diminished in several $\text{in vitro}$ liver systems preincubated in the presence of cAMP. Reductase activity in isolated, washed liver microsomes is inactivated by ATP, Mg⁺⁺, and a protein fraction separated from the liver cytosol. This effect is augmented by 3′–5′ cyclic AMP. Reductase activity in previously inactivated microsomes can be partially restored by incubation with a second protein fraction of the cytosol.     

Differences in macromolecular binding between cyclic AMP and its dibutyryl derivative in vitro

Rat liver cytosol binds ³H-cAMP and ³H-DBcAMP $\text{in vitro}$. Fractionation of bound radioactivity by DEAE-Sephadex chromatography shows that ³H-cAMP is associated with a different cytosolic protein than is ³H-DBcAMP. The pI's of the cAMP-protein and the ³H-DBcAMP-protein complexes are 6.7 and 3.9, respectively. Competition studies between ³H-cAMP and its structural analogues have shown the following order of effectiveness in competing for binding sites in rat liver cytosol: cAMP > N⁶-MBcAMP > O²′-MBcAMP. No inhibition of ³H-cAMP binding was observed with 5′-AMP, adenosine, cGMP or DBcAMP. $\text{In vitro}$ binding experiments with rat serum has shown that only ³H-DBcAMP binds to any significant extent.     

The effect of theophylline on adenosine 3′, 5′-monophosphate-dependent protein kinase and ACTH1–24 stimulated steroidogenesis in bovine adrenal cortical cells

Theophylline (theo), a known phosphodiesterase (PDE) inhibitor, was tested for its effects on ACTH_1–24 regulated steroidogenesis in isolated bovine adrenal cortical cells. Theo produced a dose related inhibition of ACTH_1–24 stimulated cortisol synthesis with half maximal inhibition occuring at 7 mM. Theo enhanced ACTH_1–24 stimulated cellular adenosine 3′, 5′-monophosphate (cAMP) levels above that produced by ACTH_1–24 alone confirming its inhibition of cAMP PDE. When tested on cAMP binding protein and cAMP-dependent protein kinase activities in cytosol prepared from bovine adrenal cortex, theo displaced ³H-cAMP binding to cAMP binding protein and inhibited cAMP-stimulated protein kinase activity. The half maximal inhibition of cAMP binding and protein kinase activity was observed at 10 and 5 mM, respectively. Inhibition of cAMP-dependent protein kinase by theo provides a possible explanation of its inhibitory effects on adrenal steroidogenesis and further implicates cAMP-dependent protein kinase in mediating ACTH stimulated steroidogenesis. Furthermore these studies suggest a novel mechanism of action for theo in addition to its known action on cAMP PDE.     

A comparison of the uptake, metabolism, and action of cyclic adenine nucleotides in cultured hepatoma cells.

Intracellular radioactivity following incubation of HTC or RLC cells in [³H]cAMP exceeds that following incubation in either [³H]mono- or dibutyryl cAMP by 30-fold, yet little [³H]cAMP is found within the cells. Even at early times (30 min) the label derived from [³H]cAMP is predominantly found in ADP or ATP, suggesting it mostly enters the cell as the nucleoside. Significant intracellular concentrations of monobutyryl cAMP (2–10 μm) result from incubation of both cell lines in either N⁶ mono- or dibutyryl cAMP. A very small percentage of this label is in cAMP, and within 2 h of incubation > 65% of the label is again found in ADP or ATP.Liver cytosol contains three major cAMP-dependent protein kinases, designated A, B, and C, as resolved by DEAE-Sephadex chromatography. cAMP is the most effective in vitro activator (10- to 16-fold stimulation) of kinases A and B, the preponderant forms, in the order cAMP > N⁶ monobutyryl cAMP ? dibutyryl cAMP. Kinase C, a minor fraction, was stimulated two to threefold with the order cAMP ≥ N⁶ monobutyryl cAMP > dibutyryl cAMP. HTC and RLC cell cytosol protein kinase has Chromatographic and cyclic nucleotide activation properties similar to those of liver fraction C.The activation state of the protein kinases of HTC and RLC cells incubated in the various cyclic nucleotides was also studied. The ability of such nucleotides to occupy regulatory protein binding sites in intact cells (as determined by the inhibition of subsequent in vitro binding of [³H]cAMP) was of the order N⁶ monobutyryl cAMP > dibutyryl cAMP > cAMP > untreated cells. Correspondingly, the ratio of basal protein kinase activity in cyclic nucleotide treated:control cells was higher in cells incubated in monobutyryl cAMP > dibutyryl cAMP > cAMP. This in vivo activation suggests that little additional stimulation would be obtained by adding cAMP to extracts prepared from such cells. This activation can be expressed as the ratio ? cAMP: + cAMP (a ratio of 1 being maximal activation). The highest such ratio was seen in cells which had been incubated in monobutyryl cAMP > dibutyryl cAMP > cAMP > untreated cells. The studies indicate that all three cyclic nucleotides are capable of activating protein kinase in intact RLC and HTC cells; however the monobutyryl derivative is the most effective, and the degree of stimulation is greater in RLC than in HTC cells.RLC cell tyrosine aminotransferase activity is increased two to threefold by butyrylated cAMP derivatives (but not by cAMP) whereas the HTC cell enzyme is not induced. The rate of replication of both lines is unaltered by the butyrylated compounds.Since HTC and RLC cells accumulate and metabolize cAMP and its derivatives equally, and since they both contain a protein kinase with similar in vivo and in vitro activation properties, it is suggested that the effects of butyrylated cAMP derivatives on cell replication and tyrosine aminotransferase induction are mediated separately, either by distinct protein kinases, or at a point distal to protein kinase, or by a mechanism independent of protein kinase.     

Nuclear matrix as acceptor for cAMP-binding proteins

Using [³²P]-8-N₃-cAMP, a photoaffinity analog of cAMP, we have established that nuclear binding of cAMP is preferentially localized in the “nuclear matrix”. Two major radioactive bands corresponded to proteins of M_r 40 K and 50 K, and three minor bands to proteins of M_r 55, 150 and 200 K. Even though the molecular weight of the major nuclear binding proteins in the matrix are similar to those of the cytosolic cAMP binding proteins, the characteristics of the binding reaction in the nucleus were markedly different from those in the cytosol.     

Hydrocortisone binding receptor in the rat pituitary GH3 cell line.

A receptor with specificity and high affinity for hydrocortisone (HC) has been found in the cytosol of GH₃ cells, a growth hormone (GH) producing culture. Scatchard analysis indicated that the interaction of [³H]HC with the receptor has an apparent dissociation constant (K_d) of about 6.0 × 10^?9M and a concentration of binding sites of approx. 1 × 10^?13 mol/mg cytosol protein. The second order association rate constant was determined to be 1.5 × 10⁶ M^?1 min^?1. The receptor activity is stable at 2°C for several hours, but is destroyed completely by heating at 37°C for 1 hour, or by treatment with pronase, only partially by RNase, but not by DNase. The binding of [³H]HC to the cytosol receptor is inhibited by unlabeled progesterone (PR) or dexamethasone to the same extent as the inhibition by unlabeled HC. However, it is only partially inhibited by testosterone, 17-methyl-testosterone, 17α and 17β-estradiol, and 4-pregnen-20β-ol-3-one, and is unaffected by 5α-pregnan-3β,20β-diol. The biological role for these receptors in the regulation of GH synthesis is supported by the observations that the HC-stimulated production of GH is antagonized by PR, which competes with the binding of HC to the receptor.     

Characterization of cyclic AMP-binding proteins in rat Sertoli cells using a photoaffinity ligand

Summary Protein-bound cyclic AMP (cAMP) levels in cultured rat Sertoli cells have been determined after exposure to follicle-stimulating hormone (FSH) and agents which elevate intracellular cAMP or mimic cAMP action. Changes in the content of protein-bound cAMP were correlated with changes in receptor availability determined by measuring [³H] cAMP binding. Using the photoaffinity analog of cAMP, 8-N₃ [³²P] cAMP, two major cAMP-binding proteins in Sertoli cell cytosol, with molecular weights of 47 000 and 53 000 daltons, were identified as regulatory subunits of type I and type II cAMP-dependent protein kinases, respectively. Densitometric analysis of autoradiograms demonstrated differential activation of the two isozymes in response to treatment with FSH and other agents. Results of this study demonstrate the value of measuring changes in protein-bound cAMP and the utility of the photoaffinity labeling technique in correlating hormone-dependent processes in which activation of cAMP-dependent protein kinase occurs.     

Cyclic AMP-binding protein and estrogen receptor: antagonism during nuclear translocation in a hormone-dependent mammary tumor

Incubation of nuclei from hormone-dependent rat mammary tumors with its cytosol activated with 5 nM 17β-estradiol resulted in a 4-fold increase of nuclear estrogen binding activity over the control nuclei. The presence of 100 nM cAMP in the activated cytosol inhibited this nuclear uptake of estrogen receptor by 50%. Conversely, incubation of the nuclei with cytosol activated with 100 nM cAMP increased nuclear cAMP binding and cAMP-dependent protein kinase activity 4-fold, while the presence of 5 nM 17β-estradiol in the activated cytosol inhibited the nuclear cAMP binding and the protein kinase activity by 50%. No competition was found between estrogen and cAMP for each other's cytoplasmic binding proteins or the nuclear acceptor sites. These data suggest that a mutual antagonism exists between the cAMP-binding protein and estrogen receptor during their nuclear translocation.     

Estramustine binding in rat,baboon and human prostate measured by high pressure liquid chromatography

High pressure liquid chromatography (HPLC) was used to determine ³H-estramustine (estradiol-17β3N-bis-[2-chlorethyl] carbamate), ³H-17β-hydroxy-5α-androstan-3-one (³H-dihydrotestosterone or ³H-DHT), ³H-estradiol-17β (³H-E₂) and ³H-3β-hydroxy-5-pregnen-20-one (³H-pregnenolone) binding in 50μ1 of cytosol utilizing a column which separates proteins in the molecular weight range of 2,000 to 70,000 daltons. The rat prostate contains a protein in considerable concentration and with the highest affinity for estramustine (375,000dpm ³H-estramustine per mg. cytosol protein) among the substances tested. Operationally, we have named this protein “estramustine binding protein” (EBP), though it is very likely similar to other previously described prostatic proteins (e.g., α-protein, prostatein, prostatic binding protein). The sensitivity of the HPLC method disclosed EBP-like proteins, but in much lesser concentrations, in some of the other tissues tested. The concentration of these proteins in the human and baboon prostates was much lower (average for the baboon cranial lobe 4800dpm/mg cytosol protein, with a somewhat higher value for the caudal lobe) than that in the rat gland. The amount of the EBP-like protein was higher in prostatic cancer than in that of benign prostatic hypertrophy (BPH) (range 9350 – 25,900 vs. 2200 – 18,900 dpm/mg cytosol protein). In the human, the highest value was found in one normal prostate tested (106,000dpm/mg cytosol protein).     

Increase of cyclic AMP-dependent protein kinase type II as an early event in hormone-dependent mammary tumor regression.

Primary, 7,12-dimethylbenz(α)anthracene (DMBA)-induced mammary carcinoma in the rat contains cyclic adenosine 3′,5′-monophosphate (cAMP)-dependent and -independent forms of protein kinase. When growth of DMBA-induced tumors was arrested by either ovariectomy or N⁶,O²′-dibutyryl cAMP treatment of the host, the activity of cAMP-dependent protein kinase type II markedly increased in the tumor cytosol, as shown by DEAE-cellulose chromatography and autophosphorylation. The increase in activity of cAMP-dependent protein kinase was also demonstrable in the tumor cytosol and nuclei following $\text{in}$$\text{vitro}$ incubation of tumor slices with cAMP. These results suggest that protein kinase type II is involved in the regression of hormone-dependent mammary tumors.     

Intestinal cytosol binders of 1,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3

The binding of 25-hydroxy-[26,27-³H]vitamin D₃ and 1,25-dihydroxy-[26,27-³H]vitamin D₃ to the cytosol of intestinal mucosa of chicks and rats has been studied by sucrose gradient analysis. The cytosol from chick mucosa showed variable binding of 1,25-dihydroxyvitamin D₃ to a 3.0S macromolecule which has high affinity and low capacity for this metabolite. However, when the mucosa was washed extensively before homogenization, a 3.7S macromolecule was consistently observed which showed considerable specificity and affinity for 1,25-dihydroxyvitamin D₃. Although 3.7S binders for 1,25-dihydroxyvitamin D₃ could also be located in other organs, competition experiments with excess nonradioactive 1,25-dihydroxyvitamin D₃ suggested that they were not identical to the 3.7S macromolecule from intestinal mucosal cytosol. As the 3.7S macromolecule was allowed to stand at 4 °C with bound 1,25-dihydroxy-[³H]vitamin D₃, the 1,25-dihydroxy-[³H]vitamin D₃ became increasingly resistant to displacement by non-radioactive 1,25-dihydroxyvitamin D₃. The 1,25-dihydroxy-[³H]vitamin D₃ remained unchanged and easily extractable with lipid solvents through this change, making unlikely the establishment of a covalent bond. Unlike the chick, mucosa from rats yielded cytosol in which no specific binding of 1,25-dihydroxy-[³H]vitamin D₃ was detected. Instead, a 5-6S macromolecule which binds both 1,25-dihydroxyvitamin D₃ and 25-hydroxyvitamin D₃ was found. This protein which was also found in chick mucosa shows preferential binding for 25-hydroxyvitamin D₃. It could be removed by washing the mucosa with buffer prior to homogenization which suggests that it may not be a cytosolic protein. Although the 3.7S protein from chick mucosa has properties consistent with its possible role as a receptor, the 5-6S macromolecule does not appear to have “receptor”-like properties.     

High affinity estrogen binding by rabbit liver

Macromolecular binding components for [³H]estradiol-17β are present to cytosol prepared from rabbit liver. When cytosol from sexually mature male liver was incubated with [³H]estradiol and analyzed for binding on low ionic strength sucrose gradients, two peaks of binding activity were detected. One peak had a sedimentation coefficient of 4–5 S and the other had a sedimentation coefficient of 8–9 S. The two components differed from each other regarding steroid specicity and various physiocochemical parameters. [³H]-estradiol binding to the 4–5 S component was not inhibited by estrogens, 5α-dihydrotestosterone, progesterone or cortisol. Binding to this component did not appera to be saturable and lavel was rapidly stripped from it by cahrcoal. Estradiol bindng to the 8–9 S component was estrogen specific, saturable and of high affinity. The specific binder dissociates on high ionic strength sucrose gradients and sediments as a 4–5 S moiety. The specific binding protein has a K_d of 3.05 · 10⁻¹⁰ M and a dissociation half-time of 33 h and there are 35.2 fmol of binding sites/mg cytosol protein. Estrogen binders are also present in liver cytosol from sexually mature female and sexually immature male rabbits. During prolonged incbuation of [³H]estradiol with mature male liver cytosol at 0–5°C polar metabolites of estradiol are produced.     

Inactivation by Na+,K+-ATPase of cytosol glucocorticoid receptors from rat brain and liver

Summary We have examined the effect of Na⁺,K⁺-ATPase on ³H-triamcinolone acetonide binding capacity of cytosol glucocorticoid receptors from rat brain and liver. Preincubation of the brain or liver cytosol with Na⁺,K⁺-ATPase (10 units/ml) at 30 °C resulted in a rapid loss of specific ³H-triamcinolone acetonide binding, with a half-life of approximately 7 min. The ATPase effect could be prevented by the addition of 10^–5 M ouabain, or substantially reduced by the omission of Na⁺,K⁺ or Mg⁺². The cytosol receptor bound with ³H-triamcinolone acetonide was totally resistant to the inactivation by the ATPase. Since there is some evidence that ATP may bind to glucocorticoid receptor, our findings indicate that an ATP-receptor complex may be essential for steroid binding. The effects of the ATPase in the inactivation of the receptor are very similar to those of alkaline phosphatase reported by others. This raises doubts about the proposal based on the phosphatase inactivation that the cytosol glucocorticoid receptor may be phosphorylated.     

Specific cytosol binding of diethylstilbestrol in rat skeletal muscle

Rat skeletal muscle cytosol proteins bound ³H-diethylstilbestrol (³H-DES). More than 90% of this binding was high capacity and low affinity. Serum albumin accounted for roughly 50–60% of the binding, as evidenced by its precipitation with anti-rat albumin IgG. About half of the binding was distinguishable from albumin and other serum proteins by its precipitation in 40% saturated ammonium sulfate. This material sedimented at 4–5S in high-salt sucrose gradients, and resolved into two components (8S and 4–5S) in low-salt. Following incubation at 23–27°C for one hour, 2% of the bound ³H-DES in whole cytosol (approximately 2 fmole/mg cytosol protein) was retained by DNA-cellulose, and was eluted with 0.6 M KCl. This small fraction of the total binding was inhibited by estrogens and DES analogues: estradiol-17β, DES, dienestrol, and hexestrol were strong inhibitors; isodienestrol, dimethylstilbestrol, estradiol-17α, estrone, tamoxifen, MER-25, CI-628, and nafoxidine were weak inhibitors; dihydrotestosterone, testosterone, and prednisone did not compete. These observations indicate that specific estrogen-binding sites exist in rat skeletal muscle.     

A novel effect of molybdate on the binding of [3H]aldosterone to gel-filtered type I receptors in brain cytosol

Recently we reported that adding molybdate to crude steroid-free cytosol at 0°C results in a dose-dependent reduction in the binding of [³H]aldosterone ([³H]ALDO), to Type I adrenocorticosteroid receptors. In the experiments outlined here, we found that addition of molybdate to steroid-free brain cytosol produces a 30–50% increase in the subsequently measured maximal specific binding capacity (B _MAX) of [³H]ALDO-Type I receptors if the cytosol is subjected to Sephadex G-25 gel filtration prior to steroid addition. These manipulations were found to have no effect on the equilibrium dissociation constant (K _d) of the receptors. In contrast, when gel filtration of steroid-free cytosol was performed in the absence of molybdate, there was a 2-fold increase in the K_d and over a 50% reduction in the subsequently measuredB _MAX of [³H]ALDO-Type I receptors. When molybdate was added to this steroid-free cytosol immediately following gel filtration, there was no reduction (or increase) in Type I receptor [³H]ALDO binding capacity compared with nongel-filtered controls. The addition of as little as 2 mM molybdate to crude steroid-free cytosol was found to stabilize the binding capacity of Type I receptors during exposure to 22°C incubations; however, when gel-filtered steroid-free cytosol was exposed to these conditions at least 10 mM molybdate was required to stabilize Type I receptor binding capacity. Adding the sulfhydryl reducing reagent, dithiothreitol, to the various steroid-free cytosols had little effect on [³H]ALDO-Type I receptor binding. The effects of molybdate, revealed in this study, on Type I receptors in brain cytosol subjected to gel filtration are clearly different from those seen with receptors in crude cytosol preparations, as well as from those reported in the literature for other steroid receptors. Possible mechanisms of action of molybdate on unoccupied Type I receptors in crude and gel-filtered cytosol are discussed.     

Steroid hormone receptor studies in melanoma model systems

The Transplantable B-16 melanotic melanoma carried in syngeneic C57B1/6J female mice and the Syrian hamster melanoma cell line, RPMI 3460, were utilized to determine whether steroid-hormone receptors are present in animal melanomas. In the B-16 melanoma, a cytoplasmic-estrogen receptor is detectable, but there is no evidence for androgen or progestin receptors. Some tumors contain a glucocorticoid-binding macromolecule. Sucrosedensity gradient centrifugation of cytosol after incubation with [³H]-estradiol revealed an 8S peak that was suppressed by excess radioinert diethylstilbesterol. Binding varied from 5–35 fmoles per mg cytosol protein. Scatchard analysis of [³H]-estradiol binding in cytosol yielded a single class of high-affinity binding sites; the dissociation constant is 6 × 10^?10 M. The receptor molecule is shown to be estrogen-specific by ligand competition assays. In contrast to B-16 melanoma, no estrogen, androgen, or progestin receptor can be found in the Syrian hamster melanoma cell line. However, a substantial level of specific binding is observed using [³H]-dexamethasone. Sucrose-gradient centrifugation of cytosol from this cell line after incubation with [³H]-dexamethasone revealed a 7S peak that was suppressed by excess radioinert dexamethasone. Scatchard analysis indicated a single class of high affinity sites with a dissociation constant of 2 × 10^?9 M. Binding levels from 70–610 fmoles per mg cytosol protein were observed. The Syrian hamster melanoma cells also exhibit a biological response to glucocorticoids: Dexamethasone causes both an inhibition of growth and a decrease in final-cell density in these cells.     

A study of adenosine 3′–5′ cyclic monophosphate binding sites of human erythrocyte membranes using 8-azidoadenosine 3′–5′ cyclic monophosphate,a photoaffinity probe

An earlier report (1a) has shown the utility of 8-N₃cAMP (8-azidoadenosine-3′, 5′-cyclic monophosphate) as a photoaffinity probe for cAMP binding sites in human erythrocyte membranes. The increased resolution obtained using a linear-gradient SDS polyacrylamide gel system now shows that: (1) both cAMP and 8-N₃cAMP stimulate the phosphorylation by [γ-³²P]-ATP of the same red cell membrane proteins; (2) the protein of approximately 48,000 molecular weight whose phosphorylation by [γ-³²P]-ATP is stimulated by cAMP and 8-N₃cAMP migrates at a solwer rate than the protein in the same molecular weight range which is heavily photolabeled with [³²P]-8-N₃cAMP; (3) other cyclic nucleotide binding sites exist besides those initailly reported; (4) the variation in the ratio of incorporation of ³²P-8-N₃cAMP into the two highest affinity binding sites appears to be the result of a specific proteolysis of the larger protein.     

Rat ovary glucocorticoid receptor: Identification and characterization

A soluble, thermolabile protein with characteristics typical of glucocorticoid receptors has been identified in the ovaries of estrogenstimulated hypophysectomized immature rats. After the incubation of ³H-dexamethasone with ovarian cytosol, fractionation on a Sephadex G-200 column reveals a peak of radioactivity which elutes at the void volume. This peak, which represents saturable ³H-dexamethasone binding, disappears following heating (4 ° C × 15 min) or treatment of the cytosol with pronase. Scatchard analysis of the ³H-dexamethasone binding to cytosol shows it to be high affinity (Kd=5.1 nM) and saturable, with 327 fmol binding sites/mg cytosol protein. Binding site number rises linearly with increasing cytosol protein concentrations. The relative abilities of various steroids to inhibit ³H-dexamethasone binding are: triamcinolone acetonide ≥ dexamethasone > cortisol = progesterone > dihydrotestosterone > estradiol. This binding protein sediments at 9 S on a sucrose gradient, has a mean Stokes radius of 105 Å on gel exclusion chromatography, and has a calculated molecular weight of 388, 000 daltons and a frictional ratio of 2.1. ³H-Dexamethasone is not metabolized and does not bind specifically to serum. We have identified a protein in the rat ovary with characteristics of a glucocorticoid receptor and propose that this protein may be responsible for mediating direct effects of glucocorticoids on the ovary.     

Hormonal effects on fat cell adenosine 3′,5′-monophosphate dependent protein kinase

Fat cell extracts were electrophoresed on polyacrylamide gels to separate the regulatory subunit and holoenzyme species of protein kinase. Gels were incubated with cyclic [³H]AMP ([³H]cAMP) and washed, and the bound [³H]cAMP was estimated. The band of [³H]cAMP found closest to the origin (Peak I) was associated with cAMP-dependent protamine kinase activity. A seond [³H]cAMP peak (Peak II) also contained protamine kinase activity. Although the kinase activity of Peak II was much less than Peak I, more [³H]-cAMP was bound in Peak II than in Peak I. The [³H]cAMP peak furthest from the origin (Peak III) was devoid of kinase activity.Incubation of extracts with cAMP prior to electrophoresis diminished or abolished kinase activity in Peaks I and II. This incubation also decreased [³H]cAMP binding in Peaks I and II, and increased binding in Peak III. When extracts were incubated with [³H]cAMP before electrophoresis, essentially all of the radioactivity was found in Peak III. It was concluded that Peak I represents a holoenzyme form and that Peak III is composed of the regulatory subunits of this enzyme. Peak II may represent a relatively inactive holoenzyme form not previously described.Incubation of adipocytes with epinephrine resulted in a dose- and time-dependent decrease in Peak I and increase in Peak III, and insulin opposed these effects of epinephrine. After 1-min incubations with epinephrine, the decreases in Peak I or increases in Peak III correlated with increases in phosphorylase a activity, decreases in glycogen synthase I activity and changes in cAMP, both in the presence and absence of insulin. However, after incubation with epinephrine for more than 2 min in the presence of insulin, phosphorylase a activity did not correlate with cAMP, suggesting that factors other than the cyclic nucleotide mediate the effects of epinephrine and insulin.