Protein phosphorylation of the smooth and rough endoplasmic reticulum in normal and neoplastic liver of the rat.

Smooth and rough endoplasmic reticulum from rat liver and hepatomas exhibited endogenous protein kinase activity independent of adenosine 3':5'-monophosphate. The phosphorylation of smooth membranes by this process was consistently higher than that of rough membranes. When histone was added along with the smooth endoplasmic reticulum, cyclic AMP stimulated protein phosphorylation. Analysis of membrane-phosphorylated proteins by gel electrophoresis showed 5 major phosphorylated bands with estimated molecular weights of 155 000, 62 000, 50 000, 46 000 and 43 000, whereas major bands having estimated molecular weights of 62 000, 50 000 and 43 000 were found in membranes of the smooth endoplasmic reticulum of the Morris hepatoma 5123 C. Since previous studies in this and other laboratories have demonstrated the similarity of the protein components of membranes of the endoplasmic reticulum of normal liver and hepatoma, our findings indicate an inability of the protein kinase of hepatoma intracellular membranes to phosphorylate protein species that are found in membranes of both liver and the neoplasm.     

Distribution of adenylate cyclase among membrane fractions of rat liver.

Adenylate cyclase activity was detected in plasma membranes, Golgi apparatus, and endoplasmic reticulum from rat liver. Adenylate cyclase activities of purified membranes were determined biochemically by two methods. In one, the synthesis of radioactive cyclic AMP from ATalpha32P was monitored. In the other, the synthesis of cyclic AMP was quantitiated using a protein which specifically binds cyclic AMP. The enzyme activity was responsive to activation by both glucagon and sodium fluoride although differences in degree of activation were noted comparing plasma membrane, Golgi apparatus, and endoplasmic reticulum. Cytochemical studies, using both whole tissue and purified cell fractions and conducted in parallel, confirmed the biochemical results. Deposition of lead phosphate, enhanced by glucagon and NaF with samples incubated with appropriate substrates, was not restricted to plasma membranes of hepatocytes but was present in intracellular membranes as well. Adenylate cyclase of rat hepatocytes appears more widely distributed among internal membranes than previously recognized.     

The selectivity and stoicheiometry of membrane binding sites for polyribosomes, ribosomes and ribosomal subunits in vitro.

Differences in the binding sites for polyribosomes, template-depleted ribosomes and large ribosomal subunits were found in microsomal derivatives of the rough endoplasmic reticulum. 1. The stoicheiometry of polyribosome and ribosome interaction in vitro with membranes was shown to be influenced by the relative concentration of interactants and the duration of their mixing. Large ribosomal subunits required a more prolonged mixing schedule to achieve saturation of membranes than did polyribosomes. 2. By using a procedure which minimized the effects on binidng by the stoicheiometric variables, competition between populations of polyribosomes, ribosomes and subunits for membrane sites showed that subunits, and to a lesser extent ribosomes, failed to block polyribosome attachment. 3. Polyribosomes isolated from liver, kidney and hepatoma 5123C entirely bound to a common membrane site, but some polyribosomes from myeloma MOPC-21 bound to other sites, perhaps influenced by their unique nascent proteins. 4. Subunit-binding sites appear on rough membranes only after endogenous polyribosomes have been removed, but no evidence that resulting changes in surface constituents are responsible was found. Large-subunit binding was largely abolished by lowering MgC12 concentration of 0.1 mM, whereas under the same conditions polyribosome binding was undiminished. 5. The large-subunit site appears to be distinct from the polyribosome site not only in the restriction of its affinity for particles but also spatially, to the extent that bound subunits do not hinder access of polyribosomes to their sites.     

Characterization of regulation of thyrotropin (TSH) receptor function in thyroid plasma membrane: interaction between TSH receptor function and activation of adenosine 3',5'-monophosphate-dependent protein kinase

The changes in the characteristics of thyrotropin (TSH) binding to thyroid plasma membranes during the activation of cyclic AMP-dependent protein kinase in the membranes were studied. Preincubation of thyroid plasma membranes with TSH or cyclic AMP reduced the maximal binding capacity but increased the association rate for TSH binding. In double reciprocal analysis, a marked reduction of the total number of binding sites and association constant was observed in the membranes treated with cyclic AMP. These reductions were also observed in the membranes preincubated with buffer alone. The degree of these reductions, however, was greater in the membranes pretreated with cyclic AMP. During incubation of the membranes with buffer alone, cyclic AMP formation (activation of adenylate cyclase) was observed though the degree of the formation was lower than that induced by TSH. The results suggested that not only TSH receptor release from thyroid plasma membrane but also the modification of TSH binding activity in the membrane is produced by cyclic AMP-dependent protein kinase.     

The insulin-stimulated cyclic AMP phosphodiesterase binds to a single class of protein sites on the liver plasma membrane.

The peripheral cycle AMP phosphodiesterase from rat liver plasma membranes binds with high affinity (2.4 nM) to a single class of receptor sites on the liver plasma membrane. These receptor sites appear to be proteins, as they are trypsin- and heat-labile. The sensitivity of these sites to denaturation by trypsin and heat is a first-order process. The presence of Ca2+ (5 mM) increases the affinity of these sites for the enzyme, but does not alter their total number. The receptor sites and the cyclic AMP phosphodiesterase occur in similar numbers, at around 2 pmol/mg of plasma-membrane protein. It is proposed that the peripheral, liver plasma-membrane cyclic AMP phosphodiesterase is attached to a specific site on the insulin receptor and that the binding of insulin to the receptor site triggers a conformational change in the enzyme such that the enzyme can be phosphorylated and activated by an endogenous cyclic AMP-dependent protein kinase.     

Subcellular localization and hormone sensitivity of adipocyte cyclic AMP phosphodiesterase.

Treatment of intact adipocytes with either or both insulin and adrenaline stimulated membrane cyclic AMP phosphodiesterase activity only in the endoplasmic reticulum subfraction. The cyclic GMP-inhibited cyclic AMP phosphodiesterase activity was also found in this fraction. Quantitative Western blotting using a specific polyclonal antibody, raised against the homogeneous 'dense-vesicle' cyclic AMP phosphodiesterase from rat liver, identified a single 63 kDa species which was localized in the adipocyte endoplasmic reticulum fraction. The ability of adrenaline to stimulate adipocyte membrane cyclic AMP phosphodiesterase was shown to be mediated via beta-adrenoceptors and not alpha 1-adrenoceptors. Membrane cyclic AMP phosphodiesterase was stimulated by glucagon but not by vasopressin, A23187 or 12-O-tetradecanoylphorbol 13-acetate (TPA). Treatment of adipocytes with either chloroquine or dansyl cadaverine failed to affect the ability of insulin to stimulate cyclic AMP phosphodiesterase activity. Treatment of an isolated adipocyte endoplasmic reticulum membrane fraction with purified protein kinase A increased its cyclic AMP phosphodiesterase activity some 2-fold. When this fraction was treated with purified protein kinase A and [32P]ATP, label was incorporated into a 63 kDa protein which was specifically immunoprecipitated with the antiserum against the liver 'dense-vesicle' cyclic AMP phosphodiesterase.     

Isolation and functional characterization of an inositol trisphosphate receptor from brain

We have identified an IP3 receptor protein in brain membranes through the binding of radiolabelled IP3. Autoradiographic studies localize the receptor to various areas of the brain with highest densities in Purkinje cells of the cerebellum. IP3 binding is inhibited by physiologic intracellular concentrations of calcium. Purification of the IP3 receptor to homogeneity reveals it to be comprised of four identical subunits of 260 kD each. Antisera to the purified receptor protein have been employed for immunohistochemical studies which, at the electron microscopic level, localize the IP3 receptor to a subdivision of the rough endoplasmic reticulum occurring in synaptic areas and in close association with the nuclear membrane. The IP3 receptor protein is selectively phosphorylated by cyclic AMP (cAMP) dependent protein kinase. This phosphorylation decreases 10-fold the potency of IP3 in releasing calcium from brain membranes.     

Characterization of gonadotropin binding sites in the intracellular organelles of bovine corpora lutea and comparison with plasma membrane sites

The specific binding of 125I-human choriogonadotropin (hCG) to plasma membranes, nuclear membranes, lysosomes, rough endoplasmic reticulum, heavy golgi, and medium and light golgi of bovine corpora lutea was dependent on the amount of protein, 125I-hCG concentration and incubation time. The bound hormone in all the organelles was able to rebind to fresh corresponding organelles. Scatchard analysis revealed a homogenous population of gonadotropin binding sites in plasma membrane, rough endoplasmic reticulum, heavy golgi, and medium and light golgi, whose binding affinities (Kd = 8.6-11.0 X 10(-11) M) were similar but whose number of available gonadotropin binding sites varied. Scatchard analyses of nuclear membranes and lysosome binding, on the other hand, were heterogenous (Nuclear membranes, 11 and 23 X 10(-11) M lysosomes, 3.4 and 130 X 10(-11) M). The rate constants for association (5.9 to 12.1 X 10(6) M-1 S-1) and dissociation (7.4 to 9.0 X 10(-4) S-1) were similar among different subcellular organelles except for nuclear membranes and lysosomes, where rate constants for association were significantly lower. The ligand binding specificity, lower effectiveness of human luteinizing hormone as compared to hCG in competition, the optimal pH, the lack of ionic requirements for binding, and the molecular size of 125I-hCG-gonadotropin binding site complexes solubilized from various intracellular organelles were similar to those observed for plasma membranes. Numerous differences were also observed between intracellular organelles and plasma membranes as well as among intracellular organelles themselves with respect to binding losses due to exposure to low and high pH values, di- and monovalent cations, increasing preincubation temperatures, and a variety of enzymes and protein reagents. The possible reasons for these similarities as well as differences observed are discussed. The differences are viewed as an additional indication that contamination cannot solely explain the presence of gonadotropin binding sites in various intracellular organelles.     

Heterogeneity of cyclic nucleotide phosphodiesterases in liver endoplasmic reticulum.

A microsomal fraction from rat liver was subfractionated into three rough endoplasmic reticulum fractions RIII, RII and RI, together with a smooth endoplasmic reticulum plus Golgi fraction. Cyclic nucleotide phosphodiesterase activity was found in all fractions. Subsequently it was shown that Golgi fractions were essentially devoid of cyclic AMP phosphodiesterase activity and the activity resided in the smooth endoplasmic reticulum fraction. The activity of the endoplasmic reticulum constituted some 20% of the homogenate activity, with the major fraction of this being associated with the RII fraction and the least with the RI fraction. With the exception of the activity of the RI fraction, which was a peripheral enzyme, all of the other enzyme activities were integral, requiring detergent or repeated freeze-thawing to effect solubilization. All of the activities appeared to be exposed at the external surface of the endoplasmic reticulum, as they were inactivated by trypsin under conditions where glucose 6-phosphatase was not. All of these activities displayed distinct sensitivities to both thermal and trypsin inactivation, yielding activity decays consistent with a single enzyme species being present in each case. The freeze-thaw-solubilized enzymes yielded single symmetrical peaks on sucrose-density-gradient centrifugation and polyacrylamide-gel electrophoresis. The sedimentation coefficients for the enzymes in the smooth-endoplasmic-reticulum-plus-Golgi, RIII, RII and RI fractions were 3.2S, 4.2S, 4.5S and 4.5S respectively. Whereas the activity in the smooth-endoplasmic-reticulum-plus-Golgi fraction exhibited normal Michaelis kinetics, those in the other fractions yielded kinetics indicative of apparent negative co-operativity. All of the enzymes exhibited low Km values towards cyclic AMP. The enzymes did not appear to be regulated by Ca2+ or calmodulin. ZnCl2 was found to be a potent non-competitive inhibitor of the enzyme in all fractions. NaF was a weak non-competitive inhibitor. The bilayer fluidizing agent benzyl alcohol exerted dissimilar effects on the enzyme activities. It is concluded that the endoplasmic reticulum displays lateral heterogeneity, with single, rather distinct, cyclic AMP phosphodiesterases being found in the different fractions.     

Characterization of inositol 1,4,5-trisphosphate receptors and calcium mobilization in a hepatic plasma membrane fraction

The distribution of hepatic binding sites for the calcium-mobilizing second messenger, inositol 1,4,5-trisphosphate (IP3), was analyzed in subcellular fractions of the rat liver by binding studies with [32P]IP3 and compared with the Ca2+ release elicited by IP3 in each fraction. Three major subcellular fractions enriched in plasma membrane, mitochondria, and endoplasmic reticulum were characterized for their 5'-nucleotidase, glucose-6-phosphatase, succinate reductase, and angiotensin II binding activities. The fraction enriched in plasma membrane showed 7- and 20-fold increases in IP3 binding capacity over those enriched in endoplasmic reticulum and mitochondria, respectively, and contained a single class of high-affinity binding sites with Kd of 1.7 +/- 1.0 nM and concentration of 239 +/- 91 fmol/mg protein. IP3 binding reached equilibrium in 30 min at 0 degrees C, and the half-time of dissociation was about 15 min. The specificity of the IP3 binding sites was indicated by their markedly lower affinities for inositol 1-phosphate, phytic acid, fructose 1,6-bisphosphate, 2,3-bisphosphoglycerate, and inositol 1,3,4,5-tetrakisphosphate. The Ca2+-releasing activity of IP3 in the subcellular fractions was monitored with the fluorescent indicator, Fura-2. All three fractions showed ATP-dependent Ca2+ uptake and rapidly released Ca2+ in response in IP3. The fraction enriched in plasma membrane was the most active in this regard, releasing 174 +/- 67 pmol Ca2+/mg of protein compared to 45 +/- 10 and 48 +/- 7 pmol/mg protein for the fractions enriched in endoplasmic reticulum and mitochondria, respectively. These data suggest that the [32P]IP3 binding sites represent specific intracellular receptors through which IP3 mobilizes Ca2+ from a storage site associated (or co-purifying) with the plasma membrane of the rat liver. It is likely that a specialized vesicular system (to which IP3 can bind and trigger the release of Ca2+) is located in close proximity with the plasma membrane and is thus adjacent to the site at which IP3 is produced during stimulation of the hepatocyte by Ca2+-mobilizing hormones.     

Kinetic characteristics of Ca2+, Mg2+-ATPases in cells of the submandibular salivary gland of rats

Kinetic properties of Ca2+, Mg2+-ATPases membranes from acinar cells of rat submandibular salivary glands have been investigated. It was found that kinetics of ATP hydrolysis dependent on Ca2+, Mg2+-ATPases corresponds to the first-order reaction during first 2 min. It was found that the initial velocity of the reaction (V0), maximal amount of the reaction product (Pmax) and characteristic time of the reaction (T) comprised 1.8 +/- 0.4 and 1.6 +/- 0.2 mmole Pi/min per 1 mg protein, 7.5 +/- 1.3 and 1.4 +/- 0.2 mmole Pi/mg protein and 4.1 +/- 0.7 min and 1.1 +/- 0.1 for Ca2+-ATPases from plasma and endoplasmic reticulum membranes, correspondingly. High- and low-affinity sites of ATP and Ca2+-binding in Ca2+-ATPases from plasma and endoplasmic reticulum membranes were identified. Negative cooperation in ATP binding to Ca2+-ATPase from plasma membrane and a positive cooperation for Ca2+-ATPase from endoplasmic reticulum has been found. Ca2+ binding to low-affinity sites of both Ca2+-ATPases showed no cooperation, while Ca2+ binding to high-affinity sites showed the positive cooperation. Using the Hill's coordinates we have found the values of the Mg2+ Michaelis constant (K(Mg)) which yielded 3.89 x 10(-5) and 3.80 x 10(-5) mole/l for Ca2+-ATPases from plasma and endoplasmic reticulum membranes, correspondingly. It is supposed that obtained data are important for further studies of molecular and membrane mechanisms involved in the regulation of intracellular calcium signalling and secretion by salivary acinar cells.     

Autophagocytosis: in vitro induction by microtuble poisons.

In the rat liver in vivo autophagy has been shown to be induced by glucagon, cyclic AMP (cAMP), dibuturyl-cyclic AMP (db-cAMP) and vinblastine administration. Vinblastine and demecolcine were shown in this study to be potent inducers of autophagy also in Ehrlich ascites tumor cells in vitro. It is suggested that this effect is rather dependent on direct effect on membranes of endoplasmic reticulum than on the breakdown of microtubules by these compounds.     

Replication of coronavirus MHV-A59 in sac- cells: determination of the first site of budding of progeny virions

During infection of sac- cells by murine coronavirus MHV A59 the intracellular sites at which progeny virions bud correlate with the distribution of the viral glycoprotein E1. Budding is first detectable by electron microscopy at 6 to 7 hours post infection in small, smooth, perinuclear vesicles and tubules in a region transitional between the rough endoplasmic reticulum and the Golgi apparatus. At later times the rough endoplasmic reticulum becomes the major site of budding and accumulation of progeny virus particles. Indirect immunofluorescence microscopy shows that E1 is confined at 6 hours post infection to the perinuclear region while at later times it also accumulates in the endoplasmic reticulum. At 6 hours post infection the second viral glycoprotein, E2, is distributed throughout the endoplasmic reticulum and is not restricted to the site at which budding begins. Core protein, the third protein in virions, can be detected 2 hours before E1 is detectable and budding begins, and at 6 hours post infection it is distributed throughout the cytosol. We conclude that the time and the site at which the maturation of progeny virions occurs is determined by the accumulation of glycoprotein E1 in intracellular membranes. Only rarely do progeny virions bud directly into the cisternae of the Golgi apparatus but at least some already budded virions are transported to the Golgi apparatus where they occur in structures some of which also contain TPPase, a trans Golgi marker.     

Hormonal regulation of inositol 1,4,5-trisphosphate receptor in rat liver

Inositol 1,4,5-trisphosphate (IP3) is a second messenger which induces Ca2+ release from an intracellular store. We have investigated the properties of the [32P]IP3 binding sites in rat liver. Two specific [32P]IP3 receptors with KD of 2.3 and 88 nM and respective capacities of 33 fmol/mg protein and 195 fmol/mg protein have been detected in a crude membrane fraction prepared from rat liver homogenate. The pretreatment of the liver with IP3-dependent hormones increased two-fold the capacity of the high affinity site. This effect was partly reversed by dibutyryl cyclic AMP. Permeabilized hepatocytes also displayed two [32P]IP3 binding sites with KD of 1.5 and 84 nM and respective capacities of 8 and 300 fmol/10(6) cells. We have measured the [32P]IP3 binding and the IP3-induced 45Ca2+ release in the same batch of permeabilized hepatocytes. In a low Mg2+ medium, the EC50 for 45Ca2+ release was in close correlation with the KD for the low affinity site. These data suggest that an equilibrium between two states of the IP3 receptor is regulated by hormone action and the low affinity state is responsible for the intracellular Ca2+ release.     

Subcellular distribution and regulation of hepatic bilirubin UDP-glucuronyltransferase

We have investigated the subcellular location and regulation of hepatic bilirubin UDP-glucuronyltransferase, which has been presumed to be located largely in the smooth endoplasmic reticulum. Purity of subcellular membrane fractions isolated from rat liver was assessed by electron microscopy and marker enzymes. Bilirubin UDP-glucuronyltransferase activity was measured by radiochemical assay using a physiologic concentration of [14C]bilirubin, and formation rates of bilirubin diglucuronide and monoglucuronides (C-8 and C-12 isomers) were determined. Activity of the enzyme was widely distributed in subcellular membranes, the majority being found in smooth and rough endoplasmic reticulum, with small amounts in nuclear envelope and Golgi membranes. No measurable activity was found in plasma membranes or in cytosol. Synthesis of bilirubin diglucuronide as a percentage of total conjugates and the ratio of C-8/C-12 bilirubin monoglucuronide isomers formed were comparable in all membranes, suggesting that the same enzyme is present in all locations. However, the regulation of bilirubin UDP-glucuronyltransferase activity differed among intracellular membranes; enzyme activity measured in the presence of the allosteric effector uridine 5'-diphospho-N-acetylglucosamine exhibited latency in smooth endoplasmic reticulum and Golgi membranes, but not in rough endoplasmic reticulum and nuclear envelope. Since rough membranes comprise 60% of hepatocyte endoplasmic reticulum and bilirubin UDP-glucuronyltransferase activity in vitro is maximal in this membrane fraction under presumed physiologic conditions, it is likely that the rough endoplasmic reticulum represents the major site of bilirubin glucuronidation in hepatocytes.     

Change in gonadotropin-binding sites in intracellular organelles and plasma membranes during luteal growth, development and regression

Changes in the gonadotropin-binding sites in plasma membranes and several intracellular organelles of bovine corpora lutea of days 3, 13 and 19 of the cycle were investigated. These three times represent periods of rapid luteal growth (early luteal phase), maturity (mid luteal phase) and the onset of regression (late luteal phase), respectively. The 5'-nucleotidase activity was highest in the fraction possessing a predominance of plasma membranes. It was undetectable in nuclear fractions and detectable to a varying extent in fractions enriched with mitochondria-lysosomes, rough endoplasmic reticulum and Golgi. The gonadotropin-binding sites, as measured by 125I-human choriogonadotropin (hCG) specific binding, were found in all the subcellular organelles. Whereas the affinities remained about the same, the total number of available gonadotropin-binding sites in all the organelles increased from day 3 to 13 and then declined by day 19 of the cycle. Occupancy of binding sites by endogenous luteinizing hormone was not detectable and therefore was unlikely to be responsible for the changes in total number of available binding sites. Thus, binding site changes observed in all the organelles of early, mid and late luteal phase corpora lutea probably reflect actual changes in the steady-state turnover of binding sites. Morphometrically determined relative membrane counts of various subcellular organelles varied with the luteal phase. The relative total gonadotropin-binding sites, calculated from the relative membrane counts and the total number of available binding sites, increased in all the organelles from early to mid and then declined by late luteal phase. Plasma membranes of all three luteal phases contained greater relative total gonadotropin-binding sites than any other single intracellular organelle. However, all the intracellular organelles combined contained 59% of the total luteal cell gonadotropin-binding sites in early luteal phase which decreased to 43 and 28% by mid and late luteal phases respectively.     

Microsomal binding sites for antioestrogens in rat liver. Properties and detergent solubilization.

The properties of an antioestrogen binding site (AEBS), which has high affinity and specificity for nonsteroidal antioestrogens and structurally related compounds, have been studied in rat liver microsomes. When subcellular organelles were separated on Percoll density gradients the distribution of the AEBS paralleled that of NADPH-cytochrome c reductase, indicating that the AEBS is associated with the endoplasmic reticulum. Saturation analysis showed that [3H]tamoxifen was bound to a single class of saturable binding sites in liver microsomes with a KD of 0.9 +/- 0.1 nM at 0 degrees C. The equilibrium KD was not significantly different at 22 degrees C. The KD calculated from the association and dissociation rate constants for [3H]tamoxifen binding at 0 degrees C and 22 degrees C was compatible with the KD measured at equilibrium. Ligand specificity studies using tamoxifen analogues showed qualitatively similar structure-affinity relationships for the AEBS from both rat liver and the MCF 7 breast cancer cell line. In general structural modifications caused correspondingly greater changes in affinity for rat liver AEBS than for MCF 7 AEBS. The AEBS was solubilized from microsomal membranes with sodium cholate. This was the only detergent of nine tested that solubilized the site in high yield without loss of activity. Solubilization using cholate was more effective in the presence of 1 M-NaCl. In the solubilized state there was an apparent loss of [3H]tamoxifen binding activity which could be restored by dilution of the detergent. Gel filtration indicated an Mr of 440,000-490,000 for the AEBS-cholate complex. These studies demonstrate that rat liver contains high concentrations of a microsomal AEBS which has similar properties and specificity to the AEBS previously described in human breast cancer cells. This site can be solubilized by sodium cholate to supply material suitable for further purification.     

Effect of ribosome stripping procedures on antigenicity and conformation of endoplasmic reticulum membrane.

The effects of 3 different procedures for stripping ribosomes from membranes on theantigeniticity and conformation of isolated rough and smooth endoplasmic reticulum from rat liver were examined by microcomplement fixation and circular dichroism. Some of the blocked antigenic binding sites in rough endoplasmic reticulum became available after stripping of ribosomes. None of the 3 methods used is capable of stripping ribosomes completely from rough endoplasmic reticulum without the concomitant removal of protein from the membrane. Such loss of membrane protein by the stripping treatments is probably involved in the observed changes in rough endoplasmic reticulum, since a marked reduction in complement fixing capacity and in ellipticity of circular dichroism is observed also in smooth endoplasmic reticulum after similar treatments.     

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.