A comparison between adenylate cyclase solubilized from normal and Rous sarcoma virus-transformed chicken embryo fibroblasts

The specific activity of adenylate cyclase in membrane preparations obtained from Rous Sarcoma virus-transformed chicken embryo fibroblasts is two to four times lower than that found in untransformed membranes. Adenylate cyclase was solubilized from normal and transformed membranes in order to evaluate the influence of the membrane phase on the properties of the enzyme. Adenylate cyclase in normal and transformed membranes differed in specific activity, V for ATP, activation entropies, sensitivity to Ca2+, and stability at 37 degrees C. Solubilization with Brij 96 abolished or greatly reduced these differences. These data suggest that the differences between adenylate cyclase activities in normal and transformed chicken embryo fibroblasts are due either to differential modulation of enzyme activity by an effector which requires intact membranes for its effects, or indirect effects due to altered membrane properties.     

Alterations in components of adenylate cyclase associated with transformation of chicken embryo fibroblasts by Rous sarcoma virus

Regulation of adenylate cyclase coincident with transformation of chicken embryo fibroblasts by Rous sarcoma virus is manifest as a 10-50% decrease in basal, Mg2+-, and forskolin-stimulated activities; activities elicited by fluoride and guanosine 5'-O-(3-thiotriphosphate) are unaltered. The level of the catalytic component of adenylate cyclase, assessed with activated stimulatory guanine nucleotide-binding protein (Gs), increases approximately 1.5-fold. The level of the beta subunit common to Gs and the inhibitory regulatory protein assessed by enzyme-linked immunotransfer blotting, increases 2.7-fold. The isoelectric behavior of the beta subunit is unaltered. The amount of radiolabel incorporated into the alpha subunit of Gs (Mr = 45,000) upon incubation of membranes with 32P-labeled NAD and cholera toxin increases 3-fold upon transformation. Detergent extracts prepared from membranes of untransformed and transformed fibroblasts nevertheless exhibit equivalent abilities to reconstitute fluoride-stimulated activities to membranes of the cyc-variant of mouse S49 lymphoma cells. Islet-activating protein catalyzes incorporation of radiolabel from 32P-labeled NAD into 39,000- and 41,000-dalton proteins; the extent of radiolabel incorporation does not change upon transformation. Modest alterations in the isoelectric behaviors of substrates for cholera toxin and islet-activating protein occur.     

Adenylate cyclase activity is decreased in chick embryo fibroblasts transformed by wild type and temperature sensitive Schmidt-Ruppin Rous sarcoma virus

Chick embryo fibroblasts (CEF) transformed by the Schmidt-Ruppin strain of Rous sarcoma virus (RSV-SR) have decreased adenylate cyclase activity. In cells infected by a temperature-sensitive mutant of this virus (RSV-SR-T5), enzyme activity is near normal when the cells are grown at the non-permissive temperature (41°C) but decreases at the permissive temperature (36°). Adenylate cyclase activity decreases slowly over a 24 hr period to one half normal levels when CEF-RSV-SR-T5 are shifted from 41° to 36°C. The low enzyme activity in CEF-RSV-SR is not due to an alteration in the K_m ATP or a change in the kinetics of Mg⁺⁺ activation, and is not observed when the enzyme is assayed in the presence of NaF. We conclude that transformation by RSV-SR reduces adenylate cyclase activity by a different mechanism than the Bryan high-titer strain of RSV.     

Proteolysis of skeletal muscle adenylate cyclase. Destruction and reconstitution of fluoride and guanylnucleotide sensitivity

Adenylate cyclase was measured in skeletal muscle plasma membranes incubated with subtilisin. Under specific conditions the protease preferentially inactivated fluoride and guanylnucleotide sensitivity. Following protease treatment, membranes were solubilized with Lubrol 12A9 and subjected to ion-exchange chromatography. Adenylate cyclase was eluted with 200 mM NaCl; the enzyme recovered was completely unresponsive to either NaF or guanylyl imidodiphosphate. Responsiveness to the two ligands was restored by adding a heart fraction in which basal activity had been destroyed by heating at 40 degrees C or by adding a soluble skeletal muscle fraction in which basal activity had been largely destroyed by N-ethylmaleimide. The solubilized subtilisin-treated skeletal muscle preparation may serve as a source of catalytic activity for the study and purification of regulatory factors for adenylate cyclase.     

Proteolysis of skeletal muscle adenylate cyclase Destruction and reconstitution of flouride and guanylnucleotide sensitivity

Adenylate cyclase was measured in skeletal muscle plasma membranes incubated with subtilisin. Under specific conditions the protease preferentially inactivated flouride and guanylnucleotide sensitivity. Following protease treatment, membranes were solubilized with Lubrol 12A9 and subjected to ion-exchange chromatography. Adenylate cyclase was eluted with 200 mM NaCl; the enzyme recovered was completely unresponsive to either NaF or guanylyl imidodiphosphate. Responsiveness to the two ligands was restored by adding a heart fraction in which basal activity had been destroyed by heating at 40°C or by adding a soluble skeletal muscle fraction in which basal activity had been largely destroyed by N-ethylmaleimide. The solubilized subtilisin-treated skeletal muscle preparation may serve as a source of catalytic activity for the study and purification of regulatory factors for adenylate cyclase.     

Solubilization of adenylate cyclase of brain membranes by lipid peroxidation

Adenylate cyclase in the membrane fractions of bovine and rat brains, but not in rat liver plasma membranes, was solubilized by treatment with Fe²⁺ (10 μM) plus dithiothreitol (5 mM). Solubilization of the enzyme by these agents was completely prevented by simultaneous addition of N,N′-diphenyl-p-phenylenediamine (DPPD), an inhibitor of lipid peroxidation. Ascorbic acid also solubilized the enzyme from the brain membranes. Lipid peroxidation of the brain membranes was characterized by a selective loss of phosphatidylethanolamine. Solubilization of membrane-bound enzymes by Fe²⁺ plus dithiothreitol was not specific for adenylate cyclase, because phosphodiesterase, thiaminediphosphatase and many other proteins were also solubilized. Solubilized adenylate cyclase had a high specific activity and was not activated by either NaF, 5′-guanylyl imidodiphosphate (Gpp[NH]p) or calmodulin. These results suggested that lipid peroxidation of the brain membranes significantly solubilized adenylate cyclase of high specific activity.     

The purification and characterization of plasma membranes and the subcellular distribution of adenylate cyclase in mouse parotid gland.

1. Plasma membranes have been purified 17-fold from mouse parotid gland homogenates prepared in hypertonic sucrose media using differential centrifugation. The method is fast and simple. The membranes were characterised by electron microscopy, enzyme composition and chemical composition. Further purification was achieved by isopycnic centrifugation in discontinuous sucrose gradients. 2. The purified membranes contain an adenylate cyclase activity which is stimulated by isoproterenol and fluoride. Only 50% of the total adenylate cyclase activity sedimented in the plasma membrane fraction. The rest of the activity resided in the crude nuclear and mitochondrial pellets. However, this adenylate cyclase activity was not associated with these organelles but with membrane fragments in the pellets. Purified nuclei did not contain adenylate cyclase activity. 3. Adenylate cyclase activity was also localised by electron microscopic cytochemistry. Besides being found at the plasma membrane, large amounts of adenylate cyclase were found in a small proportion of the vesicles within the acinar cells, which appeared to be secondary lysosomes. 4. Adenylate cyclase activities, under standard assay conditions, are proportional to the time of incubation and the concentration of enzyme. The enzyme requires both Mg-2+ and CA-2+ for activity. Isoproterenol increased activity 2-fold and this increase is abolished by beta-adrenergic blocking agents.     

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.     

Heterogeneous localization of adenylate and guanylate cyclases in R3230AC rat mammary adenocarcinoma cells

Adenylate and guanylate cyclase activities were demonstrated in R3230AC rat mammary adenocarcinomas by electron microscopic cytochemistry. Adenylate (AC) and guanylate (GC) cyclases were detected on plasma membrane of tumor epithelial cells, but not on fibroblasts and endothelial cells in the perivascular space. Both AC and GC activities were enriched in tumor epithelial cells at the periphery of the tumor lobular parenchyma rather than in cells in central core of the lobular parenchyma. Furthermore, the tumor cell plasma membranes facing the connective tissue stroma were in paucity or devoid of either enzyme activity. These heterogeneous distributions of both AC and GC among tumor epithelia suggest that R3230AC epithelial cells in different parts of the tumor mass may vary significantly in their regulation of cellular physiology.     

Activation of adenylate cyclase in cultured fibroblasts by trypsin

Adenylate cyclase activity measured in membranes of cultured normal rat kidney (NRK) fibroblasts was markedly increased by prior treatment of the intact cells with trypsin. Cell population density influenced the extent of activation observed. Trypsin treatment of sparse cells significantly enhanced adenylate cyclase activity, whereas similar treatment of confluent cells caused only a slight increase in adenylate cyclase activity. The degree of activation noted after trypsin treatment also varied depending on the adenylate cyclase function measured. Activity determined in the presence of GTP alone showed the greatest increase after trypsin treatment. Similar enhancement of adenylate cyclase activity of a washed cell membrane preparation was achieved by the addition of low concentrations of trypsin directly to the adenylate cyclase reaction mixture. The membranes of confluent NRK fibroblasts initially exhibited higher adenylate cyclase activity than did membranes of sparse cells. The present results suggest that this change in adenylate cyclase activity at cell confluence is not due to an increase in the amount of adenylate cyclase in the cell membrane but rather to a change in membrane components that regulate its activity. Proteolytic activation of adenylate cyclase appears to result from degradation of cell membrane proteins that modulate the activity of this enzyme.     

Adenylate cyclase of Dictyostelium discoideum: solubilization and Mn2+-dependency

Adenylate cyclase from Dictyostelium discoideum was solubilized under alkaline conditions using the zwitterionic detergent CHAPS. In contrast to the membrane bound adenylate cyclase, the solubilized enzyme can use only Mn2+, but is inactive in the presence of Mg2+.     

Direct evidence for co-localization of adenylate cyclase,dopamine-β-hydroxylase and cytochrome b562 to bovine chromaffin granule membranes

Adenylate cyclase, dopamine-β-hydroxylase and cytochrome b₅₆₂ have been found to co-equilibrate on equilibrium sucrose gradients of lysed chromaffin granule membranes from bovine adrenal medulla. Peak activities for these enzymes, as well as maximum membrane protein concentration, were found to coincide at d = 1.11 gm cm^?3, and the ratios of adenylate cyclase to both dopamine-β-hydroxylase and cytochrome b₅₆₂ were constant across the entire peak. Adenylate cyclase activity has been reported previously to co-purify with chromaffin granule membranes, and we conclude, on the basis of these new data, that adenylate cyclase is also an intrinsic granule membrane enzyme.     

Incorporation of rat brain adenylate cyclase into artificial phospholipid vesicles.

Adenylate cyclase was solubilized from rat brain particulate fraction with the nonionic detergent, Nonidet P-40. Incubation of detergent-solubilized adenylate cyclase with liposomes prepared from egg yolk phosphatidylcholine results in virtually quantitative incorporation of the enzyme activity into phospholipid vesicles. Incorporation of adenylate cyclase into liposomes results in an approximately 10- to 20-fold purification relative to the solubilized preparation giving a final specific activity of about 50 nmol of cAMP min-1 mg-1. The detergent-solubilized adenylate cyclase migrates as a broad band between 14 and 33% sucrose on density gradient centrifugation, separated from the endogenous phospholipid. Following overnight incubation of the solubilized enzyme with exogenous phospholipid, all enzyme activity is found in a narrow band between 7 and 9% sucrose, co-migrating with the phospholipid. The adenylate cyclase could not be released from the liposomes by extraction with high ionic strength, low ionic strength-EDTA, or sonication. Treatment of liposomal adenylates cyclase with soluble proteases or immobilized trypsin destroys enzyme activity. Thus, it is likely that a functionally important part of the enzyme molecule is exposed on the outer surface of the liposome. Optimal conditions for the incorporation of adenylate cyclase into liposomes, and some effects of manipulating the phospholipid composition on enzyme activity are reported.     

Characterization and subcellular localization of nucleotide cyclases in calf thymus lymphocytes

The role of cyclic nucleotides in the regulation of lymphocyte growth and differentiation remains controversial, as an adequate characterization of the key enzymes, adenylate cyclase and guanylate cyclase, in the plasma membrane of lymphocytes is still lacking. In this study, calf thymus lymphocytes were disrupted by nitrogen cavitation and various cellular fractions were isolated by differential centrifugation and subsequent sucrose density ultracentrifugation. As revealed by the chemical composition and the activities of some marker enzymes, the plasma membrane fraction proved to be highly purified. Nucleotide cyclases were present in the plasma membranes in high specific activities, basal activities of adenylate cyclase being 13.7 pmol/mg protein per min and 34.0 pmol/mg protein per min for the guanylate cyclase, respectively. Adenylate cyclase could be stimulated by various effectors added directly to the enzyme assay, including NaF, GTP, 5'-guanylyl imidodiphosphate, Mn2+ and molybdate. Addition of beta-adrenergic agonists only showed small stimulating effects on the enzyme activity in isolated plasma membranes. Basal activity of adenylate cyclase as well as activities stimulated by NaF or 5'-guanylyl imidodiphosphate exhibited regular Michaelis-Menten kinetics. Activation by both agents only marginally affected the Km values, but largely increased Vmax. The activity of the plasma membrane-bound guanylate cyclase was about 10-fold enhanced by the nonionic detergent Triton X-100 and high concentrations of lysophosphatidylcholine, but was slightly decreased upon addition of the alpha-cholinergic agonist carbachol. Basal guanylate cyclase indicated to be an allosteric enzyme, as analyzed by the Hill equation with an apparent Hill coefficient close to 2. In contrast, Triton X-100 solubilized enzyme showed regular substrate kinetics with increasing Vmax but unaffected Km values. Thus the lymphocyte plasma membrane contains both adenylate cyclase and guanylate cyclase at high specific activities, with properties characteristic for hormonally stimulated enzymes.     

Activation of solubilized liver membrane adenylate cyclase by nucleotides

Liver plasma membranes isolated from hypophysectomized rats were treated with 0.1 M Lubrol-PX, a nonionic detergent, and centrifuged at 165,000 × g for 1 hour. Adenylate cyclase activity remaining in the supernate had a specific activity that was at least equal to that of the particulate enzyme. The activity of the solubilized, non-sedimentable adenylate cyclase, as well as the membrane bound enzyme, was increased by GTP, ITP, and GMP-PCP at 10^?4 M. The activity of the solubilized, non-sedimentable enzyme increased linearly with GTP from 10^?6 to 10^?4 M but there was no further increase in the activity of the solubilized enzyme with 10^?3 M GTP. In contrast, the particulate liver membrane enzyme activity increased exponentially with GTP from 10^?6 to 10^?4 M and was further increased by 10^?3 M GTP. These data indicate that GTP, ITP or GMP-PCP have direct effects on solubilized adenylate cyclase. This effect is in addition to a role of nucleotides in modifying membrane structure (16).     

Hexose uptake enhancing factor released from rous sarcoma cells

Conditioned media from Rous sarcoma virus transformed chicken embryo fibroblasts stimulate the uptake of 2-deoxyglucose in normal chicken fibroblasts. The factor responsible for this effect, which is also shed in very low amount by non-transformed fibroblasts, is destroyed by trypsin and not linked to the protease and plasminogen activator activities present in the media. Its apparent molecular weight, determined by gel filtration, is about 20.000 daltons. The factor released by transformed cells might be related to the monomeric form of a family of glucose binding and transport proteins recently reported by Lee and Lipmann ('78) to be detached by detergents from normal and transformed cells.     

Solubilization and separation of the glucagon receptor and adenylate cyclase in guanine nucleotide-sensitive states.

Adenylate cyclase in liver membranes was solubilized with Lubrol PX and partially purified by gel filtration. The partially purified enzyme was susceptible to activation by guanyl-5'-yl imidodiphosphate (Gpp(NH)p). Studies on the binding of [3H]Gpp(NH)p to various fractions eluted from the gels revealed that an upper limit of 1% of the Gpp(NH)p binding sites is associated with adenylate cyclase activity stimulated by the nucleotide. The glucagon receptor, pretagged with 125I-glucagon in the membranes, solubilized with Lubrol PX, and fractionated on the same gel columns, eluted in a peak fraction that overlaps with, but is separate from, adenylate cyclase in its Gpp(NH)p-stimulated form. Addition of GTP to the solubilized glucagon-receptor complex caused complete dissociation of the complex, as has been shown with the membrane-bound form of the complex. Since the GTP-sensitive form of the glucagon receptor complex separates from the Gpp(NH)p-sensitive form of adenylate cyclase, it is concluded that the receptor and the enzyme are separate molecules, each associated with a distinct nucleotide regulatory site or component. These findings are discussed in terms of the possible structure of the hormone-sensitive state of adenylate cyclase.     

Rat testis mitochondrial adenylate cyclase. Partial purification and characterization.

1. Adenylate cyclase (EC 4.6.1.1) from rat testis mitochondria has been solubilized by treatment with the non-ionic detergent Lubrol PX. The soluble enzyme was further purified by DEAE-cellulose chromatography. 2. The specific activity of the adenylate cyclase eluted from the DEAE-cellulose column was found to be four times higher than that of an intact mitochondrial preparation. At this step the enzyme shows a sedimentation coefficient of 4.2 S and a diffusion coefficient (D) of 3.12 - 10- minus 7 cm-2/sec. 3. Solubilization of the adenylate cyclase resulted in loss of responsiveness to gonadotrophic hormones. Addition of phosphatidylserine to the soluble preparation partially restored the activation of adenylate cyclase by human chorionic gonadotrophin. 4. The results of this study suggest that the activity of the adenylate cyclase may be dependent on the membrane-bound phospholipids and that the enzyme attached to the mitochondrial membranes has some properties which are similar to the adenylate cyclase found to be associated with other membrane systems of the cell.?     

The increase in hormone-stimulated adenylate cyclase activity following Rous sarcoma virus transformation

Rous sarcoma virus (RSV)-infected chicken embryo cells were used to study the effect of viral transformation on the hormone-stimulated synthesis of cyclic AMP. Transformation by RSV greatly increased the cells' ability to synthesize and accumulate cyclic AMP in response to the beta-adrenergic agonist isoproterenol as compared to untransformed cells. This enhancement was observed in both intact cells and in membranes prepared from these cells. The inclusion of guanosine 5'-0-(3-thiotriphosphate), a nonhydrolyzable analogue of GTP, in assays of adenylate cyclase activity did not abolish the quantitative differences between the transformed and normal cell membranes. Infection of cells by Rous-associated virus, which lacks the oncogene src, did not induce this hyperresponsiveness thus indicating the probable involvement of the src gene product in this phenomenon. The duration of the isoproterenol-induced cyclic AMP elevation was longer in the transformed than in the untransformed cells; transformed cells, unlike untransformed cells, required at least 120 min before full desensitization became established. Membranes prepared from transformed cells specifically bound more than 5 times the quantity of the beta-adrenergic radiolabeled antagonist (-)3H-dihydroalprenolol and 125I-iodocyanopindolol compared to the untransformed cell membranes. Thus, it appears that major differences between the transformed and normal phenotypes reside in the concentration of membrane beta-adrenergic receptors and the inability of RSV-transformed cells to self-limit their response to specific external stimuli.     

Association of pp60src and src protein kinase activity with the plasma membrane of nonpermissive and permissive avian sarcoma virus-infected cells.

The intracellular localization of pp60src and src protein kinase activity in avian sarcoma virus (ASV)-infected chicken embryo fibroblasts and transformed and morphologically reverted field vole cells was examined by subcellular fractionation procedures. Fractionation by differential centrifugation of Dounce-homogenized cellular extracts prepared from vole cells showed that 83 to 91% of pp60src sedimented with particulate subcellular components from both transformed and revertant vole cells. A slightly lesser amount (60 to 70%) of pp60src was found associated with the particulate fraction from ASV-infected chicken embryo fibroblasts. The distribution of src protein kinase activity in the cytosol and particulate cell fractions was identical to that of pp60src, indicating no detectable differences in the activity of cytosol- and particulate-associated pp60src. When subcellular components of the cell were fractionated by discontinuous sucrose gradient centrifugation, similar amounts of both pp60src and src protein kinase activity cosedimented with the plasma membrane fractions from both transformed and revertant vole cells, as well as from ASV-infected chicken embryo fibroblasts. src protein kinase activity associated with plasma membrane fractions prepared from vole cells and ASV-infected chicken embryo fibroblasts was resistant to extraction with high salt concentrations, but partial elution was achieved with nonionic detergent. Thus, in both transformed and morphologically reverted vole cells, pp60src is intimately associated with the plasma membrane. Since transforming virus can be rescued from revertant vole cells by fusion to chicken embryo fibroblasts, revertant vole cell pp60src is capable of inducing morphological transformation. Thus, although the data presented herein suggest that transformation requires the association of pp60src with the plasma membrane, the binding of pp60src to the plasma membrane per se is insufficient to induce morphological transformation and requires the additional interaction with a specific target membrane protein which appears to be defective in revertant vole cells.