guanosine-5--o-(3-thiotriphosphate) and Lymphoma

Mammals express nine membranous adenylyl cyclase isoforms (ACs 1-9), a structurally related soluble guanylyl cyclase (sGC) and a soluble AC (sAC). Moreover, Bacillus anthracis and Bacillus pertussis produce the AC toxins, edema factor (EF), and adenylyl cyclase toxin (ACT), respectively. 2'(3')-O-(N-methylanthraniloyl)-guanosine 5'-[gamma-thio]triphosphate is a potent competitive inhibitor of AC in S49 lymphoma cell membranes. These data prompted us to study systematically the effects of 24 nucleotides on AC in S49 and Sf9 insect cell membranes, ACs 1, 2, 5, and 6, expressed in Sf9 membranes and purified catalytic subunits of membranous ACs (C1 of AC5 and C2 of AC2), sAC, sGC, EF, and ACT in the presence of MnCl(2). N-Methylanthraniloyl (MANT)-GTP inhibited C1.C2 with a K(i) of 4.2 nm. Phe-889 and Ile-940 of C2 mediate hydrophobic interactions with the MANT group. MANT-inosine 5'-[gamma-thio]triphosphate potently inhibited C1.C2 and ACs 1, 5, and 6 but exhibited only low affinity for sGC, EF, ACT, and G-proteins. Inosine 5'-[gamma-thio]triphosphate and uridine 5'-[gamma-thio]triphosphate were mixed G-protein activators and AC inhibitors. AC5 was up to 15-fold more sensitive to inhibitors than AC2. EF and ACT exhibited unique inhibitor profiles. At sAC, 2',5'-dideoxyadenosine 3'-triphosphate was the most potent compound (IC(50), 690 nm). Several MANT-adenine and MANT-guanine nucleotides inhibited sGC with K(i) values in the 200-400 nm range. UTP and ATP exhibited similar affinities for sGC as GTP and were mixed sGC substrates and inhibitors. The exchange of MnCl(2) against MgCl(2) reduced inhibitor potencies at ACs and sGC 1.5-250-fold, depending on the nucleotide and cyclase studied. The omission of the NTP-regenerating system from cyclase reactions strongly reduced the potencies of MANT-ADP, indicative for phosphorylation to MANT-ATP by pyruvate kinase. Collectively, AC isoforms and sGC are differentially inhibited by purine and pyrimidine nucleotides.

Topics: Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Algorithms; Animals; Binding Sites; Brain; Catalytic Domain; Cattle; Cell Line; Cell Membrane; Dose-Response Relationship, Drug; Enzyme Inhibitors; Escherichia coli; GTP Phosphohydrolases; Guanosine 5'-O-(3-Thiotriphosphate); Guanylate Cyclase; Inhibitory Concentration 50; Insecta; Kinetics; Lymphoma; Magnesium; Models, Chemical; Models, Molecular; Models, Statistical; Nucleotides; Phosphorylation; Protein Isoforms; Purines; Pyrimidines; Rats; Receptors, Cytoplasmic and Nuclear; Soluble Guanylyl Cyclase; Thionucleotides

Topics: Animals; Autoradiography; Cell Line; Cell Membrane; Chromatography; Chromatography, Gel; Chromatography, Ion Exchange; Durapatite; Electrophoresis, Polyacrylamide Gel; Erythrocyte Membrane; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Indicators and Reagents; Lymphoma; Macromolecular Substances; Mice; Molecular Weight; NAD; Sulfur Radioisotopes; Swine; Tumor Cells, Cultured; Virulence Factors, Bordetella

We have assessed the possible interaction between the microtubular component of the cytoskeleton and signal transducing GTP-binding (G) proteins by examining the ability of colchicine and vinblastine (two microtubule disrupters) to alter Gs and Gi protein activity in S49 lymphoma cells. Treatment of wild type S49 cells with cholchicine and vinblastine increased beta-adrenergic agonist- and prostaglandin (PG) E1-stimulated formation of cAMP. The microtubular inhibitor nocodazole also enhanced isoproterenol-stimulated cAMP accumulation, whereas the inactive analog of colchicine, beta-lumicolchicine, did not have this action. Based on data obtained with wild type, cyc-, and UNC S49 cells, we determined that enhancement in cyclic AMP accumulation is proximal to the catalytic (C) unit of adenylylcyclase, distal to hormone receptors, and seems to be located on Gs. Treatment with colchicine increased guanosine 5'-(gamma-thio)triphosphate-stimulated accumulation of cAMP in permeabilized wild type cells. The increase in activity of Gs appeared not to result from a change in the intracellular concentration of GTP. Treatment of cells with colchicine or vinblastine also increased the amount of the alpha s-C complex, as assessed by the binding of [3H]forskolin to intact cells at 37 degrees C. In contrast to the observed effect on Gs, treatment of wild type S49 cells with colchicine failed to modify the degree of inhibition of cAMP formation produced by somatostatin, which acts via the activation of Gi. These data suggest that microtubules regulate the ability of Gs to interact with and activate the catalyst of adenylylcyclase.

Topics: Adenylyl Cyclases; Catalysis; Cholera Toxin; Colchicine; Colforsin; Cyclic AMP; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Isoproterenol; Lymphoma; Microtubules; Signal Transduction; Somatostatin; Tumor Cells, Cultured; Vinblastine

Glycosylphosphatidylinositol (GPI) is biosynthesized by the sequential addition of carbohydrates to phosphatidylinositol (PI). In the first two reactions, GlcNAc is transferred from UDP-GlcNAc to PI and then deacetylated to form GlcNAc-PI and GlcN-PI, respectively. In this paper, stimulation of GlcNAc-PI deacetylation by GTP, is reported. Addition of this nucleotide triphosphate to incubations in which GPI precursors were synthesized from UDP-[6-3H]GlcNAc by microsomes prepared from the lymphoma cell line EL4 resulted in a shift in the relative amount of each intermediate formed such that [6-3H]GlcN-PI was the predominant product. GTP also increased the total synthesis of the first GPI intermediate, GlcNAc-PI, by inhibiting reactions that metabolize UDP-[6-3H]GlcNAc into non-GPI-related products. However, unlike the stimulation of GlcNAc-PI deacetylation, ATP was equally effective in increasing the formation of GlcNAc-PI. An additional product, tentatively identified as [6-3H]GlcN-PI(acyl-inositol), was also detected when GTP was present in the incubation. The synthesis of this GPI precursor, which is proposed to be the third intermediate in GPI biosynthesis in mammals, was increased by GTP because the level of GlcN-PI, the substrate for acylation, was elevated. To isolate the effects of GTP on the GlcNAc-PI deacetylation, this reaction was studied directly by using [6-3H]GlcNAc-PI as the substrate. The stimulation was found to be specific for the guanosine-containing nucleotide triphosphate and optimal with approximately 1 mM GTP. Both the reaction rate at early time points and the total amount of deacetylated product formed in 60 min were increased by GTP. The effect on the second reaction of the pathway does not appear to be coupled to the first reaction because GlcNAc-PI deacetylation was increased by GTP in microsomes from cells defective in the GlcNAc-PI synthesis. Finally, 0.5 mM GTP gamma S (guanosine 5'-O-(thiotriphosphate)) completely inhibited the stimulation of GlcNAc-PI deacetylation caused by 1 mM GTP, indicating that hydrolysis of the nucleotide triphosphate was required for this effect. Although the mechanism and role of the GTP stimulation of GlcNAc-PI deacetylation is not clear, this regulation could influence the biosynthesis of mature GPI precursors and the subsequent expression of GPI-anchored proteins.

Topics: Acetylglucosamine; Animals; Glycosylphosphatidylinositols; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Kinetics; Lymphoma; Mice; Microsomes; Phosphatidylinositol Diacylglycerol-Lyase; Phosphatidylinositols; Phosphoric Diester Hydrolases; Ribonucleotides; Tumor Cells, Cultured; Uridine Diphosphate N-Acetylglucosamine

We reported [Ransnäs, Svoboda, Jasper & Insel (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7900-7903] that in intact S49 lymphoma cells the beta-adrenergic-receptor agonist isoprenaline dissociates the stimulatory guanine-nucleotide-binding protein, Gs, into its alpha s and beta gamma subunits, leading to redistribution of alpha s from plasma membranes to the cytoplasm. In the present studies we investigated the kinetics of Gs dissociation and membrane release in plasma membranes from S49 lymphoma cells. We analysed cholate extracts of membranes for alpha s levels by a competitive e.l.i.s.a. with a polyclonal antibody that selectively recognizes monomeric alpha s and we assayed supernatant fractions using both competitive e.l.i.s.a. and immunoblotting. The plasma membranes contained 19.3 +/- 1.4 pmol of alpha s/mg of membrane protein and lacked significant dissociation of Gs and activity of adenylate cyclase in the absence of guanine nucleotides. Mg2+ ions were obligatorily required for isoprenaline-induced dissociation of Gs in plasma membranes and for membrane release of alpha s. At a physiological concentration of free Mg2+ ions (100 microM), 100 microM-GTP induced a slow first-order (k = 0.038 +/- 0.004 min-1) dissociation of 17.8 +/- 1.2 pmol of Gs/mg of membrane protein. A substantial increase in the dissociation rate of Gs was achieved by addition of 1 microM-isoprenaline and 100 microM-GTP; 18.4 +/- 0.9 pmol of Gs/mg of membrane protein was dissociated, with a kappa of 1.49 +/- 0.22 min-1. The effect of isoprenaline on the dissociation rate and on membrane release of Gs was completely blocked by the beta-adrenergic receptor antagonist propranolol. The concentration-response relationship for isoprenaline-induced dissociation during the first 1 min after addition of hormone yielded a kappa act. of 16 +/- 5 nM, whereas the kappa act. for isoprenaline-induced membrane release was 10 nM. We conclude that release of alpha s from plasma membranes is likely to accompany Gs-subunit dissociation and constitutes a potentially important facet of Gs action.

Topics: Animals; Cell Line; Cell Membrane; Enzyme-Linked Immunosorbent Assay; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Isoproterenol; Kinetics; Lymphoma; Macromolecular Substances; Magnesium; Mice; Receptors, Adrenergic, beta; Tumor Cells, Cultured

After ADP-ribosylation by cholera toxin which promotes dissociation of the subunits, the alpha-subunit of Gs (Gs alpha) remained strongly associated with plasma membranes of wild-type S49 cells, since its interaction with the membrane was insensitive to 1 M KCl. Its association with the membrane was partially disrupted by 6 M urea and totally abolished by treatment with alkali at pH greater than or equal to 11.5. In vitro translated Gs alpha could interact with plasma membranes from the cyc- mutant of S49 cells as revealed by its cosedimentation with the membrane fraction and incubation of reconstituted membranes with GTP gamma S did not alter anchorage of Gs alpha. The characteristics of the association of in vitro translated Gs alpha with cyc- membranes after GTP gamma S treatment, i.e. sensitivity to 1 M KCl, 6 M urea and alkali treatment, were very similar to those described for the ADP-ribosylated form in wild-type membranes. Restoration of the coupling between the adrenergic receptor and adenylate cyclase further confirmed the vectorial reconstitution of cyc- membranes by in vitro translated alpha-subunit of Gs.

Topics: Adenosine Diphosphate Ribose; Adenylyl Cyclases; Animals; Cell Membrane; Enzyme Activation; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Hydrogen-Ion Concentration; Isoproterenol; Lymphoma; Mice; Molecular Weight; Mutation; NAD; Osmolar Concentration; Protein Biosynthesis; RNA, Messenger; Thionucleotides; Tumor Cells, Cultured

The role of a specific guanine nucleotide binding (G protein) protein in coupling murine B lymphocyte receptor immunoglobulin to inositol phospholipid hydrolysis was investigated. Using an in vitro system with isolated membranes, we have observed specific enhancement of GTP binding subsequent to ligand-induced receptor crosslinking. Induced increases were inhibited by pretreatment with pertussis toxin which catalyzed ADP-ribosylation of a 43 kDa substrate. Involvement of this G protein with receptor immunoglobulin-induced inositol phospholipid hydrolysis was evidenced by the ability of pertussis toxin to block this response. This report, then, indicates that the B lymphocyte antigen receptor belongs to a family of receptors which are linked to inositol phospholipid hydrolysis through a G protein.

Topics: Adenosine Diphosphate Ribose; Animals; B-Lymphocytes; Cell Membrane; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Hydrolysis; Immunoglobulin M; Immunoglobulin mu-Chains; Lymphoma; Mice; Pertussis Toxin; Phosphatidylinositols; Receptors, Immunologic; Thionucleotides; Tumor Cells, Cultured; Virulence Factors, Bordetella

GTP-binding proteins have been implicated as transducers of a variety of biological signaling processes. These proteins share considerable structural as well as functional homology. Due to these similarities, it was thought that a monoclonal antibody that inhibits the light activation of the rod outer segment GTP-binding protein, tranducin (Gt), might exert some functional effect upon the G proteins that regulate the adenylate cyclase system. Antibody 4A, raised against the alpha subunit of Gt, cross-reacted (by hybridization on nitrocellulose) with purified alpha subunits of other G proteins (Gi and Gs, regulatory guanyl nucleotide-binding proteins that mediate inhibition and stimulation of adenylate cyclase, respectively) as long as they were not denatured. This antibody, which interferes with rod outer segment cGMP phosphodiesterase activation by blocking interaction between rhodopsin and Gt, also interfered with actions of both the stimulatory and inhibitory G proteins of adenylate cyclase from rat cerebral cortex membranes. Effects of monoclonal antibody (mAb) 4A were dose-dependent and not reversed by washing. mAb 4A also blocked the Gi-mediated inhibition of adenylate cyclase in the cyc- variant of S49 lymphoma and in doing so raised the level of adenylate cyclase activity in both the cyc- variant and the S49 wild type. There was no effect of mAb 4A on adenylate cyclase activity of the resolved catalytic subunit. These results suggest that the well known sequence homologies among the G proteins involved in cellular signal transduction may extend to the sites that interact with other members of signal-transducing cascades (receptors and effector molecules). Therefore, antibody 4A may serve as a useful tool to probe the similarities and differences among the various systems.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Antibodies, Monoclonal; Antibody Specificity; Cell Membrane; Cerebral Cortex; Enzyme Activation; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Guanylyl Imidodiphosphate; Immunoblotting; Light; Lymphoma; Mice; Photoreceptor Cells; Pineal Gland; Rats; Rhodopsin; Rod Cell Outer Segment; Thionucleotides; Transducin; Tumor Cells, Cultured

The effects of ethanol on the beta adrenergic receptor-coupled adenylate cyclase system were examined in vitro using membranes prepared from S49 lymphoma cells. Ethanol caused a dose-dependent increase in adenylate cyclase activity in membranes prepared from wild-type cells when the activity was measured in the presence of GTP. Activity measured in the presence of isoproterenol was also increased by ethanol, but the fold-stimulation by isoproterenol was lower in the presence of ethanol. Ethanol also shifted the dose-response curve for stimulation of the enzyme by isoproterenol to the right. This shift was due to a decrease in the affinity of the beta adrenergic receptor for isoproterenol. A decrease in the affinity of the receptor for the antagonists [125I]iodopindolol and propranolol was also observed, but the magnitude of this effect was less than that seen with the agonist isoproterenol. The density of binding sites for [125I]iodopindolol was not affected by ethanol. Dose-response curves for NaF and guanosine-5'-O-(3-thiotriphosphate), both of which stimulate adenylate cyclase activity through an effect on the stimulatory guanine nucleotide-binding protein (Gs), were shifted to the left by the addition of ethanol. In membranes prepared from the CYC- variant of S49 cells, which lacks the alpha subunit of Gs, guanosine-5'-O-(3-thiotriphosphate) inhibited forskolin-stimulated adenylate cyclase activity. The inhibition by guanosine-5'-O-(3-thiotriphosphate) was not affected by ethanol. In membranes prepared from both wild-type and CYC- S49 cells, ethanol inhibited forskolin-stimulated adenylate cyclase activity. Whereas the inhibition of this activity by GTP was greatly attenuated in membranes prepared from CYC- S49 cells which had been pretreated with pertussis toxin, the inhibition by ethanol was not affected by pretreatment with pertussis toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenylyl Cyclases; Animals; Cell Line; Dose-Response Relationship, Drug; Ethanol; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Isoproterenol; Lymphoma; Molecular Weight; Propranolol; Receptors, Adrenergic, beta; Thionucleotides

We have recently reported (Ransnäs, L.A., and Insel, P.A. (1988) J. Biol. Chem. 263, 9482-9485) development of antipeptide antibodies to the alpha s protein of the stimulatory guanine nucleotide binding regulatory protein, Gs, and use of one of these antibodies, GS-1, to quantitate Gs levels in S49 lymphoma cell membranes. Another of these antibodies, termed GS-2, appears to detect only dissociated alpha s, but not the heterotrimer alpha s beta gamma. Using a competitive enzyme-linked immunosorbent assay, we have found that the guanine nucleotides GTP and guanosine 5'-O-(thiotriphosphate) (GTP gamma S) (but not GDP) and the beta-adrenergic receptor agonist isoproterenol activate Gs in native S49 cell membrane by subunit dissociation. Evidence for this includes detection of dissociated alpha s in membrane extracts and release of alpha s from S49 cell membranes treated with GTP gamma S or isoproterenol. Moreover, the estimates of apparent stoichiometry for this dissociation indicate that each beta-adrenergic receptor is able to activate greater than or equal to 100 molecules of Gs in native membranes. Thus, receptor-mediated dissociation of Gs is likely to be the major site of amplification of signal transduction by agonists active at hormone receptors that link to Gs.

Topics: Animals; Cell Line; Cell Membrane; Enzyme-Linked Immunosorbent Assay; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Isoproterenol; Kinetics; Liver; Lymphoma; Macromolecular Substances; Rabbits; Thionucleotides

Treatment of intact human platelets and S49 lymphoma cyc- cells with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, impairs GTP-dependent and hormone-induced inhibition of adenylate cyclase, an action mediated by the inhibitory coupling protein Ni. In contrast, receptor-independent activation of Ni with subsequent adenylate cyclase inhibition induced by the stable GTP analog, guanosine 5'-[gamma-thio]triphosphate, was affected in neither the potency nor onset of Ni activation by the stable GTP analog, in both membrane systems studied. The data indicate that modification of Ni following phorbol ester treatment does not impair its activation by stable GTP analogs.

Topics: Adenosine Diphosphate Ribose; Adenylyl Cyclase Inhibitors; Blood Platelets; Epinephrine; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Hydrolysis; Lymphoma; Phorbols; Phosphorylation; Protein Kinase C; Tetradecanoylphorbol Acetate; Thionucleotides

Four members of a family of GTP-binding proteins (G-proteins) which translate stimulation of extracellular receptors into regulation of intracellular enzymes were isolated from the bovine central nervous system. These proteins were examined for functional similarities and cross-reactivity with antibodies to the G-protein (transducin, Gt) from the photoreceptor system. Two proteins, Gs and Gi, can be distinguished by their respective abilities to stimulate or inhibit adenylate cyclase. The activated alpha subunits of Gt and a fourth member of the family, Go, did not affect this enzyme. Gt was shown to be unique in its ability to stimulate cGMP-dependent phosphodiesterase. While functionally diverse, the G-proteins were shown to have some common antigenic properties. Antibodies directed against the beta subunit of Gt recognize the beta 36 subunits of all preparations but not a putative second beta 35 subunit. Antibodies specific for the alpha subunit of Gt did not recognize other alpha subunits when immune blots from sodium dodecyl sulfate gels were examined. However, Go alpha, but not Gs alpha or Gi alpha, reacted strongly with the antibodies when the native subunit was spotted directly. This suggests that Go alpha and Gt alpha have homologous structural determinants. An antiserum that recognized Gt gamma did not recognize gamma subunits from other sources. These data support the proposed diversity of function and similarity of structure among the four G-proteins. The alpha and potentially gamma subunits appear to be responsible for the specificity of function.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Adenylyl Cyclases; Animals; Brain Chemistry; Cattle; Cell Line; Cell Membrane; Chromatography; Durapatite; Electrophoresis, Polyacrylamide Gel; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Hydroxyapatites; Kinetics; Lymphoma; Mice; Molecular Weight; Photoreceptor Cells; Rod Cell Outer Segment; Thionucleotides

The influence of sodium was studied on hormone and guanine nucleotide-induced stimulation and inhibition of adenylate cyclase and on beta-adrenoceptor binding in various membrane systems. Sodium exerted almost identical effects on stimulation and inhibition of adenylate cyclase by various stimulatory and inhibitory hormones in all of the systems studied. The potencies of the hormones and of GTP to increase or to decrease the enzyme activity were reduced by sodium ions, without changing the maximal degree of adenylate cyclase stimulation or inhibition. Stimulation and inhibition of adenylate cyclase by the stable GTP analog, GTP gamma S, was affected in an identical manner by sodium, causing a retardation in the onset without a change in final stimulation or inhibition by the analog. Similar to the well-known reduction in alpha 2-adrenoceptor affinity for agonists, sodium also reduced the apparent affinity of beta-adrenoceptors for the agonist, isoproterenol. It is concluded that sodium exerts identical effects on Ns and Ni, inhibiting the activation process of these two coupling components of the adenylate cyclase.

Topics: Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Adipose Tissue; Alprostadil; Animals; Blood Platelets; Cricetinae; Cyclic AMP; Epinephrine; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; In Vitro Techniques; Isoproterenol; Lymphoma; Prostaglandins E; Receptors, Adrenergic, beta; Receptors, Cell Surface; Sodium Chloride; Thionucleotides

The inhibitory and stimulatory guanine nucleotide-binding regulatory components (Gi and Gs) of adenylate cyclase both have an alpha X beta subunit structure, and the beta subunits are functionally indistinguishable. GTP-dependent hormonal inhibition of adenylate cyclase and that caused by guanine nucleotide analogs seem to result from dissociation of the subunits of Gi. Such inhibition can be explained by reduction of the concentration of the free alpha subunit of Gs as a result of its interaction with the beta subunit of Gi in normal Gs-containing membranes. However, inhibition in S49 lymphoma cyc- cell membranes presumably cannot be explained by the Gi-Gs interaction, since the activity of the alpha subunit of Gs is not detectable in this variant. Several characteristics of Gi-mediated inhibition of adenylate cyclase have been studied in both S49 cyc- and wild type membranes. There are several similarities between inhibition of forskolin-stimulated adenylate cyclase by guanine nucleotides and somatostatin in cyc- and wild type membranes. 1) Somatostatin-induced inhibition of the enzyme is dependent on GTP; nonhydrolyzable GTP analogs are also effective inhibitors. 2) The effect of guanosine-5'-(3-O-thio)triphosphate (GTP gamma S) is essentially irreversible, and somatostatin accelerates GTP gamma S-induced inhibition. 3) Inhibition of adenylate cyclase by somatostatin or Gpp(NH)p is attenuated by treatment of cells with islet-activating protein (IAP). 4) Both cyc- and wild type membranes contain the substrate for IAP-catalyzed ADP-ribosylation (the alpha subunit of Gi). 5) beta Subunit activity in detergent extracts of membranes is liberated by exposure of the membranes to GTP gamma S. The alpha subunit of Gi in such extracts has a reduced ability to be ADP-ribosylated by IAP, which implies that this subunit is in the GTP gamma S-bound form. The resolved subunits of Gi have been tested as regulators of cyc- and wild type adenylate cyclase under a variety of conditions. The alpha subunit of Gi inhibits forskolin-stimulated adenylate cyclase activity in cyc-, while the beta subunit stimulates; these actions are opposite to those seen with wild type membranes. The inhibitory effects of GTP plus somatostatin (or GTP gamma S) and the alpha subunit of Gi are not additive in cyc- membranes. In wild type, the inhibitory effects of the hormone and GTP gamma S are not additive with those of the beta subunit.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Adenylyl Cyclases; Animals; Antihypertensive Agents; Cell Line; Cell Membrane; Colforsin; Diterpenes; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Kinetics; Lymphoma; Mice; Molecular Weight; Mutation; Receptors, Cell Surface; Somatostatin; Thionucleotides

Pertussis toxin catalyzes the ADP-ribosylation of a single 41-kDa peptide of membranes prepared from rat hepatocytes, S49 mouse lymphoma wild-type and cyc-mutant cells. This 41-kDa peptide has been shown to be the alpha-subunit of the inhibitory, guanine nucleotide binding regulatory component of adenylate cyclase (Ni). Incubating membranes of rat fat cells with pertussis toxin and [32P]NAD+ radiolabels a 41- and a 40-kDa peptide. Possible homologies between these peptides were investigated by comparing the electrophoretic patterns of proteolytic fragments derived from each of them that are radiolabeled by [32P]NAD+ and pertussis toxin. The 40-kDa substrate for pertussis toxin-catalyzed ADP-ribosylation and the alpha-subunit of Ni in rat fat cells appear to be homologous, but non-identical peptides.

Topics: Adenosine Diphosphate Ribose; Adenylate Cyclase Toxin; Adipose Tissue; Animals; Bacterial Toxins; Cholera Toxin; Electrophoresis, Polyacrylamide Gel; Female; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Liver; Lymphoma; Membrane Proteins; Molecular Weight; NAD; Nucleoside Diphosphate Sugars; Peptide Hydrolases; Pertussis Toxin; Rats; Rats, Inbred Strains; Thionucleotides; Virulence Factors, Bordetella

The mechanism by which Ns and Ni, the stimulatory and inhibitory regulatory components of adenylyl cyclases, regulate the activity of the catalytic component (C) of adenylyl cyclase was investigated using cyc-S49 cell membranes which contain a functional inhibitory regulatory protein (Ni) but not the active subunit of the stimulatory regulatory protein (Ns). To this end, purified Ns protein was preactivated (Ns) in solution with guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) and Mg2+, and then added to cyc- membranes under conditions where Ni was either unactivated or activated (Ni) by GTP gamma S and Mg2+. Activation of Ni in cyc- membranes resulted in a lowered expression of Ns activity under all conditions tested. Upon dilution of the reactants (Ns and cyc- membranes) the reconstituted activity declined in proportion to the dilution with an approximate t 1/2 of 30-45 min, being unaffected by activation of Ni. Postactivation of Ni after reconstitution of cyc- membranes with Ns resulted in a time-dependent decline in Ns activity to a level that was the same as that obtained when Ns was added to cyc- membranes with preactivated Ni. These data indicated that the effects of Ns on C are of a reversible type. The following indicated that Ns and Ni affect C activity in a noncompetitive manner: (a) the per cent reduction in Ns activity due to activation of Ni was constant and independent of the concentration of Ns, (b) double reciprocal plots of activities reconstituted in control and Ni-containing cyc- membranes versus Ns concentration were linear with an unaltered apparent Km for Ns, and (c) the onset of inhibition of C prereconstituted with Ns was much faster (approximate t 1/2 = 2-5 min) than expected if it were due to occupancy of a common site on C left vacant by Ns.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Adenylyl Cyclases; Animals; Cell Line; Cell Membrane; Enzyme Activation; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Lymphoma; Magnesium; Mice; Sodium Fluoride; Thionucleotides

N-Ethylmaleimide treatment of rat liver plasma membranes results in an adenylyl cyclase (EC 4.6.1.1) system that shows no measurable cyclizing activity but retains both an active glucagon receptor and a receptor-sensitive regulatory component N as assessed by reconstitution into cyclase-negative (cyc-) membranes from S49 murine lymphoma. Treatment of such N-ethylmaleimide-treated membranes, termed C- liver membranes, with guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S] ) and Mg2+, followed by the removal of GTP[gamma S] by washing, yields an activated N which upon mixing with cyc- S49 membranes reconstitutes the cyc- S49 membrane adenylyl cyclase in the absence of added GTP[gamma S]. It was found that GTP[gamma S] activation of the N at saturating concentrations of GTP[gamma S] is slow at low Mg2+ concentration and accelerated by increasing Mg2+ concentrations. Addition of glucagon during the activation results in a lowering of the Mg2+ requirement for full activation from 25 mM to around 10 muM and in concomitant increases in both the rate and the extent of N activation. In contrast to its dramatic effect on Mg2+ requirement, glucagon has little (less than 2-fold) effect on the GTP[gamma S] requirement of N activation. These experiments indicate that the glucagon receptor facilitates activation of N by: (i) decreasing the apparent Km of N for Mg2+, and (ii) increasing the extent of activation that can be elicited by saturating concentrations of guanine nucleotide. It is postulated that the mechanism by which Mg2+ and receptors facilitate N activation involves dissociation of n alpha activated ADP-ribosylatable subunits (with guanine nucleotide bound to them) from n beta non-ADP-ribosylatable subunits (with receptor and Mg2+ bound to them).

Topics: Adenylyl Cyclases; Animals; Cell Line; Cell Membrane; Ethylmaleimide; Glucagon; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Kinetics; Liver; Lymphoma; Magnesium; Mice; Neoplasms, Experimental; Receptors, Cell Surface; Receptors, Glucagon; Thionucleotides

Cyc- S49 mouse lymphoma cell membranes contain an adenylyl cyclase system which is deficient in the regulatory properties characteristic of a stimulatory guanine nucleotide-binding regulatory component (Ns), such as enhancement of activity in the presence of GTP, GTP analogues, and NaF. Detailed kinetic analysis of cycl- adenylyl cyclase showed, however, that it is not unresponsive to these agents, for their addition to assays elicited inhibitory effects. Inhibition of cyc- activity was seen in 11 different membrane preparations obtained from two different strains and was observable both in the absence and presence of the strongly stimulatory diterpene forskolin. The GTP analogues GTP gamma S and guanyl-5'-yl imidodiphosphate caused a maximum of 60% inhibition with IC50 values of 2 and 12 nM, respectively. GTP itself was less potent than its analogues, with an IC50 of 100 mM, and elicited less inhibition as well (a maximum of 25%). Cholera toxin treatment of cyc- S49 cell membranes, under conditions which appeared to maximally activate the adenylyl cyclase system of wild type S49 cell membranes, had no effect on inhibition of catalytic activity. Our results indicate the presence in cyc- S49 cell membranes of a guanine nucleotide-binding component which is inhibitory to adenylyl cyclase activity (Ni). This suggests (a) that these membranes cannot be considered as completely deficient of adenylyl cyclase guanine nucleotide-binding regulatory components, and (b) that these membranes offer a unique opportunity to study Ni-mediated effects of guanine nucleotides and fluoride in the functional absence of Ns-mediated effects of these agents.

Topics: Adenylyl Cyclase Inhibitors; Animals; Cell Line; Cell Membrane; Guanine Nucleotides; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Guanylyl Imidodiphosphate; Kinetics; Lymphoma; Mice; Neoplasms, Experimental; Sodium Fluoride; Thionucleotides