guanosine-triphosphate and 3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate

Under reducing conditions of SDS-polyacrylamide gel electrophoresis, the CB(1) receptor exists in its monomeric form as well as in an SDS-resistant high molecular weight form that appears to be devoid of G proteins. The CB(1) cannabinoid receptor was immunoprecipitated from 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate-solubilized rat brain membranes using an antibody against the CB(1) receptor N terminus. The CB(1) receptor was coimmunoprecipitated with its associated G proteins, specifically those of the Galpha(i/o) family, but not Galpha(s), Galpha(q), or Galpha(z). The CB(1) receptor-Galpha(i/o) complex existed in the absence of exogenous agonists, and the cannabinoid receptor agonist desacetyllevonantradol failed to alter the stoichiometry of the receptor-Galpha(i/o) interaction. Guanosine-5'-O-(3-thio)triphosphate could disrupt the interaction. A peptide derived from the CB(1) receptor juxtamembrane C-terminal domain, peptide CB(1)401-417, autonomously activates G(i/o) proteins. Peptide CB(1)401-417 competitively disrupted the CB(1) receptor association with Galpha(o) and Galpha(i3) but not Galpha(i1) or Galpha(i2). This G protein specificity was also observed in detergent extracts from membranes of the frontal cortex, striatum, and cerebellum. Alternative peptides, including peptides from the CB(1) receptor third intracellular loop and the G protein activating peptide mastoparan-7, failed to promote uncoupling from Galpha(o). A CB(2) receptor juxtamembrane C-terminal peptide failed to disrupt the CB(1) receptor-Galpha(o) complex. These studies illustrate that the CB(1) receptor can exist as an SDS-resistant multimer. In 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate detergent, the CB(1) receptor exists in a complex with G proteins of the G(i/o) family in the absence of exogenous agonists. Furthermore, this study provides the first description of domain specificity for interaction with a selective set of G proteins.

Topics: Binding, Competitive; Brain; Cannabinoids; Cholic Acids; GTP-Binding Protein alpha Subunit, Gi2; GTP-Binding Protein alpha Subunits, Gi-Go; GTP-Binding Proteins; Guanosine Triphosphate; Heterotrimeric GTP-Binding Proteins; Membrane Proteins; Peptide Fragments; Polymers; Precipitin Tests; Proto-Oncogene Proteins; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, Drug; Signal Transduction

Rab3A small G protein is implicated in Ca2+-dependent exocytosis. It undergoes posttranslational lipid-modifications at its C-terminal region. These lipid moieties are important for the actions of the regulators of Rab3A, but not for the interaction with its downstream target.. We have found another function of the C-terminal lipid moieties of Rab3A. GTP rapidly associates with the guanine nucleotide-free form of unmodified Rab3A, but not with the same form of modified Rab3A. Moreover, GTP rapidly dissociates from the GTP-bound form of modified Rab3A, but not from the same form of unmodified Rab3A. The association of GTP with the guanine nucleotide-free form of modified Rab3A is stimulated by the Rab3 GDP/GTP exchange protein (Rab3 GEP), and the dissociation of GTP from the GTP-bound form is markedly reduced by synaptic vesicle phospholipid.. These results suggest that the interaction of the lipid moieties of Rab3A with Rab3 GEP or synaptic vesicles is required for the interaction of modified Rab3A with GTP. Moreover, these results - together with the fact that Rabphilin-3A associated with synaptic vesicles inhibits the activity of Rab3 GTPase-activating protein - suggest that the GTP-bound form of modified Rab3A is associated with synaptic vesicles through both Rabphilin-3A and the vesicle phospholipid.

Topics: Adaptor Proteins, Signal Transducing; Animals; Calcium; Cholic Acids; Drug Stability; Exocytosis; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Hot Temperature; Kinetics; Nerve Tissue Proteins; Phospholipids; Proto-Oncogene Proteins; rab GTP-Binding Proteins; rab3 GTP-Binding Proteins; Rabphilin-3A; Rats; Sulfur Radioisotopes; Synaptic Vesicles; Vesicular Transport Proteins

Biochemical, structural, and functional properties of Rab5 wild-type (WT) protein were compared with those of Q79L and N133I mutants. The detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate increased guanine nucleotide binding to Rab5 WT approximately 10-fold. The single-step catalytic rate of Rab5 WT exceeded that of Q79L 12.2-fold, but the steady-state GTPase rate was only 2.8-fold greater because GDP dissociation was rate-limiting and GDP dissociation was 3.6-fold slower than for Q79L. In contrast, dissociation rates of GTP were indistinguishable. Binding to Rab5 N133I was not detectable. GTP protected Rab5 WT and Q79L from any apparent proteolysis by trypsin. A 20-kDa fragment was the major product of digestion in the presence of GDP, and 12- and 8-kDa fragments were the major products in the absence of added guanine nucleotides. Rab5 N133I underwent no apparent proteolysis with 10 mM GTP or GDP, suggesting a "triphosphate" conformation may be induced in Rab5 N133I by either GTP or GDP. Partially geranylgeranylated Rab5 WT stimulated endosome fusion in vitro, whereas unmodified Rab5 WT did not. Processed Rab5 Q79L failed to inhibit endosome fusion, and Rab5 N133I could not be geranylgeranylated. These findings identify biochemical and structural features of Rab5 proteins, providing data for the interpretation of functional assays.

Topics: Amino Acid Sequence; Base Sequence; Cholic Acids; Detergents; Escherichia coli; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Humans; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Oligodeoxyribonucleotides; Point Mutation; rab5 GTP-Binding Proteins; Recombinant Proteins

The neutrophil NADPH oxidase activation factors, p47, p67 and the small guanosine-nucleotide-binding regulatory (G) protein Rac1, were expressed in a baculovirus/insect cell system and purified. In coinfection experiments in which Sf9 cells overexpressed concomitantly p47, p67 and Rac1, the latter was not detected in the p47-p67 complex. The propensity of p47 and p67 to associate together was used to purify recombinant p67 from baculovirus-infected Sf9 cells. 20% of the overexpressed Rac1 in infected Sf9 cells was prenylated and was extracted with low doses of detergent from membranes. Elicitation of full oxidase activity on crude neutrophil membranes using a cell-free system required addition of recombinant p47 and p67, but not that of Rac. In contrast, in the case of KCl-washed membranes, addition of Rac, prenylated or unprocessed, together with p47 and p67 was found to enhance oxidase activation up to fivefold. In all experiments, the amount of added arachidonic acid was optimized. In contrast to prenylated Rac, non-prenylated Rac had to be loaded with guanosine 5'-(3-thiotriphosphate) (GTP[S]) to exhibit full activation efficiency. In the cell-free system used, Rac was shown to be the mediator of the GTP[S] effect. The results suggest that the plasma membrane of resting neutrophils contains a sufficient amount of prenylated Rac for efficient oxidase activation. We therefore propose that Rac has a membrane-associated role and helps to dock and position p47 and p67 on the flavocytochrome b component of the oxidase complex.

Topics: Animals; Cattle; Cell Membrane; Cell-Free System; Cholic Acids; Enzyme Activation; Gene Expression; GTP-Binding Proteins; Guanosine Triphosphate; Indicators and Reagents; NADH, NADPH Oxidoreductases; NADPH Dehydrogenase; NADPH Oxidases; Neutrophils; Phosphoproteins; Potassium Chloride; Protein Prenylation; rac GTP-Binding Proteins; Recombinant Proteins; Retroviridae; Spodoptera; Superoxides

PGE2 binding sites or receptors of porcine ciliary nonpigmented epithelial (NPE) and pigmented epithelial (PE) membranes were solubilized with detergents (CHAPS and Triton X100). From the Scatchard plots of PGE2 binding to CHAPS-solubilized proteins, the Kd and Bmax values were calculated to be 35 nM and 470 fmol/mg protein for NPE protein and 65 nM and 430 fmol/mg protein for PE protein, respectively. On the basis of the Kd and Bmax values, the solubilized receptor proteins correspond to PGE2 binding sites of the membranes which have previously been shown to be coupled to adenylate cyclase inhibition. For both NPE and PE proteins, the order of binding potency was PGE2 > PGF2 alpha > PGD2. By gel filtration chromatography of NPE and PE proteins, the molecular mass of the major PGE2 binding peak was estimated to be about 150 KDa when solubilized in CHAPS and 46 KDa for Triton X100 extracts. The Bmax values of membrane-associated binding proteins were increased by GTP, indicating a close association of the PGE2 binding sites with a GTP-binding protein. However, GTP did not affect the Bmax values of detergent-solubilized receptor proteins.

Topics: Animals; Binding, Competitive; Cholic Acids; Ciliary Body; Dinoprostone; Guanosine Triphosphate; Membrane Proteins; Octoxynol; Pigment Epithelium of Eye; Receptors, Prostaglandin E; Swine

1. The characteristics of membrane bound GABAB receptors in pig brain are similar to those in rat brain as judged by in vitro binding experiments and sensitivity to GTP. The rank order of affinity of GABAB receptor ligands was CGP 54626 > GABA approximately (-)-baclofen >> CGP 35348 = CGP 36742 > (+)-baclofen in membranes from both species. 2. For solubilization of GABAB receptors from pig brain, washed membranes were preincubated with 5 mM MgSO4 and subsequently incubated with various detergents. 3-[(3-Cholamidopropyl)dimethyl-ammoniol]-1-propane sulphonate (CHAPS) (0.5%) proved to be the most successful, solubilizing 22.7 +/- 4.7% (mean +/- s.e. mean, n = 6) of GABAB receptors. 3. Binding of [3H]-GABA to GABAB receptors solubilized with 0.5% CHAPS exhibited similar characteristics to the binding at membrane bound receptors since, firstly, the Kd and Bmax values (around 30 nM and 450 fmol mg-1 protein, respectively) were comparable; secondly, stereospecific binding for baclofen was obtained in both forms; thirdly, the affinity for the agonists GABA and (-)-baclofen and the antagonists CGP 35348, CGP 36742 and CGP 54626 were the same; fourthly, comparable sensitivity to Ca2+ (2.5 mM) was observed and finally, a similar sensitivity to GTP was apparent. 4. Saturation experiments performed with the GABAB antagonist, [3H]-CGP 54626, indicated a higher Kd value and a lower Bmax value for solubilized (7.7 +/- 2.6 nM and 1033 +/- 41 fmol mg-1 protein, mean +/- s.e. mean, n = 3) than for membrane bound receptors (1.35 +/- 0.08 nM, 1171 +/- 20 fmol mg-1 protein, n = 3).

Topics: Animals; Binding Sites; Brain Chemistry; Cholic Acids; GABA Antagonists; gamma-Aminobutyric Acid; Guanosine Triphosphate; Receptors, GABA; Swine; Synaptic Membranes

The present investigation was undertaken to characterize cannabinoid receptor binding in the absence of the membrane environment, inasmuch as cannabinoid drugs have been noted to influence the behavior of integral membrane proteins. The zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) was able to solubilize the cannabinoid receptor from rat brain membranes, with the greatest yield and specific activity being obtained at a detergent/protein ratio of 0.5:1. [3H]CP-55940 bound to a single class of binding sites in the CHAPS extract, which exhibited a Kd of 0.94 nM as determined by nonlinear regression analysis of equilibrium binding data. The order of potency for cannabinoid agonists in heterologous equilibrium binding studies was CP-55244 > or = desacetyllevonantradol > delta 9-tetrahydrocannabinol > cannabinol >> cannabidiol, consistent with the relative affinities for these agonists in brain membrane preparations. CP-55243, the biologically inactive enantiomer of CP-55244, competed for binding of [3H]CP-55940 by < 50% at 1 microM, similar to its poor affinity for the receptor in membranes. The CHAPS-solubilized cannabinoid receptor exhibited functional interactions with guanine nucleotide-binding proteins (G proteins). GTP and nonhydrolyzable analogs decreased [3H]CP-55940 binding by 75%. The concentration-effect curves for guanine nucleotides exhibited a potency order similar to that observed for other G protein-linked receptors. Kinetic analyses indicated that GTP analogs increased the rate of agonist dissociation, decreasing the t1/2 from 60 min at 0-4 degrees to a multiphasic dissociation that exhibited a component having a t1/2 of < 1 min. The cannabinoid agonist desacetyllevonantradol was able to reduce pertussis toxin-catalyzed ADP-ribosylation of G proteins by 50%, demonstrating a receptor effect on G protein functions. These studies demonstrate that the membrane environment is not necessary for agonist binding to the cannabinoid receptor. Furthermore, the cannabinoid receptor maintains its functional interactions with pertussis toxin-sensitive G proteins in detergent solution.

Topics: Animals; Brain Chemistry; Cholic Acids; Cyclohexanols; GTP-Binding Proteins; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Phenanthridines; Rats; Receptors, Cannabinoid; Receptors, Drug; Solubility

Binding of 2-[125I]iodomelatonin to 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS)-solubilized sites from chick forebrain was rapid. reversible, saturable, of high affinity, and of pharmacological selectivity. Scatchard analyses showed that 2-[125I]iodomelatonin binds to a single site with equilibrium dissociation constant (KD) values of 328 +/- 22 (n = 4) and 302 +/- 26 pM (n = 3) and a maximal number of binding sites (Bmax) of 36.2 +/- 2.0 and 49.5 +/- 6.6 fmol/mg of protein in solubilized and membrane fractions, respectively. The KD values obtained from the ratio of kinetic constants (k2/k1) in solubilized and membrane preparations were 228 and 216 pM, respectively. Inhibition studies indicated the following order of pharmacological affinities for both membrane and solubilized sites: 2-iodomelatonin greater than melatonin greater than 6-chloromelatonin much greater than prazosin greater than N-acetylserotonin much greater than serotonin greater than metergoline greater than ketanserin greater than propranolol greater than phentolamine greater than cyproheptadine. Guanyl nucleotides inhibited binding of 2-[125I]iodomelatonin to solubilized and membrane fractions, by converting binding sites from a high-affinity to a low-affinity state. These findings show that solubilized binding sites for melatonin exhibit the specific binding and pharmacological characteristics present in membrane-bound sites. Moreover, the retention of sensitivity to guanine nucleotides in fractions solubilized with CHAPS suggests that this solubilization procedure is suitable for further studies aimed at the isolation, purification, and molecular characterization of active melatonin binding sites.

Topics: Animals; Binding Sites; Brain; Cell Membrane; Chickens; Cholic Acids; Cytosol; Detergents; Guanine Nucleotides; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Iodine Radioisotopes; Kinetics; Male; Melatonin; Receptors, Melatonin; Receptors, Neurotransmitter; Solubility

Vasoactive intestinal peptide (VIP) receptors were solubilized from porcine liver membranes using CHAPS. The binding of 125I-VIP to solubilized receptors was reversible, saturable and specific. Scatchard analysis indicated the presence of one binding site with a Kd of 6.5 +/- 0.3 nM and a Bmax of 1.20 +/- 0.15 pmol/mg protein. Solubilized and membrane-bound receptors displayed the same pharmacological profile since VIP and VIP-related peptides inhibited 125I-VIP binding to both receptor preparations with the same rank order of potency e.g. VIP greater than helodermin greater than rat GRF greater than rat PHI greater than secretin greater than human GRF. GTP inhibited 125I-VIP binding to membrane-bound receptors but not to solubilized receptors supporting functional uncoupling of VIP receptor and G protein during solubilization. Affinity labeling of solubilized and membrane-bound VIP receptors with 125I-VIP revealed the presence of a single molecular component with Mr 55,000 in both cases. It is concluded that VIP receptors from porcine liver can be solubilized with a good yield, in a GTP-insentive, G protein-free form. This represents a major advance towards the purification of VIP receptors.

Topics: Animals; Cell Membrane; Cholic Acids; GTP-Binding Proteins; Guanosine Triphosphate; Iodine Radioisotopes; Kinetics; Liver; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Substrate Specificity; Succinimides; Swine; Time Factors; Vasoactive Intestinal Peptide

A simple method for solubilization and reconstitution of the A1 adenosine receptor from bovine brain is presented. Solubilization with CHAPS-phosphatidylcholine (CHAPS/PC) mixture did not alter the binding properties of the A1 adenosine receptor antagonist [3H]-DPCPX. The solubilized receptors were chromatographed on hydroxyapatite or DEAE-cellulose to remove native membrane lipids and part of non-receptor proteins. Elution of the receptor fractions was obtained from DEAE-cellulose column with a linear gradient of KCl (0-0.4 M). The fractions corresponding to the peak of [3H]-DPCPX binding activity were then reconstituted in phosphatidylcholine by dialysis. The reconstituted receptor retained all the binding characteristics and the same rank order of competition potency (R-PIA greater than S-PIA greater than NECA) as the native receptor, although its thermal stability was remarkably reduced. The binding of [3H]-DPCPX to A1 adenosine receptors was increased by GTP, probably as result of interactions with coeluted G-proteins.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Binding, Competitive; Brain Chemistry; Cattle; Cholic Acids; Chromatography, DEAE-Cellulose; Chromatography, Ion Exchange; GTP-Binding Proteins; Guanosine Triphosphate; Hydroxyapatites; In Vitro Techniques; Membranes; Nerve Tissue Proteins; Phenylisopropyladenosine; Phosphatidylcholines; Receptors, Purinergic; Xanthines

The 23.5-kDa Sec4 protein is required for vesicular transport between the Golgi apparatus and the plasma membrane in Saccharomyces cerevisiae. In order to analyze its biochemical properties, we have purified the soluble pool of the wild-type protein from an overproducing yeast strain. At 30 degrees C, Sec4p bound [35S] guanosine 5'-O-(thiotriphosphate) (GTP gamma S) with a rate of 0.18 min-1 in a reaction requiring micromolar concentration of free magnesium ions. The protein had high affinity for guanine nucleotides with Kd values for GTP gamma S and GTP of 3.7 nM and 3.5 nM, respectively, and that for GDP of 77 nM. The dissociation of [3H] GDP from Sec4p occurred with a rate of 0.21 min-1 suggesting that the association of GTP gamma S was the result of exchange for prebound GDP. The release of GTP from Sec4p was slow and correlated with a low inherent GTPase activity of 0.0012 min-1. By analogy with other classes of GTP binding proteins, both the nucleotide exchange and hydrolysis activities of Sec4p may be modulated in vivo to facilitate its role in the regulation of intercompartmental membrane traffic.

Topics: Binding, Competitive; Biological Transport; Cell Membrane; Cholic Acids; Cloning, Molecular; Edetic Acid; Fungal Proteins; Gene Expression; Golgi Apparatus; GTP Phosphohydrolases; GTP-Binding Proteins; Guanine Nucleotides; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Hydrolysis; Kinetics; Magnesium; rab GTP-Binding Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Thionucleotides

The rate of release of guanine nucleotides from the ras proteins (Ras) is extremely slow in the presence of Mg2+. It seemed likely, therefore that a factor might exist to accelerate the release of guanosine diphosphate (GDP), and hence the exchange of GDP for guanosine triphosphate (GTP). Such a factor has now been discovered in rat brain cytosol. Brain cytosol was found to catalyze, by orders of magnitude, the release of guanine nucleotides from recombinant v-H-Ras protein bound with [alpha-32P]GDP. This effect occurred even in the presence of a large excess of Mg2+, but was destroyed by heat or by incubation of the cytosol for an hour at 37 degrees C in the absence of phosphatase inhibitors. The effect was observed with either v-H-Ras or c-H-Ras, but not with p25rab3A, a small G protein with about 30% similarity to Ras. The effect could not be mimicked by addition of recombinant Ras-GAP or purified GEF, a guanine nucleotide exchange factor involved in the regulation of eukaryotic protein synthesis. By gel filtration chromatography, the factor appears to possess a molecular size between 100,000 and 160,000 daltons. This protein (Ras-guanine nucleotide-releasing factor, or Ras-GRF) may be involved in the activation of p21ras.

Topics: Animals; Binding, Competitive; Brain; Cholic Acids; Cytosol; Guanine Nucleotides; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Hot Temperature; Immunosorbent Techniques; Kinetics; Magnesium Chloride; Molecular Weight; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); Rats; Thionucleotides

Despite numerous reports of solubilization of adenosine A1 receptors, little progress has been made in isolating or purifying the receptor, owing to the extreme lability of the preparations. The present solubilization strategies recognized the possible role of endogenous adenosine to produce adenosine-receptor-N-protein complexes, which are intrinsically unstable, and instead attempted to use caffeine to solubilize free adenosine receptors, which might be more stable. Endogenous adenosine was removed from membranes by using adenosine deaminase along with GTP to accelerate the release of receptor-bound adenosine. The receptors were then occupied with caffeine and solubilized with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate (CHAPS) in the presence of glycerol. These soluble preparations exhibited the characteristics of free adenosine receptors. They bound the A1-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPDPX) with high affinity to a single class of binding sites, which were insensitive to GTP. The binding activity was extremely stable, with a half-life of about 5 days at 4 degrees C; there was little change in either receptor number or affinity during 3 days at 4 degrees C. This methodology should greatly facilitate the characterization, isolation and purification of the adenosine A1 receptor.

Topics: Animals; Brain; Cell Membrane; Cholic Acids; Guanosine Triphosphate; Rats; Rats, Inbred Strains; Receptors, Purinergic; Solubility; Xanthines

The specific binding protein for substance P (SP) was solubilized in an active form from the crude mitochondrial (P2) fraction of bovine brainstem. After incubation with 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) and 0.1 M NaCl at 0 degrees C for 30 min, the SP binding to the supernatant fraction (100,000 g, 60 min) was determined by the glass fiber filtration method reported by Bruns et al. (1983). The specific [3H]SP binding to the solubilized fraction was highly specific for SP and was displaced by nanomolar concentrations of SP and physalaemin, but only by micromolar concentrations of eledoisin. In addition, the binding was inhibited by GTP (approximately 40% of the specific binding decreased by 10 microM GTP) in both preparations. These results were virtually identical to those of P2 membrane preparations and suggested that this high-affinity SP binding site belongs to the SP-P type. Scatchard analyses of SP binding to the solubilized fraction revealed a single saturable component with a Bmax of 22.0 +/- 5.10 fmol/mg protein and a KD of 0.79 nM, and these values are almost the same as those obtained in the P2 fraction (Bmax = 31.3 +/- 3.56 fmol/mg protein, KD = 0.82 nM). Gel filtration analysis showed that the detergent-SP binding protein complex has two calculated molecular weights of greater than 1,000,000 and 55,000-60,000 (a corresponding Stokes radius of 35.5 nm).

Topics: Animals; Brain Stem; Cattle; Cholic Acids; Chromatography, High Pressure Liquid; Eledoisin; Guanosine Triphosphate; Intracellular Membranes; Mitochondria; Molecular Weight; Physalaemin; Receptors, Neurokinin-1; Receptors, Neurotransmitter; Solubility; Substance P

Topics: Animals; Atrial Function; Cholic Acids; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Heart; Heart Atria; Humans; Ion Channels; Potassium; Thionucleotides

The cardiac muscarinic receptor stimulates a potassium-selective ionic current (IK.ACh) through activation of a guanine nucleotide-binding regulatory protein. Purified alpha and beta gamma subunits of the guanine nucleotide-binding regulatory protein have each been reported to open the K+ channel. We have reported that nanomolar concentrations of purified brain beta gamma subunits activated IK.ACh in chicken embryonic atrial patches. In contrast, J. Codina, A. Yatani, D. Grenet, A.M. Brown, and L. Birnbaumer [(1987) Science 236, 442-445] subsequently reported that picomolar concentrations of activated erythrocyte alpha subunits (i.e., the 40-kDa alpha subunit that the authors call alpha K) opened K+ channels in guinea pig atrial patches. In this paper, we further explore the specificity of various beta gamma and alpha subunits in embryonic chicken and neonatal rat atrial patches. Beta gamma subunits from either human placenta (beta 35 gamma) or bovine brain (beta 35,36 gamma) activated IK.ACh whereas transducin beta gamma (beta 36 gamma) did not. The beta gamma activation was consistent in rat and chicken patches [118 of 123 patches (97%)]. Beta gamma subunits opened K+ channels at concentrations greater than or equal to 200 pM and maximally activated the channel at 10 nM. Beta gamma or guanosine 5'-[gamma-thio]triphosphate (GTP[gamma-S]) channel activation could be reversed by alpha 41-GDP. The purified brain beta gamma preparation was contaminated with less than 0.01% unactivated alpha. The detergent (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; CHAPS), used to suspend the hydrophobic beta gamma, did not activate IK.ACh alone, with buffer, with heat-inactivated beta gamma, or with transducin beta gamma. Unactivated alpha subunits did not open K+ channels. Activated, alpha subunits purified from human erythrocytes (alpha 40-GTP[gamma-S]) or bovine brain (alpha 39-GTP[gamma-S]) at concentrations of 10 pM or higher (up to 1 nM) opened K+ channels less frequently in chicken atrial patches [5 of 27 patches (19%) and 9 of 35 patches (26%), respectively] than in rat atrial patches [5 of 11 patches (45%) and 11 of 19 patches (58%), respectively]. Negative results were not due to patch vesicle formation. Other experiments indicated that alpha and beta gamma activated the same population of channels. Activation of the channel by both beta gamma and alpha subunits implies a more complicated scheme for guanine nucleotide-binding regulatory protein act

Topics: Animals; Cattle; Chick Embryo; Cholic Acids; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Ion Channels; Myocardium; Potassium; Rats; Receptors, Muscarinic; Thionucleotides

Topics: Adenylyl Cyclases; Animals; Cell Membrane; Cholic Acids; Cyclic AMP; Enzyme Activation; Growth Hormone-Releasing Hormone; GTP-Binding Proteins; Guanosine Triphosphate; Guinea Pigs; Lung; Phospholipases A; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Solubility; Type C Phospholipases; Vasoactive Intestinal Peptide

We report the reconstitution of the smooth muscle vasopressin V1 receptor functionally coupled to a pertussis toxin-insensitive guanine nucleotide-binding protein. This V1 receptor was spontaneously coupled to this guanine nucleotide-binding protein upon solubilization in the absence of agonist, in contrast to our earlier report on the liver V1 receptor, which required agonist for coupling. The smooth muscle V1 receptor was reconstituted as a high affinity receptor (Kd = 5 nM), with a slow rate of agonist dissociation. Upon the addition of guanosine 5'-thiotriphosphate, there was a decrease in receptor affinity (Kd = 30 nM) concomitant with an increase in the rate of ligand dissociation. The ability of the smooth muscle V1 receptor to spontaneously couple to a guanine nucleotide-binding protein(s) suggests that in the absence of agonist it exists as a high affinity receptor. The smooth muscle V1 receptor may, therefore, be more sensitive to plasma concentrations of vasopressin than its liver homologue.

Topics: Animals; Cholic Acids; GTP-Binding Proteins; Guanosine Triphosphate; Kinetics; Liver; Male; Molecular Weight; Muscle, Smooth; Rats; Rats, Inbred Strains; Receptors, Angiotensin; Receptors, Vasopressin; Solubility

Cells from Dictyostelium discoideum carry chemotactic cAMP receptors on their surface. Kinetic studies have revealed the existence of two slowly dissociating, high affinity receptor forms (SS and S) and one or more fast dissociating, low affinity forms (F) (Van Haastert, P.J.M., and De Wit, R.J.W. (1984) J. Biol. Chem. 259, 13321-13328). We have studied the interaction of these different cAMP-receptor types with a detergent-insoluble membrane residue. Isolated D. discoideum membranes were extracted with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS), which was previously shown to be the only detergent in the presence of which cAMP receptor binding is completely preserved (Janssens, P. M. W., and Van Driel, R. (1986) Biochim. Biophys. Acta 885, 91-101). The protein composition of the CHAPS-insoluble membrane residue appeared to be similar to that of the Triton X-100-insoluble membrane skeleton. Cyclic AMP binding studies revealed a specific association of the slowly dissociating cAMP receptors (SS and S forms) with this CHAPS-insoluble residue. All fast dissociating (F type) receptors were solubilized by CHAPS. GTP induced a transition of 75% of the SS and S receptors to faster dissociating forms. This transition was accompanied by the release of an equal number of receptors from the residue. These effects of GTP required that the cAMP receptor was occupied, and were completely reversible. After removal of the guanine nucleotide SS and S type receptors reappeared, bound to the residue, with a t1/2 of 5-10 min at 0 degrees C. We conclude that a detergent-insoluble membrane residue is involved in signal transduction via the chemotactic cAMP receptor. Both receptor occupation and a guanine nucleotide binding protein control receptor-residue interaction.

Topics: Chemotaxis; Cholic Acids; Dictyostelium; Guanine Nucleotides; Guanosine Triphosphate; Kinetics; Microscopy, Electron; Receptors, Cyclic AMP; Solubility

The zwitterionic detergent CHAPS was used to solubilize functional receptors for vasoactive intestinal peptide (VIP) from guinea pig lung. The solubilized receptors were resolved by high performance gel filtration in 3 mM CHAPS into two active fractions with apparent Stokes radii of 5.9 +/- 0.1 and 2.3 +/- 0.1 nm. The binding of 125I-VIP to the two receptor fractions was time-dependent, reversible, and saturable. Trypsin destroyed the binding activity of the receptor fractions, indicating their proteinic nature. Unlabeled VIP competitively displaced the binding of 125I-VIP to the 5.9-nm fraction (IC50 = 240 pM) and the 2.3-nm fraction (IC50 = 1.2 microM). Scatchard analysis indicated a single class of binding sites in each receptor fraction, with Kd values 300 pM and 0.97 microM for the 5.9- and 2.3-nm Stokes radii fractions, respectively. When the high affinity, 5.9-nm Stokes radius fraction was rechromatographed in 9 nM CHAPS, 46% of the binding activity eluted in the low affinity, 2.3-nm Stokes radius fraction, indicating that the latter is a product of dissociation of the high affinity receptor complex. GTP inhibited the binding of 125I-VIP to the high affinity complex but not the low affinity species. Scatchard plots of VIP binding by the high affinity receptors treated with GTP suggested the presence of two distinct binding sites (Kd 4.4 and 153 nM), compared to a single binding site (Kd = 0.3 nM) obtained in untreated receptors. The nonhydrolyzable GTP analog, guanyl-5'-yl-imidodiphosphate, inhibited VIP binding by the high affinity receptor fraction with potency nearly equivalent to that of GTP. These observations suggest that GTP-binding regulatory proteins are functionally coupled to the VIP-binding subunit in the high affinity receptor complex. The peptide specificity characteristics of the two receptor fractions were different. Peptide histidine isoleucine and growth hormone releasing factor, peptides homologous to VIP, were 87.5- and 22.9-fold less potent than VIP in displacing 125I-VIP binding by the high affinity receptor complex, respectively. On the other hand, growth hormone-releasing factor was more potent (22.7-fold) and peptide histidine isoleucine was less potent (31.3-fold) than VIP in displacing the binding by the low affinity species.

Topics: Animals; Binding, Competitive; Chemical Phenomena; Chemistry, Physical; Cholic Acids; Chromatography, Gel; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Guinea Pigs; Lung; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Solubility; Trypsin; Vasoactive Intestinal Peptide

Membranes prepared from mammalian brain or intestine contain two types of specific binding sites for neurotensin that differ by their affinity and by their sensitivity to sodium ions, GTP, and the antihistamine drug levocabastine. Only the high affinity sites are present in cell cultures and in soluble extracts of CHAPS-treated membranes. These sites represent functional neurotensin receptors coupled to GTP-binding proteins that regulate intracellular levels of cAMP, cGMP and inositol phosphates in neuroblastoma N1E115 cells. The molecular weight of neurotensin receptors in cells and membrane preparations of various origin is about 110,000.

Topics: Animals; Brain Chemistry; Cell Membrane; Cells, Cultured; Cholic Acids; Cyclic AMP; Guanosine Triphosphate; Intestines; Kinetics; Neuroblastoma; Neurotensin; Receptors, Neurotensin; Receptors, Neurotransmitter

The specific binding protein for prostaglandin (PG) E2 solubilized from porcine brain was sensitive to guanine nucleotides. GTP inhibited the association and enhanced the dissociation of the specific [3H]PGE2 binding. Scatchard analyses showed that GTP (10 microM) decreased the binding affinity more than 3-fold without major change in the number of binding site. Gel filtration separated the binding site from GTP-regulatory component (N). The separated binding protein had a reduced affinity to PGE2 and lost its sensitivity to GTP. The addition of the separated N restored its responsiveness to GTP, and also increased the binding affinity to the original level. These results provide direct evidence for the molecular interaction between the PGE2 binding protein and N in the brain.

Topics: Animals; Brain; Cholic Acids; GTP-Binding Proteins; Guanosine Triphosphate; Kinetics; Macromolecular Substances; Protein Binding; Receptors, Cell Surface; Receptors, Prostaglandin; Receptors, Prostaglandin E; Swine

Pertussis toxin is composed of an enzymatically active A subunit and a binding component (B oligomer). Both the holotoxin and the isolated A subunit have previously been shown to exhibit NAD glycohydrolase activity although the A subunit is more active on a molar basis than the holotoxin. We have investigated the mechanism by which ATP stimulates the activity of this toxin. Since dissociation of pertussis toxin subunits would result in increased NAD glycohydrolase activity, the ability of ATP to promote release of the A subunit from the B oligomer was examined. In the presence of the zwitterionic detergent 3-(3-cholamidopropyldimethyl)-1-ammonio)-propanesulfonate, concentrations of ATP as low as 1 microM promoted subunit dissociation. The concentration of ATP required for release of the A subunit was similar to that required for stimulation of NAD glycohydrolase activity. Both ATP and ADP promoted subunit dissociation and stimulated NAD glycohydrolase activity. In contrast, AMP and adenosine did not alter NAD glycohydrolase activity or affect subunit structure. The ability of ATP to decrease the affinity of the A subunit for the B oligomer may play a role in nucleotide stimulation of pertussis toxin activity.

Topics: Adenine Nucleotides; Adenosine Triphosphate; Cholic Acids; Cytidine Triphosphate; Electrophoresis, Polyacrylamide Gel; Guanosine Triphosphate; Macromolecular Substances; Molecular Weight; NAD+ Nucleosidase; Pertussis Toxin; Thymine Nucleotides; Virulence Factors, Bordetella

The specific binding of [3H]spiperone was determined in membrane preparations of rat striatum and following solubilization treatment with the zwitterionic detergent CHAPS. Membrane protein solubilization was confirmed by ultrafiltration, gel filtration and increased heat sensitivity. Specific binding of [3H]spiperone to the solubilized preparation was saturable and of high affinity, although solubilization led to an approximate 10 fold decrease in receptor affinity for [3H]spiperone. The drug displacement profile of binding to the CHAPS solubilized preparation corresponded to that of the dopamine D-2 receptor; binding was stereoselectively displaced by the isomers of butaclamol. The sodium dependence of sulpiride displacement of specific [3H]spiperone binding was retained in the CHAPS solubilized preparation. GTP (100 microM) only altered the ability of dopamine to displace [3H]spiperone binding to the solubilized preparation in the presence of 120 mM sodium chloride. The GTP effect was small compared with that observed in the membranes. Specific [3H]spiperone binding sites in the solubilized preparation were preferentially retained by a wheat germ agglutinin affinity column and subsequently eluted with N-acetyl-D-glucosamine. Gel filtration of the solubilized preparation using a Sepharose column resulted in two peaks of specific [3H]spiperone binding, the larger component had a Stokes radius of 7.7 nm. CHAPS treatment of rat striatal membranes results in solubilization of the D-2 receptor in an active form. The D-2 site appears to be a glycoprotein of high molecular weight.

Topics: Adsorption; Animals; Cholic Acids; Chromatography, Affinity; Chromatography, Gel; Corpus Striatum; Detergents; Female; Guanosine Triphosphate; In Vitro Techniques; Lectins; Rats; Rats, Inbred Strains; Receptors, Dopamine; Sodium; Solubility; Spiperone; Surface-Active Agents; Wheat Germ Agglutinins

Alpha 2-adrenergic receptors labeled by [3H]clonidine (alpha 2-agonist) can be solubilized from the rat brain with a zwitterionic detergent, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS). CHAPS-solubilized receptors have the same characteristics of membrane-bound alpha 2-receptors in the brain, and the regulation of receptor binding by guanine nucleotides is retained in the soluble state.

Topics: Adenosine Triphosphate; Animals; Brain; Cholic Acids; Clonidine; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Rats; Receptors, Adrenergic, alpha; Solubility