Solubilization of the nitrendipine receptor from skeletal muscle transverse tubule membranes. Interactions with specific inhibitors of the voltage-dependent Ca2+ channel

The nitrendipine receptor associated with the voltage-dependent calcium channel from rabbit skeletal muscle transverse tubule membranes has been solubilized by detergent extraction. A highly stable solubilized receptor preparation was obtained using 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate as detergent with phospholipids or glycerol present as stabilizing agents. Binding of [3H]nitrendipine to the solubilized receptor was reversible and saturable. At 4 degrees C the equilibrium dissociation constant of the [3H]nitrendipine X receptor complex was 7 +/- 3 nM and was close to that determined from the rate constants of association (k1 = 1.3 10(5) M-1 s-1) and dissociation (k-1 = 1.10 X 10(-3) s-1) of 8.4nM. The nitrendipine concentration that gave a half-maximal inhibition of [3H]nitrendipine binding to the solubilized receptor was 10 nM, which was similar to the values for the dissociation constant determined for the radiolabelled ligand. [3H]Nitrendipine binding to its solubilized receptor was also inhibited by other antiarrythmic drugs, such as bepridil and verapamil, and enhanced by d-cis-diltiazem. Since these drugs are apparent non-competitive inhibitors of [3H]nitrendipine binding it was concluded that these different binding sites are tightly coupled. Sucrose density sedimentation of solubilized nitrendipine receptor resulted in the separation of three [3H]nitrendipine binding activities with apparent sedimentation coefficients of 11.4 S, 14.4 S and 21 S.     

Characterization of high-affinity [3H]TBZOH binding to the human platelet vesicular monoamine transporter.

The present study indicates that human platelets can be used as an accessible peripheral model not only for the plasma membrane serotonin transporter, but also for the vesicular monoamine transporter. The vesicular monoamine transporter (VMAT2) is responsible for the accumulation of monoamines in the synaptic vesicles. VMAT2 differs from the plasma membrane transporters in its capability to recognize serotonin, histamine, norepinephrine and dopamine with almost the same affinity. Dihydrotetrabenazine (TBZOH) is a very potent inhibitor of VMAT2 that binds with high affinity to this transporter. [3H]TBZOH has been used as a ligand to label VMAT2 in human, bovine and rodent brain. In this study we characterized the pharmacodynamic and pharmacokinetic parameters of [3H]TBZOH binding in human platelets as compared to rat brain. The density (Bmax) and affinity (Kd) of [3H]TBZOH specific binding was assessed by Scatchard analysis. Association and dissociation rate constants (k(on), K(off)) were assessed by kinetic binding studies. In this study high-affinity and saturable binding sites for [3H]TBZOH were demonstrated in human platelets. Both the affinity of [3H]TBZOH to its binding site in platelets (Kd = 3.2+/-0.5 nM) and the kinetic rate constants (K(on) = 2.8 x 10(7) M(-1) min(-1); K(off) = 0.099 min(-1)) were similar to that in rat brain (Kd(striatum) = 1.5 nM; Kd(cerebral cortex) = 1.35 nM; K(on) = 2 x 10(7) M(-1) min(-1); K(off) = 0.069 min(-1)). Only the VMAT2 blockers tetrabenazine and reserpine inhibited [3H]TBZOH specific binding.     

Dopamine D1 receptors characterized with [3H]SCH 23390. Solubilization of a guanine nucleotide-sensitive form of the receptor

Dopamine D1 receptors were solubilized from canine and bovine striatal membranes with the detergent digitonin. The receptors retained the pharmacological characteristics of membrane-bound D1 receptors, as assessed by the binding of the selective antagonist [3H]SCH 23390. The binding of [3H]SCH 23390 to solubilized receptor preparations was specific, saturable, and reversible, with a dissociation constant of 5 nM. Dopaminergic antagonists and agonists inhibited [3H]SCH 23390 binding in a stereoselective and concentration-dependent manner with an appropriate rank order of potency for D1 receptors. Moreover, agonist high affinity binding to D1 receptors and its sensitivity to guanine nucleotides was preserved following solubilization, with agonist dissociation constants virtually identical to those observed with membrane-bound receptors. To ascertain the molecular basis for the existence of an agonist-high affinity receptor complex, D1 receptors labeled with [3H] dopamine (agonist) or [3H]SCH 23390 (antagonist) prior to, or following, solubilization were subjected to high pressure liquid steric-exclusion chromatography. All agonist- and antagonist-labeled receptor species elute as the same apparent molecular size. Treatment of brain membranes with the guanine nucleotide guanyl-5'-yl imidodiphosphate prior to solubilization prevented the retention of [3H]dopamine but not [3H]SCH 23390-labeled soluble receptors. This suggests that the same guanine nucleotide-dopamine D1 receptor complex formed in membranes is stable to solubilization and confers agonist high affinity binding in soluble preparations. These results contrast with those reported on the digitonin-solubilized dopamine D2 receptor, and the molecular mechanism responsible for this difference remains to be elucidated.     

Solubilization of prostacyclin membrane receptors from human platelets

Prostacyclin (PGI2) receptors have been identified on platelets and other tissues but their physicochemical properties remain unknown due to difficulties in obtaining active solubilized receptors. We evaluated the ability of several detergents to release the receptors from platelet membrane preparations. In contrast to the results of Dutta-Roy and Sinha (Dutta-Roy, A. K., and Sinha, A. K. (1987) J. Biol. Chem. 262, 12685-12691) which revealed selective solubilization of PGE1/PGI2 receptors by 0.05% Triton X-100, we found that CHAPS (3-[(3-chlamidopropyl)dimethylammonio]-1-propanesulfonic acid) (10 mM) was far superior in releasing the PGI2 receptors. In fact, Triton X-100 failed to release detectable PGI2 binding activity into the supernatant. The CHAPS-solubilized receptor degraded rapidly unless 30% glycerol was added which greatly enhanced its stability. By employing an improved binding assay using [3H]iloprost as the ligand and selective membrane filters (AP-15 or GF/B) pretreated with polyethyleneimine for achieving a higher trapping efficiency, we showed by equilibrium binding measurements that the solubilized receptors exhibited a single class of binding sites with a KD of 18.5 nM and Bmax 0.5 pmol/mg. These values were similar to those of the membrane receptors, i.e. KD of 16.6 nM and Bmax 1.0 pmol/mg. Kinetic binding measurements of the solubilized receptors revealed an association rate constant of 0.51 x 10(6) M-1 s-1 and dissociation rate constant of 0.0041 s-1 yielding a calculated KD of 8.0 nM. Displacement of [3H]iloprost (Ki values) from the solubilized and the membrane receptors by diversified eicosanoids was parallel. Our data demonstrate for the first time a successful solubilization of platelet PGI2 receptors. The solubilized receptors retained almost identical binding characteristics as the native membrane receptors.     

Characterization of a Digitonin-Solubilized Bovine Brain H3 Histamine Receptor Coupled to a Guanine Nucleotide-Binding Protein

The H3 receptor is a high-affinity histamine receptor that inhibits release of several neurotransmitters, including histamine. We have characterized H3 receptor binding in bovine brain and developed conditions for its solubilization. Particulate [3H]histamine binding showed an apparently single class of sites (KD = 4.6 nM; Bmax = 78 fmol/mg of protein). Of the detergents tested, digitonin at a detergent/protein ratio of 1:1 (wt/wt) yielded the greatest amount of solubilized receptors, typically 15-30% of particulate binding. Neither equilibrium binding of [3H]histamine to receptors (KD = 6.1 nM; Bmax = 92 fmol/mg of protein) nor the inhibitor profile was substantially altered by digitonin solubilization. However, solubilization did increase the rate of [3H]histamine association with and dissociation from the receptor. Size-exclusion chromatography indicated an apparent molecular weight of 220,000 for the solubilized receptor, and peak binding from this column retained its guanine nucleotide sensitivity. These last two observations are consistent with the solubilized receptor occurring in complex with a guanine nucleotide-binding protein.     

Binding of N-[propionyl-3H]propionylated alpha-bungarotoxin and L-[benzilic-4,4'-3H] quinuclidinyl benzilate to CNS extracts of the cockroach Periplaneta americana

The nerve cord of the cockroach (Periplaneta americana) contains distinct saturable components of specific binding for the ligands N-[propionyl-3H]propionylated alpha-bungarotoxin and L-[benzilic-4,4'-3H]quinuclidinyl benzilate. N-[Propionyl-3H]propionylated alpha-bungarotoxin bound reversibly to homogenates with a Kd of 4.8 nM and Bmax of 910 fmol mg-1. The association rate constant (1.9 X 10(5) M-1 s-1) and dissociation rate constant (1.2 X 10(-4) s-1) yielded a Kd of 0.6 nM. Nicotinic ligands were found to displace toxin binding most effectively. The binding sites characterized in this way showed many similarities with the properties of the vertebrate neuronal alpha-bungarotoxin binding site. For a range of cholinergic ligands, inhibition constants calculated from toxin binding studies closely corresponded to their effectiveness in blocking the depolarizing response to acetylcholine recorded by electrophysiological methods from an identified cockroach motoneurone. The N-[propionyl-3H]propionylated alpha-bungarotoxin binding component therefore appears to be a constituent of a functional CNS acetylcholine receptor. Binding of L-[benzilic-4,4'-3H]quinuclidinyl benzilate was reversible with a Kd of 8 nM and Bmax of 138 fmol mg-1, determined from equilibrium binding experiments. The Kd calculated from the association rate constant (2.4 X 10(5) M-1 s-1) and dissociation rate constant (1.3 X 10(-4) s-1) was 1.9 nM. Muscarinic ligands were the most potent inhibitors of quinuclidinyl benzilate binding. The characteristics of this binding site resembled those of vertebrate CNS muscarinic cholinergic receptors. In contrast with vertebrate CNS, the nerve cord of Periplaneta americana contains more (approximately X 7) alpha-bungarotoxin binding sites than quinuclidinyl benzilate binding sites.     

Characterization of Neurotensin Binding Sites in Intact and Solubilized Bovine Brain Membranes

Analysis of the equilibrium binding of [3H]-neurotensin(1-13) at 25 degrees C to its receptor sites in bovine cortex membranes indicated a single population of sites with an apparent equilibrium dissociation constant (KD) of 3.3 nM and a density (Bmax) of 350 fmol/mg protein (Hill coefficient nH = 0.97). Kinetic dissociation studies revealed the presence of a second class of sites comprising less than 10% of the total. KD values of 0.3 and 2.0 nM were obtained for the higher and lower affinity classes of sites, respectively, from association-dissociation kinetic studies. The binding of [3H]neurotensin was decreased by cations (monovalent and divalent) and by a nonhydrolysable guanine nucleotide analogue. Competition studies gave a potency ranking of [Gln4]neurotensin greater than neurotensin(8-13) greater than neurotensin(1-13). Smaller neurotensin analogues and neurotensin-like peptides were unable to compete with [3H]neurotensin. Stable binding activity for [3H]neurotensin in detergent solution (Kd = 5.5 nM, Bmax = 250 fmol/mg protein, nH = 1.0) was obtained in 2% digitonin/1 mM Mg2+ extracts of membranes which had been preincubated (25 degrees C, 1 h) with 1 mM Mg2+ prior to solubilization. Association-dissociation kinetic studies then revealed the presence of two classes of sites (KD1 = 0.5 nM, KD2 = 3.6 nM) in a similar proportion to that found in the membranes. The solubilized [3H]-neurotensin activity retained its sensitivity to cations and guanine nucleotide.     

Solubilization and reassembly of the mitochondrial benzodiazepine receptor

We have solubilized and reassembled the peripheral-type benzodiazepine receptor, a component of the mitochondrial outer membrane, from rat adrenal gland mitochondria. The ligand binding site of this receptor undergoes denaturation during solubilization in digitonin, Triton X-100, or 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate at detergent concentrations above 0.1%, which is evident from the loss of high-affinity binding of [3H]PK11195, a ligand selective for the mitochondrial benzodiazepine receptor. The conformation of the binding site for PK11195 can be stabilized during solubilization in sodium cholate by relatively low concentrations of supplementary soybean lipid. Drug displacement studies demonstrate that the pharmacological properties of the receptor are preserved under these conditions. Electron micrographs of the solubilized preparation show a heterogeneous population of many small particles (less than 100 A) and some larger membranous aggregates (up to 500 A). Sucrose gradient centrifugation indicates that these lipoprotein complexes are of high buoyant density. They can be incorporated in liposomes via cholate dialysis in the presence of additional supplementary lipid. The behavior of the mitochondrial benzodiazepine receptor during solubilization and reassembly suggests that it is an integral protein of the outer membrane.     

Solubilization and Characterization of a [3H]Hemicholinium-3 Binding Site in Rat Brain

A sodium-dependent high-affinity [3H]-hemicholinium-3 ([3H]HCh-3) binding site was solubilized from rat striatal synaptic plasma membranes by 0.2% deoxycholate. Deoxycholate solubilization of the [3H]HCh-3 binding site was dependent upon both detergent concentration and ionic strength of the solubilization medium. Specific [3H]HCh-3 binding to the solubilized preparation was both sodium- and chloride-dependent and saturable, exhibiting an affinity of 14.2 nM and a capacity (Bmax) of 695 fmol/mg protein. Choline and other analogs inhibited specific [3H]HCh-3 binding to the solubilized preparation in a concentration-dependent manner with the similar rank order of potency observed in crude synaptic membranes. Treatments known to disrupt both protein and lipid moieties resulted in diminished specific [3H]HCh-3 binding. These results suggest that the characteristics of the solubilized [3H]HCh-3 binding site are similar to those of the membrane-bound site.     

Solubilization and Characterization of σ-Receptors from Guinea Pig Brain Membranes

The sigma-receptor, a distinct binding site in brain tissue that may mediate some of the psychotomimetic properties of benzomorphan opiates and phencyclidine, has been solubilized using the ionic detergent sodium cholate. Binding assays were performed with the solubilized receptor using vacuum filtration over polyethyleneimine-treated glass fiber filters. The pharmacological specificity of the solubilized binding site for sigma-receptor ligands is nearly identical to the membrane-bound form of the receptor, with the order of potencies for displacement of the selective sigma-ligand [3H]di-o-tolylguanidine ([3H]DTG) closely correlated. The stereoselectivity for (+)-benzomorphan opiate enantiomers was retained by the solubilized receptor. The soluble receptor retained high affinity for binding of [3H]DTG (KD = 28 +/- 0.5 nM) and (+)-[3H]3-(3-hydroxyphenyl)-N-(1-propyl)piperidine [(+)-[3H]3-PPP] (KD = 36 +/- 2 nM). Photoaffinity labeling of the solubilized receptor by [3H]p-azido-DTG, a sigma-selective photoaffinity label, resulted in labeling of a 29-kilodalton polypeptide identical in size to that labeled in intact membranes. Estimation of the Stokes radius of the [3H]DTG binding site was obtained by Sepharose CL-6B chromatography in the presence of 20 mM cholate and calculated to be 8.7 nm. This value was identical to the molecular size found for the binding sites of the sigma-selective ligands (+)-[3H]3-PPP and (+)-[3H]SKF-10,047, supporting the hypothesis that all three ligands bind to the same macromolecular complex.     

Solubilization of Peripheral-Type Benzodiazepine Binding Sites from Cat Cerebral Cortex

The present study demonstrates for the first time the solubilization of peripheral-type benzodiazepine binding sites (PBS) from cat cerebral cortex. Of all detergents tested [digitonin, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), Tween 20, deoxycholate, and Triton X-100] in the presence of NaCl, the best solubilization (15% of initial activity) was obtained using 0.5% of the zwitterionic detergent CHAPS plus 2 M NaCl. Specific binding of [3H]PK 11195 to membrane-bound and solubilized PBS was saturable, yielding equilibrium dissociation constants (KD) of 1.3 +/- 0.2 and 1.9 +/- 0.3 nM, respectively, and maximal numbers of binding sites of 1,435 +/- 150 and 980 +/- 126 fmol/mg protein, respectively. The KD value of PK 11195 binding to solubilized PBS obtained from experimental kinetic analysis was 0.95 +/- 0.09 nM. The relative potencies of various compounds (PK 11195, Ro 5-4864, diazepam, flunitrazepam, clonazepam, methyl-beta-carboline-3-carboxylate, and Ro 15-1788) in displacing [3H]PK 11195 specific binding from membrane-bound and solubilized PBS were similar. Most of the solubilized binding activity was destroyed by heating at 60 degrees C for 30 min or by treatment with 2 M guanidinium chloride, which indicates the presence of a protein-binding site in the solubilized preparation. Over 85% of the solubilized binding activity was retained after 1 week at 4 degrees C, which will enable future application of purification procedures without major concern for stability of the material.     

Solubilization of an alpha-bungarotoxin-binding component from rat brain.

Binding of [125I]-alpha-bungarotoxin to rat brain was investigated. Picomole quantities of specific toxin binding sites per gram of fresh tissue were found in particulate preparations as well as detergent extracts of whole brain. The toxin-binding macromolecules can be solubilized in low concentrations of Triton X-100. Specific binding occurs to a single class of sites with a dissociation constant of 5.6 X 10(-11) M. The association rate constant in 10 mM sodium phosphate, pH 7.4, was determined to be 6.8 X 10(5) M-1 s-1; the half-life of the complex was found to be 5.1 h, corresponding to a dissociation rate constant of 3.8 X 10(-5) s-1. The binding macromolecules resemble peripheral nicotinic acetylcholine receptors in toxin binding kinetics, solubility, isoelectric point, and hydrodynamic properties.     

Characterization of the Solubilized A1 Adenosine Receptor from Rat Brain Membranes

A1 adenosine receptors from rat brain membranes were solubilized with the zwitterionic detergent 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate. The solubilized receptors retained all the characteristics of membrane-bound A1 adenosine receptors. A high and a low agonist affinity state for the radiolabelled agonist (R)-N6-[3H]phenylisopropyladenosine([3H]PIA) with KD values of 0.3 and 12 nM, respectively, were detected. High-affinity agonist binding was regulated by guanine nucleotides. In addition agonist binding was still modulated by divalent cations. The solubilized A1 adenosine receptors could be labelled not only with the agonist [3H]PIA but also with the antagonist 1,3-diethyl-8-[3H]phenylxanthine. Guanine nucleotides did not affect antagonist binding as reported for membrane-bound receptors. These results suggest that the solubilized receptors are still coupled to the guanine nucleotide binding protein Ni and that all regulatory functions are retained on solubilization.     

Solubilization of the receptor for the neuropeptide gastrin-releasing peptide (bombesin) with functional ligand binding properties

The receptor for the neuropeptide gastrin-releasing peptide, the mammalian homologue of bombesin, was solubilized from rat brain and Swiss 3T3 cells by using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) and the cholesteryl hemisuccinate ester (CHS). Only the combination of the detergent CHAPS and the cholesteryl ester CHS in a glycerol-containing buffer satisfactorily preserved the binding activity upon solubilization. Specific binding activity was only solubilized from cell lines and tissue preparations known to express the GRP receptor. The dissociation constant (Kd) for the receptor solubilized from rat brain and Swiss 3T3 cells was 0.6 nM, similar to the value of 0.8 nM calculated for the membrane-bound receptor. Binding was saturable and reached equilibrium after approximately 2 h at 4 degrees C. The identity of the solubilized receptor with the membrane-bound one was further confirmed by the concordance of the relative binding affinities of various established bombesin analogues.     

Solubilization of phencyclidine receptors from rat cerebral cortex in an active ligand binding state

A phencyclidine (PCP) receptor binding site has been solubilized in an active ligand-binding state from rat cerebral cortical membranes with sodium deoxycholate. Optimal receptor solubilization occurs at a detergent/protein ratio of 0.5 (w/w); for 5 mg protein/ml solubilized with 0.25% sodium deoxycholate, about 60% of the protein and 25% of the receptor is solubilized. Specific binding of either [3H]-N-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) or [3H]MK-801 is measurable by filtration through Sephadex G-50 columns or glass fiber filters; more than 60% of the binding activity is stable after 48 h at 4 degrees C. In the presence of detergent, [3H]TCP binding exhibits a Kd of 250 nM, a Bmax of 0.56 pmol/mg protein, and a pharmacological profile consistent with that of the membrane-bound PCP receptor, although most drugs bind with affinities 2 to 8 fold lower than in membranes. Upon reduction of detergent concentration, binding parameters approximate those for the membrane-bound receptor ([3H]TCP binding: Kd = 48 nM, Bmax = 1.13 pmol/mg protein).     

Molecular pharmacology of the catecholamine transporter of chromaffin granules from the bovine adrenal medulla

Tetrabenazine (TBZ) and reserpine are two inhibitors of the catecholamine uptake system of the chromaffin granule membrane. They are structural analogs of the substrates dopamine and serotonin and they inhibit the monoamine transporter, which catalyzes a H+/neutral amine antiport. [3H]Dihydrotetrabenazine ([3H]TBZOH) is bound by chromaffin granule membranes on one class of site (T sites, KD = 3 nM); [3H]reserpine is bound on T sites and a second class of site (R1 sites, KD = 0.7 nM). The two sites are involved in monoamine translocation. The substrates displace the ligands with different efficiency: noradrenaline (Km = 10 microM) displaces reserpine efficiently (EC50 = 30 microM), but TBZOH poorly (EC50 = 2000 microM); m-iodobenzylguanidine, which has recently been shown to be a substrate of the monoamine uptake system (Km = 5 microM), displaces TBZOH efficiently (EC50 = 25 microM), but reserpine inefficiently (EC50 = 300 microM). Since both substrates are translocated by the same transporter, this result confirms the existence of two sites with different properties. T sites are characterized by a linear relationship between the reciprocal of the dissociation constants of various drugs displacing [3H]TBZOH and their partition coefficient in octanol/H2O mixtures. This relationship, which indicates a hydrophobic environment of T sites, does not exist for R1 sites. T sites have been identified by covalent labeling with a derivative of TBZ coupled to an arylazido group. The labeled sites are borne by a 65,000 dalton protein. The kinetics of reserpine binding are accelerated in the presence of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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

Solubilization of receptors for thyrotropin-releasing hormone from GH4C1 rat pituitary cells: demonstration of guanyl nucleotide sensitivity

TRH receptors have been solubilized from GH4C1 cells using the plant glycoside digitonin. Solubilized receptors retain the principal binding characteristics exhibited by the TRH receptor in intact pituitary cells and their membranes. The binding of the methylhistidyl derivative of TRH [( 3H]MeTRH) attained equilibrium within 2-3 h at 4 C, and it was reversible, dissociating with a t1/2 of 7 h. Analysis of [3H]MeTRH binding to soluble receptors at 4 C yielded a dissociation constant (Kd) of 3.8 nM and a total binding capacity (Bmax) of 3.9 pmol/mg protein. Peptides known to interact with non-TRH receptors on GH cells failed to interfere with the binding of [3H]MeTRH, indicating that the TRH binding was specific. Chlordiazepoxide, a competitive antagonist for TRH action in GH cells, inhibited TRH binding to soluble receptors with an IC50 of 11 microM. When [3H]MeTRH was bound to membranes and the membrane proteins were then solubilized, we found enhanced dissociation of the prebound [3H]MeTRH from its solubilized receptor by guanyl nucleotides. Maximal enhancement of [3H]MeTRH dissociation by 10 microM GTP gamma S occurred within about 45 min at 22 C. GTP gamma S, GTP, GDP beta S, and GDP were all effectors of [3H]MeTRH dissociation, exhibiting EC50s in the range of 14-450 nM. The rank order of potency of the tested nucleotides was GTP gamma S greater than GTP congruent to GDP beta S greater than GDP much greater than ATP gamma S greater than GMP. We conclude that TRH receptors have been solubilized from GH cells with digitonin and retain the binding characteristics of TRH receptors in intact pituitary cells. Furthermore, prebinding [3H]MeTRH to GH4C1 cell membranes results in the solubilization of a complex in which the TRH receptor is linked functionally to a GTP binding protein.     

Purification and characterization of the recombinant human dopamine D2S receptor from Pichia pastoris

The human dopamine D2S receptor was expressed in the methylotrophic yeast Pichia pastoris, where the receptor with a molecular mass of approximately 40kDa exhibited specific and saturable binding properties. The dopamine antagonist [3H]spiperone showed an average dissociation constant K(d) of 0.6+/-0.17 nM for the dopamine D2S receptor. The receptor was solubilized using the non-ionic detergent dodecylmaltoside and purified by affinity chromatography using a Ni(2+) chelate (His-Trap) column or by batch extraction with an anti-FLAG M1 affinity resin. The receptor maintained its biological activity after solubilization and purification from the membrane protein fraction. A 244- or 185-fold enrichment, as judged by an increase in specific binding, was obtained after adsorption to the His-Trap or anti-FLAG materials, respectively.     

N-methyl-D-aspartate/phencyclidine receptor complex of rat forebrain: purification and biochemical characterization

The N-methyl-D-aspartate (NMDA)/phencyclidine (PCP) receptor from rat forebrain was solubilized with sodium cholate and purified by affinity chromatography on amino-PCP-agarose. A 3700-fold purification was achieved. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol revealed four major bands of Mr 67,000, 57,000, 46,000, and 33,000. [3H]Azido-PCP was irreversibly incorporated into each of these bands after UV irradiation. The dissociation constant (Kd) of [1-(2-thienyl)cyclohexyl]piperidine [( 3H]TCP) binding to the purified NMDA/PCP receptor was 120 nM. The maximum specific binding (Bmax) for [3H]TCP binding was 3.3 nmol/mg of protein. The pharmacological profile of the purified receptor complex was similar to that of the membranal and soluble receptors. The binding of [3H]TCP to the purified receptor was modulated by the NMDA receptor ligands glutamate, glycine, and NMDA.