Irreversible labelling of rat brain opioid receptors by enkephalin chloromethyl ketones

Chloromethyl ketone derivatives of leucine enkephalin (LE), D-Ala2-Leu5-enkephalin (DALE) and D-Ala2-D-Leu5-enkephalin (DADLE) were synthesized. They all show high affinity for rat brain opioid binding sites. Preincubation of the membrane fraction with enkephalin chloromethyl ketones causes a significant inhibition of /3H/-naloxone binding which cannot be reversed by extensive washing. It was found that the irreversible inhibition is selective for the high affinity (KD less than 1 nM) /3H/-naloxone binding site (putative mu-1 site). The irreversible blockade of opioid binding was partially protected by opiate alkaloids and opioid peptides, suggesting that non-specific labelling also occurs. Affinity of enkephalin chloromethyl ketones toward the mu sites is greater than that of the parent compounds. It was also found that the covalent inhibition of mu sites (/3H/-dihydromorphine and /3H/-DAGO binding) is more effective than that of delta sites (/3H/-DALE binding). We conclude that these chloromethyl ketone derivatives can be used as affinity labels for the opioid receptors, allowing us to study the structure of the mu receptor subtype.     

Synthesis of 3H-Tyr-D-Ala-Gly-N(Me)Phe chloromethyl ketone--an opioid affinity label

A radioactive enkephalin affinity reagent, selective for the mu opioid receptor subtype, was synthesized by a fragment condensation method. 3H-BOC-Tyr-D-Ala-Gly-OH was prepared by catalytic tritiation of the protected iodinated tripeptide. The protected tritiated tripeptide and N(Me)Phe-CH2Cl were condensed by the mixed anhydride method. The protecting group was removed by HCl/acetic acid. The tritiated tetrapeptide has a specific radioactivity of 56.8 Ci/mmole (2.1 TBq/mmole).     

Interaction of beta-funaltrexamine with [3H]cycloFOXY binding in rat brain: further evidence that beta-FNA alkylates the opioid receptor complex.

beta-Funaltrexamine (beta-FNA) is an alkylating derivative of naltrexone. In addition to acting as an irreversible inhibitor of mu-receptor-mediated physiological effects, intracerebroventricular (i.c.v.) administration of beta-FNA to rat attenuates the ability of selective delta receptor antagonists and naloxone to reverse delta receptor-mediated effects. Moreover, recent work demonstrated that i.c.v. administration of beta-FNA alters the conformation of the opioid receptor complex, as inferred by a decrease in the Bmax of the lower affinity [3H][D-ala2,D-leu5]enkephalin binding site. Consistent with the decreased potency of naloxone as an inhibitor of delta receptor mediated effects, beta-FNA doubled the naloxone IC50 for displacing [3H][D-ala2,D-leu5]enkephalin from its lower affinity binding site. These data collectively support the hypothesis that the opioid receptor complex postulated to mediate mu-delta interactions in vivo is identical to the opioid receptor complex as defined by vitro ligand binding studies. A direct prediction of this hypothesis is that beta-FNA should increase the Kd of antagonists for the mu binding site (mu cx) of the receptor complex. The data reported in this paper demonstrate that beta-FNA doubled the IC50 of the potent narcotic antagonist, 6-desoxy-6 beta-fluoronaltrexone (cycloFOXY) for displacing [3H][D-ala2,D-leu5]enkephalin from its lower affinity binding site, and doubled the Kd of [3H]cycloFOXY for its mu binding site, providing additional data that the mu binding site labeled by [3H]cycloFOXY is the mu binding site of the opioid receptor complex. beta-FNA also altered the kappa binding site labeled by [3H]cycloFOXY, and when administered intrathecally to mice, beta-FNA produced a longlasting antinociception in the acetic acid writhing test.     

Mu-receptor binding in physiological media: comparison with isolated tissue data

Mu-receptor affinities have been determined for a number of opioid drugs using a combination of isolated tissue and receptor binding techniques. The affinities of antagonists and partial agonists were determined by their ability to antagonise responses to [D-Ala2,MePhe4,Glyol5]enkephalin (GLYOL) on the rat vas deferens preparation. There was little correlation between these results and affinities measured by displacement of [3H]-GLYOL from guinea-pig brain membranes incubated in 50mM Tris buffer. By contrast, affinities measured by displacement of [3H]-naloxone from rat brain membranes incubated in a Krebs/HEPES buffer containing a non-hydrolysable analogue of GTP, agreed very closely with the isolated tissue data.     

Regional distribution of μ, δ and κ opioid receptors in human brains from controls and parkinsonian subjects

The binding properties of mu and delta opioid receptors were investigated in several areas of human brain by using [3H]Tyr-D-Ala-Gly-(Me)Phe-Gly-ol and [3H]Tyr-D-Thr-Gly-Phe-Leu-Thr as respective selective ligands, while the totality of opioid receptors was measured by using [3H]etorphine as a non-selective agonist. Receptor densities were highest in cerebral cortex, amygdala and striatum, and lowest in the substantia nigra (pars compacta). In the different brain areas of patients with Parkinson's disease, the density and the proportion of the various opioid receptors were not significantly different from control subjects.     

Binding characteristics of [3H]opioid ligands to active opioid binding sites solubilized from rat brain membranes by glycodeoxycholate and NaCl: the recovery of binding activity by dilution

This paper describes the binding properties of [3H]peptidergic opioid ligands to binding sites solubilized from rat brain membranes by the treatment with 0.125% sodium glycodeoxycholate and 1 M NaCl. The highest amount of the specific binding of [3H]-[D-Ala2-, Met5]enkephalinamide was obtainable when 10-fold diluted solubilized preparations were incubated in the presence of 0.1 mM MnCl2 and 100 mM NaCl at 0 degree C (on ice) for 3 h. With this assay condition, the significant binding of following [3H]opioid ligands, which have been thought to be selective for receptor types, was also observed: [3H]-[D-Ala2, MePhe4, Gly-ol5]enkephalin (mu-type), [3H]-[D-Ala2, D-Leu5]enkephalin (delta-type) and [3H]dynorphin1-9 (kappa-type). The number of binding sites in solubilized preparations for each [3H]ligand corresponded to 40-50% recovery of original membrane-bound binding sites. The Scatchard plot of the concentration-saturation binding curve showed only one class of binding sites, with a high affinity for each [3H]ligand. Apparent dissociation constants between solubilized receptors and [3H]ligands were the same as membrane-bound ones, but the ligand specificity for each receptor-type, which was examined by binding inhibition tests with unlabeled ligands, decreased. Present results indicate that heterogeneous opioid receptors in rat brain membranes seem to be transformed into less heterogeneous forms through the treatment with glycodeoxycholate and NaCl and the dilution process.     

Characterization of N,N(Me)2-Dmt-Tic-OH, a delta selective opioid dipeptide antagonist

N,N(Me)2-Dimethyl-tyrosine-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid-OH (N,N(Me)2-Dmt-Tic-OH) is a very selective delta opioid dipeptide with elevated antagonist activity. We have radiolabelled this compound by catalytic tritiation of the N,N(Me)2-Dmt(3',5'-I2)-Tic-OH precursor. The ligand labelled rat brain membranes with a Kd value of 0.42 nM and a Bmax of 63.12 fmol/mg protein. The new tritiated ligand showed high affinity for the delta opioid receptor whereas its binding at mu and kappa opioid receptors was weak. N,N(Me)2-Dmt-Tic-OH was able to inhibit the agonist-stimulated binding of the non-hydrolysable GTP analogue ?35SGTPgammaS, thus attenuating the activation of G proteins via opioid receptors. This simple opioid dipeptide in both normal and labelled form may serve as a useful tool to study delta opioid receptors in vitro and in vivo.     

Characterization of a polyclonal antibody to the mu opioid receptor

Active opioid receptors have been solubilized from bovine striatal synaptosomal membranes and purified approximately 4000-fold using a combination of affinity and hydroxyapatite chromatography. The affinity column was constructed by attaching hybromet, a newly synthesized opioid ligand with high affinity for the mu receptor, to a solid support matrix. A polyclonal antibody was generated to opioid receptors by injection of the purified receptor preparation into female New Zealand rabbits. The specificity of the antiserum was demonstrated by receptor competition and immunoprecipitation studies. Immunological titration of opioid binding activity from rat brain showed that the antibody was able to displace specific binding of [3H]etorphine (universal opioid) and [3H]dihydromorphine (mu opioid) from rat membranes, but was ineffective against the binding of [3H]ethylketocyclazocine (kappa [3H]D-Ala2,D-Leu5-enkephalin (delta opioid) or [3H]phencyclidine (phencyclidine/sigma receptor ligand). The antibody was able to precipitate the Mr 94,000 component of the 125I-labeled affinity-purified receptor, a finding which suggests that this subunit may be an opioid recognition component. By indirect immunofluorescence, the antibody was shown to bind specifically to the plasma membranes of the neurotumor cell line NCB-20 (neuroblastoma X Chinese hamster brain hybrid cells), which has high affinity opioid receptors. The observed fluorescence in the neuroblastoma cells was prevented by pre-adsorption of the antibody with purified receptor from rat brain. These results indicate that the antibody is specific for opioid receptors and may prove useful in the precise localization of opioid receptors in the central and peripheral nervous systems by immunohistochemical procedures.     

Capsaicin inhibits the in vitro binding of peptides selective for mu- and kappa-opioid, and nociceptin-receptors

Capsaicin inhibited the equilibrium specific binding of endogenous opioid-like peptide ligands such as endomorphin-1, nociceptin, and dynorphin((1-17)) in rat brain membrane preparations. We studied the in vitro effect of capsaicin (1-10 microM) on homologous and heterologous competitive binding of opioid ligands, using unlabeled synthetic peptides and the following tritiated compounds: [(3)H]endomorphin-1, [(3)H]endomorphin-2, [(3)H]nociceptin((1-17)) and [(3)H]dynorphin((1-17)). Capsaicin-dependent inhibition was also observed in [(35)S]GTPgammaS stimulation assays in the presence of certain opioid peptides. The inhibition of opioid binding was further investigated using other synthetic and natural mu-opioid ligands such as [D-Ala(2),(NMe)Phe(4),Gly(5)-ol]enkephalin (DAMGO), morphine and naloxone. The decrease in opioid ligand affinity upon capsaicin treatments was most apparent with endomorphin-1, followed by nociceptin and dynorphin. The binding of other investigated opioids were not affected in the presence of capsaicin. In [(3)H]endomorphin-1 binding assays, capsazepine antagonized the inhibitory effect of capsaicin in rat brain membranes suggesting the involvement of TRPV1 receptors. In Chinese hamster ovary (CHO) cells stably expressing mu-opioid receptors, but lacking vanilloid receptors, the inhibition by capsaicin on the binding of [(3)H]endomorphin-1 was not present. It is concluded that the inhibitory effect of capsaicin on the receptor binding affinity of endogenous opioid peptides in brain membrane preparations seems not to be a direct effect, it is rather a negative feedback interaction with opioid receptors.     

Characterization of dermorphin binding to membranes of rat brain and heart

Binding of dermorphin to the two major opioid receptor types, mu and delta, in rat brain membranes was examined by displacement of [3H] [D-Ala2, MePhe4, Gly-(ol)5]enkephalin (DAGO) and [3H]-[D-Ala2,D-Leu5]-enkephalin (DADLE) binding. Affinity of dermorphin binding to mu sites, Kd = 1.24 nM, was almost 3 times greater than that of DAGO, Kd = 3.35 nM. In contrast, the Kd value of dermorphin binding to delta sites was 78 nM only, as compared to Kd = 2.27 nM for DADLE. Dermorphin was ineffective in displacing [3H]ethylketocyclazocine (EKC) binding to kappa receptors after prior blocking of [3H]EKC binding to mu and delta sites. Studies of dermorphin binding to mu sites revealed that the potency of dermorphin increased in the presence of Na+ (+31%) but decreased in the presence of Mn2+ (-81%) or Gpp(NH)p (-44%). Displacement of bound [3H]diprenorphine (DPN) by dermorphin from atrial membranes of the rat heart, left side, was detectable, suggesting the presence of mu sites in this section of the heart.     

Autoradiographic study of irreversible binding of [3H]beta-funaltrexamine to opioid receptors in the rat forebrain: comparison with mu and delta receptor distribution.

beta-Funaltrexamine (beta-FNA) is an irreversible mu antagonist and a reversible kappa agonist in in vivo and in vitro tests. However, whether it produces irreversible delta antagonism is controversial. In binding studies, it is clear that beta-FNA does not bind irreversibly (it does reversibly) to kappa receptors. Yet there is no consensus as to whether beta-FNA binds irreversibly to mu and/or delta receptors. In this study, irreversible binding of [3H]beta-FNA to opioid receptors was examined in rat forebrain sections in the presence of 200 mM NaCl and its distribution compared with those of mu and delta opioid receptors, labeled by [3H][D-Ala2,MePhe4,Gly-ol5]enkephalin ([3H]DAMGO) and [3H][D-Pen2,D-Pen5]enkephalin ([3H]DPDPE), respectively. Irreversible binding of [3H]beta-FNA was determined as the binding that remained following 5 washes at room temp. for 1, 5, 20, 20, and 20 min each. Non-specific binding was defined by including 10 microM naloxone, beta-chlornaltrexamine (beta-CNA), or beta-FNA in the incubation mixture. At 37 degrees C, specific irreversible binding of [3H]beta-FNA to opioid receptors reached a plateau at 10 nM in 60 min, and constituted 50-70% of total irreversible binding. Series of 4 sections of similar anatomical levels were labeled with [3H]DAMGO, [3H]beta-FNA, [3H]beta-FNA + 10 microM naloxone, beta-CNA, or beta-FNA, and [3H]DPDPE, resp., and exposed to [3H]-Ultrofilm. The distribution of [3H]beta-FNA (5 nM) irreversible labeling is very similar to that of [3H]DAMGO, i.e. patches and subcallosal streaks in caudate-putamen, patches in nucleus accumbens, dense labeling in thalamus, and more binding in the rostral than caudal striatum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Irreversible labelling of delta-opioid receptors in rat brain and neuroblastoma cells by [3H]azido-DTLET: characterization of subunits and autoradiographic visualization of the covalent binding

[3H]Az-DTLET (Tyr-D-Thr-Gly-Phe(pN3)-Leu-Thr), a photoaffinity probe for delta opioid receptors binds to a single class of sites in rat brain membranes with a high affinity (KD = 1.66 nM). The selectivity index of Az-DTLET (KI delta/KI mu = 0.036) is better than that of its precursor DTLET (0.053). Rat brain or neuroblastoma glioma cells membranes were incubated with 10 nM [3H]Az-DTLET, washed and irradiated with U.V. After irradiation a fraction (20-30%) of specific binding was found to remain indissociable after 10 min at 60 degrees C and was considered as irreversible. This fraction increased as a function of the irradiation time. The radioactivity irreversibly bound to rat brain membranes, solubilized by sodium cholate, was associated with high molecular weight species (200,000 daltons). In denaturing conditions (SDS 2%), the [3H]Az-DTLET specific binding was associated with molecular components of 45-50 K and 90-100 K daltons. In contrast, when opioid receptors were prelabelled by [3H]Az-DTLET, solubilized by Na-cholate and irradiated, the radioactivity was only recovered with subunits of 45-50 K daltons. The autoradiographic localization of the irreversibly bound [3H]Az-DTLET in rat brain was identical to that of reversibly bound [3H]DTLET or [3H]Az-DTLET. These results suggest that [3H]Az-DTLET represents an adequate specific probe for studies on the structure, function and anatomical distribution at light and even electron microscopic level of delta-opioid receptors.     

Microtubule disassembly increases the number of opioid receptor binding sites in rat cerebrum membranes

The role of microtubules in opioid receptor binding was studied by using microtubule assembly inhibitors. Preincubation of rat cerebrum membranes with podophyllotoxin or colchicine provoked a marked increase in the number of binding sites as judged by [3H]-naloxone, [3H]-morphine and [3H]-D-Ala2-Leu5-enkephalin binding experiments. These results indicate microtubule involvement in regulation of opioid receptor expression.     

Zeta (zeta), a growth-related opioid receptor in developing rat cerebellum: identification and characterization.

Endogenous opioids and opioid receptors (i.e. endogenous opioid systems) are expressed during neural ontogeny, and play a role in the development of the nervous system. Using [3H][Met5]-enkephalin, a potent ligand involved in neural growth, particularly cell proliferation, specific and saturable binding was detected in homogenates of 6-day-old rat cerebellum; the data were consistent with a single binding site. Scatchard analysis yielded a binding affinity (Kd) of 2.2 nM and a binding capacity (Bmax) of 22.3 fmol/mg protein. Binding was linear with protein concentration, dependent on time, temperature, and pH, and was sensitive to Na+, Mg2+, and guanyl nucleotides. Optimal binding required protease inhibitors, and pretreatment of the homogenates with trypsin markedly reduced binding, suggesting that the binding site was proteinaceous in character. The [Met5]-enkephalin binding site was an integral membrane protein located in the nuclear fraction. Competition experiments indicated that [Met5] enkephalin was the most potent displacer of [3H][Met5]-enkephalin, and that binding was stereospecific. In the adult rat cerebellum, non-opioid receptor binding of [3H][Met5]-enkephalin was recorded, mu and kappa receptors were also found in the developing rat cerebellum, while mu, delta, and kappa receptors were recorded in adult cerebellar tissue. The function, pharmacological and biochemical characteristics, subcellular distribution, and temporal expression of the [Met5]-enkephalin binding site suggest the presence of a unique opioid receptor, termed zeta (zeta), in the developing nervous system.     

Anatomical distribution of sodium-dependent [(3)H]naloxone binding sites in rat brain

The sulfhydryl alkylating reagent N-ethylmaleimide (NEM) blocks opioid receptor binding and receptor/G-protein coupling. Sodium partially restores [(3)H]naloxone binding after inhibition by NEM to reveal sodium-dependent [(3)H]naloxone sites, defined as binding in the presence of 50-100 mM NaCl after treatment of membranes or sections with 750 microM NEM. In the present study, receptor autoradiography of [(3)H]naloxone binding in control and NEM-treated tissue was used to examine the anatomical distribution of sodium-dependent [(3)H]naloxone sites in rat brain. In brain membranes, the pharmacology of sodium-dependent [(3)H]naloxone sites was consistent with that of mu opioid receptors. Relatively high IC(50) values for agonists and lack of effect of Gpp(NH)p on DAMGO displacement of [(3)H]naloxone binding in NEM-treated membranes indicated that the sodium-dependent sites were low affinity sites, presumably uncoupled from G-proteins. Autoradiograms revealed that NEM treatment dramatically reduced [(3)H]naloxone binding in all brain regions. However, [(3)H]naloxone binding was increased in specific regions in NEM-treated sections in the presence of sodium, including bed nucleus of the stria terminalis, interpeduncular nucleus, periaqueductal gray, parabrachial nucleus, locus coeruleus, and commissural nucleus tractus solitarius. Sodium-dependent [(3)H]naloxone binding sites were not found in other areas that exhibited [(3)H]naloxone binding in control tissue, including the striatum and thalamus. These studies revealed the presence of a subpopulation of [(3)H]naloxone binding sites which are sodium-dependent and have a unique regional distribution in the rat brain.     

Purification and mass spectrometric analysis of the mu opioid receptor

A mouse mu opioid receptor was engineered to contain a FLAG epitope at the amino-terminus and a hexahistidine tag at the carboxyl-terminus to facilitate purification. Selection of transfected human embryonic kidney (HEK) 293 cells yielded a cell line that expressed the receptor with a B(max) of 10 pmol/mg protein. 3[H]Bremazocine exhibited high affinity binding to the epitope-tagged mu opioid receptor with a KD of 1.0 nM. The agonists [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO), morphine and [D-Ala(2),D-Leu(5)]enkephalin (DADL) competitively inhibited bremazocine binding to the tagged mu receptor with KI's of 3.5, 17 and 70 nM, respectively. Chronic treatment of cells expressing the epitope-tagged mu receptor with DAMGO resulted in down-regulation of the receptor, indicating that the tagged receptor retained the capacity to mediate signal transduction. The mu receptor was solubilized from HEK 293 cell membranes with n-dodecyl-beta-D-maltoside in an active form that maintained high affinity bremazocine binding. Sequential use of wheat germ agglutinin (WGA)-agarose chromatography, Sephacryl S300 gel filtration chromatography, immobilized metal affinity chromatography, immunoaffinity chromatography, and sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) permitted purification of the receptor. The purified mu opioid receptor was a glycoprotein that migrated on SDS/PAGE with an apparent molecular mass of 80 kDa. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry was used to identify and characterize peptides derived from the mu opioid receptor following in-gel digestion with trypsin or chymotrypsin, and precursor-derived tandem mass spectrometry (ms/ms) confirmed the identity of several peptides derived from enzymatic digestion of the mu opioid receptor.     

Selective opioid agonist and antagonist competition for [3H]-naloxone binding in amphibian spinal cord

Opioids elicit antinociception in mammals through three distinct types of receptors designated as mu, kappa and delta. However, it is not clear what type of opioid receptor mediates antinociception in non-mammalian vertebrates. Radioligand binding techniques were employed to characterize the site(s) of opioid action in the amphibian, Rana pipiens. Naloxone is a general opioid antagonist that has not been characterized in Rana pipiens. Using the non-selective opioid antagonist, [3H]-naloxone, opioid binding sites were characterized in amphibian spinal cord. Competitive binding assays were done using selective opioid agonists and highly-selective opioid antagonists. Naloxone bound to a single-site with an affinity of 11.3 nM and 18.7 nM for kinetic and saturation studies, respectively. A B(max) value of 2725 fmol/mg protein in spinal cord was observed. The competition constants (K(i)) of unlabeled mu, kappa and delta ranged from 2.58 nM to 84 microM. The highly-selective opioid antagonists yielded similar K(i) values ranging from 5.37 to 31.1 nM. These studies are the first to examine opioid binding in amphibian spinal cord. In conjunction with previous behavioral data, these results suggest that non-mammalian vertebrates express a unique opioid receptor which mediates the action of selective mu, kappa and delta opioid agonists.     

Evidence for multiple "Kappa" binding sites by use of opioid peptides in the guinea-pig lumbo-sacral spinal cord

Binding properties of [3H]-etorphine and [3H]-ethylketocyclazocine have been studied in the lumbo-sacral spinal cord of guinea-pig which does not contain mu or delta binding sites. [3H]-etorphine binds to a single class of high affinity sites, whereas [3H]-ethylketocyclazocine interacts with a high and a low affinity component. Using a discriminative procedure, 5 microM (D-Ala2, D-Leu5) enkephalin (DAL), the high affinity component of [3H]-ethylketocyclazocine can be resolved in two classes of sites, (D-Ala2, D-Leu5) enkephalin sensitive sites (DALS sites) and (D-Ala2, D-Leu5) enkephalin insensitive sites (DALI sites). In these conditions, there is a total loss of [3H]-etorphine sites, whose binding capacity and properties strictly correspond to the DALS sites labelled by [3H]-ethylketocyclazocine. Pharmacological investigations indicate that DALI sites for which dynorphin (1 leads to 17) is the best ligand, can be related to kappa sites previously described in guinea-pig brain, whereas DALS sites for which (Arg6, Phe7) Met-enkephalin possesses a good affinity, closely correspond to benzomorphan sites recently characterized in rat brain and spinal cord. [3H]-ethylketocyclazocine interacts additionally with "non opiate" low affinity sites, for which only benzomorphan drugs exhibit a good affinity, whereas morphine, naloxone, phencyclidine or endogenous opioid peptides do not present any affinity for them. On the basis of these data, a new subdivision of "kappa" sites is discussed.     

Mu-receptor specificity of the opioid peptide irreversible reagent, [H] DALECK

A novel affinity reagent DALECK, i.e. D-Ala2-Leu5-enkephalin with a C-terminal chloromethyl ketone group, was previously synthesized in normal and in tritiated form and shown to react irreversibly at opioid receptors, with some evidence for selectivity for the mu subtype. DALECK tritiated in its phenolic group has been synthesized at 13-fold higher specific radioactivity than in the previous study. In the irreversible reaction of this product at pH 8.1 with rat brain membranes it was confirmed that only one polypeptide there is labelled, of apparent Mr 58,000. Competition between this reaction and ligands highly selective for the mu, delta or kappa binding sites yielded curves demonstrating the very high selectivity of the DALECK irreversible reaction for the mu site. The results provide evidence that the mu opioid receptor protein contains only one type of binding subunit, whose apparent Mr is 58,000, this size being dependent upon the conditions used in the gel electrophoresis and being higher when stringent conditions which would reduce all internal disulphide bonds are applied.     

Guanine nucleotide regulation of [125I]beta-endorphin binding to NG108-15 and SK-N-SH cell membranes: specific cation requirements

Regulation of [125I]beta h-endorphin binding by guanine nucleotides was investigated in membrane preparations from two opioid receptor-containing cell lines: NG108-15, which contains only delta opioid receptors, and SK-N-SH, which contains predominantly mu opioid receptors. In contrast to the binding of the delta-selective agonist [3H][D-penicillamine2,D-penicillamine5]enkephalin to NG108-15 cell membranes, and of the mu-selective agonist [3H][D-Ala2,MePhe4,Gly-ol5]enkephalin to SK-N-SH cell membranes, [125I]beta h-endorphin binding to NG108-15 and SK-N-SH cell membranes was not altered by guanosine triphosphate (GTP) or guanylyl-5'-imidodiphosphate (Gpp(NH)p) in the absence of cations. However, in the presence of NaCl, [125I]beta h-endorphin binding to both cell lines was inhibited by GTP and Gpp(NH)p in a concentration-dependent manner. In SK-N-SH cell membranes, the ability of sodium to promote regulation of [125I]beta h-endorphin binding by GTP was mimicked by the monovalent cations lithium and potassium, but not by the divalent cations magnesium, calcium, or manganese. In NG108-15 cell membranes, only sodium was effective in promoting inhibition of [125I]beta h-endorphin binding by GTP. The effect of GTP or Gpp(NH)p in the presence of sodium was also observed with guanosine diphosphate, but not guanosine monophosphate or any of the non-guanine nucleotides tested. These results indicate that the presence of monovalent cations is required for regulation of [125I]beta h-endorphin binding by guanine nucleotides, and that the specificity of this cation requirement differs between the mu and delta receptor-containing cell lines.