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WO1993019086A1 - Recepteur d'opioides purifie - Google Patents

Recepteur d'opioides purifie Download PDF

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Publication number
WO1993019086A1
WO1993019086A1 PCT/US1993/002539 US9302539W WO9319086A1 WO 1993019086 A1 WO1993019086 A1 WO 1993019086A1 US 9302539 W US9302539 W US 9302539W WO 9319086 A1 WO9319086 A1 WO 9319086A1
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Prior art keywords
receptor
endorphin
ligand
binding
opioid
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PCT/US1993/002539
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English (en)
Inventor
Cecil Mark Eppler
Hong-Ming Shieh
John Ronald Zysk
Martin John Corbett
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American Cyanamid Company
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Publication of WO1993019086A1 publication Critical patent/WO1993019086A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • the present invention relates to substantially pure opioid receptors.
  • Opioids are a chemically diverse group of com ⁇ pounds which includes naturally occurring peptides and alkaloids as well as a large number of synthetic analogs.
  • the physiological effects of opioid agonists include anal ⁇ gesia, drowsiness, changes in mood, respiratory depression, decreased gastrointestinal motility, nausea, vomiting and alterations in the endocrine and autonomic nervous systems (Jaffe and Martin, in The Pharmacological Basis of Thera ⁇ peutics, Gilman, A.6. et al. - ⁇ eds.; MacMillan, Mew York, pages 491-531, 1985) .
  • opiate receptors are the mu ( ⁇ ) , delta (5) and kappa ( ⁇ c) classes, based on clear differences in their ligand selectivities and pharma ⁇ cological effects (Lord et al.. Mature, 267;495-499. 1977). Sigma ( ⁇ ) (Jaffe and Martin, supra), and epsilon e, (Schulz et al., J. Pharmacol. Exp. Th ⁇ r. 216x604-606, 1981) are also thought to exist, based on differential pharmacology and ligand binding. There is also evidence for receptor subtypes within these major classes (Jaffe and Martin, supra) .
  • the present invention relates to a substantially pure opioid receptor protein, and biologically active fragments thereof.
  • substantially pure as used through ⁇ out the present specification and claims, is meant a protein free of other non-opioid receptor cellular proteins with which it would normally be associated in its membrane-bound state, such a protein is essential in order to successfully obtain accurate sequence information.
  • a purified opioid receptor is isolatable by binding a biotinylated opioid ligand with membranes derived from an appropriate tissue source, i.e., one expected to express opioid receptors, to form a receptor:ligand complex.
  • the membranes are then solubilized in a bile-salt like detergent composition, and contacted with an avidin or streptavidin containing affinity substrate, to which the biotinylated receptor:ligand complex will bind.
  • the receptor is eluted from the bound complex by contact with a GTP analog-containing eluant.
  • the eluate is then contacted with a lectin affinity column which specifi ⁇ cally binds glycoproteins.
  • a receptor is identified by its binding a ⁇ -endorphin ligand.
  • three species are identifiable by this characteristic in the method described.
  • a primary species has a molecular weight of about 66,000, while two minor species have molecular weight of 140-160,000 and 50-55,000. Based on the affinity for /3-endorphin, and other pharmacological data, these species are believed to represent a ⁇ opioid receptor type.
  • the purified receptor in addition to its use in sequencing and ultimate cloning of the receptor gene, the purified receptor, or biologically active fragments thereof, can be used in production of monoclonal or polyclonal anti-receptor anti ⁇ bodies.
  • "Biologically active” in the present context refers not only to fragments which retain ligand binding activity, but also refers to fragments capable of raising an antibody response when injected into a host animal.
  • Such antibodies poly- or monoclonal
  • Such antibodies can be used in manipulation of periph ⁇ eral opioid receptors involved in gut motility and growth hormone secretion.
  • Such antibodies can also be utilized in drug delivery to specific tissues or for tumor imaging.
  • Receptor clones isolated utilizing sequence information obtained from the purified protein are useful in identifying other receptor subtypes, in screening for new opioid ligands, and for understanding mechanisms of opioid action, for example, drug addiction.
  • Figure l Purification of Biotinylated ⁇ - Endorphin.
  • Biotinylated and Nonbiotinylated ⁇ -endorphins Rat brain membranes (30 ⁇ g per well) are incubated with [ 125I] - endorphin (100,000 cpm per well) in the microtiter plate assay as described in Experimental Procedures. 2.A. Binding incubations are for 1 hour. Figure 3. Efficacy of Biotinyl- ⁇ -Endorphin in Receptor Purification.
  • Rat brain membranes (each containing 30 g of protein) are incubated with: (a) No ligand, (b) 100 nM biotinyl-/9-endorphin Fl or (c) 100 nm biotinyl- -9-endorphin F2 and solubilized in 0.15% D:L (all procedures as in Experimental Methods. 1.-3.: one exception in this particular experiment is that the binding and wash step is done in 50 mM potassium phosphate [pH 7.4] + 0.1% BSA) .
  • Figure 4 im unoreactivity of 30-40,000 MW Material Eluted by GTP from Streptavidin-Agarose Previously Incubated with R:L Complexes Made with Biotinylated So ato- statin or -Endorphin.
  • WGA-A nonbound fractions of GTP eluates from streptavidin from receptor purifications done with biotinyl-somatostatin 28 (bio-S28) or biotinyl- ⁇ - endorphin are separated by SDS-PAGE and analyzed by Western blotting. The electroblotted samples are first reacted with an anti-Gi. ⁇ rabbit antiserum (1:400 dilution).
  • the second antibody is a peroxidase-coupled, donkey antirabbit Ig antiserum (1:104 dilution).
  • the final complex is detected by enhanced chemiluminscence (ECL; Whitehead, T.P. et al. , Nature 305:158-159, 1983).
  • ECL is based on the perox- idase-catalyzed oxidation of luminol and subsequence en ⁇ hanced chemiluminescence where the probe is bound (Amersham Life Science Products catalog, 1989/90 edition, page 5) .
  • Lanes A-c contain 200, 100 and 50 ng of recombinant G. .
  • Lanes D and E are from a purification of SRIF receptor by the method of Eppler et al. (as described in U.S. ser. No. 07/677,003) with 20 nM bio-s28, Lane E, binding step done with 20 nM bio-S28 + 20 ⁇ M S14.
  • Lanes F-I are from a purification of opioid receptor by the method described herein.
  • F and G are eluates with 500 mM Naci.
  • H and I are eluates with 100 ⁇ M GTP-g-S.
  • F and H are from a sample without ligand in the binding step.
  • G and I are from a sample with 100 nM biotinyl-9-endorphin in the binding step.
  • FIG. 5 Effects of Competition by Mon-Biotin- ylated Opioid Ligands on Purification of Receptor Bands by Bio- ⁇ -Endorphin.
  • the purification procedure is as described in Experimental Procedures. 6.
  • Competition with biotinyl- /3-endorphin in the binding step is by 50 ⁇ M ⁇ -endorphin + 50 ⁇ M met-enkephalin (A) or by 40 ⁇ M naloxone (B) .
  • A met-enkephalin
  • B 40 ⁇ M naloxone
  • 20 ⁇ M naloxone is also added to the 100,000 x g supernatant before incubation with streptavidin-agarose because of the relatively high rate of dissociation of naloxone from opioid receptors.
  • Elution from SA-A is with 500 mM NaCl.
  • Eluates are processed by adsorption to WGA, eluted from WGA by TAC and analyzed by SDS-page as
  • Receptor purification is carried out as described for Figure 6 up to the point of washing the SA-A columns. Elution is by 100 ⁇ M GTP-100 mM NaCl (1) and then by 500 M NaCl (2). Further processing of the eluates is as described for Figure 6.
  • FIG. 8 Competitive Binding of Mu and Delta Receptor-Specific Peptide with ⁇ -Endorphin for Binding with Opioid Receptor.
  • This 11% SDS-PAGE gel illustrates the mu-subtype identity of the isolated receptor protein. Details of the procedures and complete analysis are found in Results, section 4.
  • Lanes 3 and 4 demonstrate the ability of a peptide having preferential binding with a mu-subtype receptor to competitively inhibit binding of a biotinylated ⁇ -endorphin to the receptor, thereby preventing isolation of the 66 kDa protein from rat membranes using the biotin- avidin affinity chromatography.
  • Lanes 5, 6 and 7 represent competitive binding using a delta-subtype specific peptide which permits recovery of the 66 kDa protein.
  • the purified receptors of the present invention are isolated by a receptor purification method disclosed in copending U.S. Serial No. 07/677,003, the contents of which are incorporated herein by reference.
  • Opioid receptors can be found in a wide variety of tissue types (Jaffe and Martin, supra. the contents of which is incorporated herein by reference) .
  • the ⁇ , ⁇ , and ⁇ classes of receptors are found in brain, as well as other tissues; the e type is found in vas deferens. and the K type is plentiful in placenta (Ahmed et al. , supra) .
  • the opiate receptor is isolated initially as a complex with its associated G proteins.
  • a number of opiate or opioid analogs are commercially available that can be used for receptor binding. For example. Research Bio- chemicals, Incorporated, 1991 Catalog, page XV, identifies a number of opioid ligands by their subtype specificity. The ligand used will generally be selected based on its affinity for a particular receptor subtype. In a preferred method for purification, a biotinylated opiate analog is used. In the following examples, the ligand used for isolation of receptor is a biotinylated ⁇ -endorphin. In the preferred isolation method, the ligand is first bound to intact cell membranes, thereby forming a receptor:ligand (R:L) complex.
  • R:L receptor:ligand
  • the membranes are solubilized in detergent and intact receptor:ligand complexes are obtained.
  • a useful detergent for this purpose is a combination of deoxycholate and lysophosphatidylcholine in a 1:1 ratio, preferably at a concentration of 0.2% w/v or less.
  • the complex consists of the receptor and its associ ⁇ ated G protein subunits. The association of the receptor with G proteins is confirmed by the rapid dissociation of the complex in the presence of a stable GTP analog.
  • the solubilized complex is then contacted with an appropriate high affinity binding column.
  • the column used is preferably streptavidin- agarose (SA-A), whereby the biotinylated portion of the R:L complex will tightly bind to the streptavidin.
  • streptavidin is preferred, due to its lower non-specific binding; how ⁇ ever, free and immobilized avidin is also available (Pierce, vector) and may be suitable for some purposes.
  • SA-A streptavidin- agarose
  • streptavidin is preferred, due to its lower non-specific binding; how ⁇ ever, free and immobilized avidin is also available (Pierce, vector) and may be suitable for some purposes.
  • the column is eluted with a GTP analog, such as GTP- ⁇ -S.
  • the GTP analog serves to dissociate G protein subunits from the receptor, thereby lowering the affinity of the receptor for its ligand, and thus indirectly causing dissociation from the ligand.
  • the elution with GTP analog is combined with elution with at least 25 mM NaCl, preferably 50-100 mM, up to a maximum of about 500 mM NaCl.
  • dissociation will occur with GTP alone, it occurs at a relatively low level (about 30%) , and the use of NaCl enhances this dissociation.
  • a high level, i.e., 500 mM of salt can be used alone, but may result in an unacceptable level of non-specific elution.
  • the eluate or eluates contain a receptor that is about 80-90% pure, and is apparently still associated with one of its G protein subunits.
  • the eluate from the streptavidin column is then incubated with a lectin affinity chromato ⁇ graphy substrate, such as wheat germ agluttinin (WGA)- agarose, which will separate glycoproteins from nonglycopro- teins.
  • a lectin affinity chromato ⁇ graphy substrate such as wheat germ agluttinin (WGA)- agarose, which will separate glycoproteins from nonglycopro- teins.
  • WGA wheat germ agluttinin
  • the eluate containing the glycosylated material shows a protein with a molecular weight of about 66,000; this protein is also seen in material eluted by GTP- ⁇ - ⁇ and/or with NaCl, but is not seen in eluates from samples not previously bound with the biotinylated ⁇ -endorphin, indicating its ligand dependence.
  • This band appears to represent an opioid receptor, presumably a "mu” or “delta” type opioid receptor, based on ⁇ -endorphin , s known preferen ⁇ tial binding to "mu” or “delta” receptor types, and the pharmacological data discussed below.
  • the purified 66 kDa glycoprotein is subjected to Lys-C endoprotease digestion, SDS polyacrylamide gel elec- trophoresis and electroblotting, producing a 15 kDa peptide band.
  • This peptide yields 20 cycles of high quality amino acid sequence.
  • the N-terminus of this band overlaps by 4 amino acid residues with a 7-amino acid residue sequence obtained from a cyanogen bromide digest, giving a total sequence length of 23 amino acid residues.
  • the sequence (Sequence ID No. 1) obtained is as follows:
  • the novel sequence information obtained provides the basis for isolation and cloning of the corresponding gene encoding the receptor.
  • Primers based on the sequence shown above, as well as primers based on a 12-amino acid residue sequence near the N-terminus of the delta opioid receptor can be used in a PCR cloning strategy.
  • the delta opioid sequence in this region is nearly identical to the same region of SSTRl, and seems to be highly conserved in a set of 5 or 6 receptors, and preliminary data indicates homology in the mu receptor as well.
  • the combination of primers, including the mu specific-based primer, in PCR of whole brain mRNA, is expected to selectively yield the mu receptor.
  • the purified receptor can be used for a number of purposes.
  • the purified material in glycosylated or nonglycosylated form, can be used to create monoclonal or polyclonal antibodies having specificity for the opioid receptor.
  • the technology for creation of monoclonal anti ⁇ bodies is well known in the art (see, e.g., Goding, Mono ⁇ clonal Antibodies: Principle and Practice, 2nd Ed., 1986).
  • Such antibodies may have utility in, e.g. manipulating purified opioid receptors involved in gut motility and growth hormone secretion, or in drug delivery to specific tissues or for tumor imaging.
  • General techniques for preparing anti-receptor antibodies are found in U.S. Patent No. 4,857,637, the contents of which are incorporated herein by reference.
  • the isolated receptor protein itself can be used in screening assays to identify compounds that act as analogs.
  • the receptor protein can be immobil ⁇ ized by any means which does not interfere with opiate binding activity.
  • the immobilized receptor is then contact ⁇ ed with a specific compound or mixture and its ability to compete with radiolabelled opiate for binding to the recep ⁇ tor is evaluated. Variations on this method will be appar ⁇ ent to those skilled in the art.
  • the present invention encompasses the opiate receptor protein and its biologically active fragments produced by any means, whether synthetically, recombinantly. or by purification of the native protein.
  • the isolated opiate receptor as described above, is pure enough to be used in protein sequencing procedures which are well known in the art, and such sequencing is routinely accomplished using such methods.
  • the protein sequence in turn is used to design oligonucleotide probes which are used to screen ⁇ gtlO libraries containing the relevant cDNA (copies of RNA) , e.g., from brain cells. Hybridization of oligos with the library identifies the clone(s) containing the SRIF receptor gene or portions thereof.
  • the gene or gene fragments are isolated from the clones, the whole gene reconstructed and then ligated into an appropriate vector by known methods.
  • the vector is chosen based upon the choice of preferred host cell.
  • the host cell may be prokaryotic, e.g., E ⁇ . coli or other bacteria; or eukaryotic, e.g., yeast, insect, or mammalian cells.
  • Rat Brain M*""*"**"** Whole male rat brains frozen in liquid 2 are purchased from Pel-Freez (Rogers, AR) . All procedures for membrane preparation are carried out at a temperature of 2-6°C. The brains are homogenised in a Waring blender in a buffer containing l mM Na-bicarbonate (pH 7.2), 1 mM EDTA, l mM EGTA (all chemicals from Sigma Chemical, St. Louis, MO) and 0.7% (vol./vol.) of the 100X 4Pase protease inhibitor mixture (see “Protease Inhibitors” below). The ratio of tissue/homogenization medium is from 25-35 gm of brain/500 ml.
  • the blender is controlled through a variable output rheostat (Staco Energy Products, Dayton, OH; type 3PN1010) at a setting of 40.
  • the homogenate is centrifuged for 10 minutes at 1,000 x g pellet is rehomeginized in 500 ml of homogenization medium and rec ⁇ ntrifuged for 10 minutes at 1,000 x g.
  • the 1,000 x g pellet is discarded.
  • the 1,000 x g supernatants are combined and centrifuged for 30 minutes at 20,000 x g.
  • the 20,000 x g membrane pellet is washed by being resuspended with a Dounce homogenizer in 500 ml of homogenization medium supplemented with 10 mM EDTA (pH readjusted to 7.4) and then washed twice by being resuspended in 25 mM Tris buffer (Sigma Chemical Co. ; pH 7.4) and centrifuged for 25 minutes at 20,000 x g.
  • the final membrane pellet is resuspended in 25 mM inhibitor mixture to a protein concentration of 4-12 mg/ml.
  • the resuspended membranes are aliquot ⁇ d, frozen on dry ice and stored at -90°C.
  • Binding of [ I]-labelled ⁇ -endorphin, ⁇ - endorphin and other ⁇ -endorphin analogs and opioids is done in a binding buffer containing 50 mM HEPES (Sigma; pH 7.4; pHed with OH) , 0.1% (w/v) bovine serum albumin (Miles Laboratories, Elkhart, IN) and protease inhibitors as specified below for specific applications. All binding incubations are carried out at room temperature (20-23°C) .
  • ligands for example ⁇ -endorphin or biotinylated b-endorphin
  • ligands for example ⁇ -endorphin or biotinylated b-endorphin
  • Preparative - Rat brain membranes are diluted to a concentration of 0.5 mg of membrane protein/ml in binding buffer containing 1/400 (vol./vol.) of the 400X P/B/Bz protease inhibitor mixture (see “Protease Inhibitors” below) .
  • Biotinyl- ⁇ -endorphin (synthesized and purified as described below; 1:1 mixture of HPLC fractions 1 and 2 is added, most commonly to a concentration of 60 nM.
  • the mixture is incubated either by stirring in a large poly ⁇ propylene beaker (1-2 liters volume) or by rotation on a tube rotator (100-250 ml per polypropylene centrifuge tube).
  • Control incubations designed to show ligand specificity of purified proteins are done by various means as follows: i. No ligand. Rat brain membranes are incubated as above except with no biotinyl- ⁇ -endorphin or other opioid analog, ii. Blocking ligand. Binding of biotinyl- ⁇ -endorphin is blocked by a large molar excess (500-1,000 fold) of a non-biotinylated opioid ligand such as ⁇ -endorphin , met- enkephalin or naloxone. In this case, the blocking ligand is added from 5-15 minutes prior to the addition of bio ⁇ tinyl- ⁇ -endorphin. In some cases only the blocking ligand is added.
  • the receptor sites may simply be saturated with naloxone.
  • the binding reactions (l hour) are terminated by centrifugation for 10-15 minutes at 20,000 x g.
  • the supernatants are decanted and the membrane pellets are washed with a volume of binding buffer (minus bovine serum albumin) equal to the original incubation volume.
  • binding buffer minus bovine serum albumin
  • the membranes are dispersed in the wash buffer in a Dounce homogenizer, diluted out in the wash buffer and recentrifuged at 20,000 x g. This final membrane pellet is then solubilized in—detergent as described in part 3, below, and used to characterize soluble R:L complex
  • This step is carried out in a solubilization buffer containing 25 mM Tris (pH 8.0) and 10% glycerol. All procedures are at 4 C or on ice.
  • Protease inhibitors 100X 4Pase; 1% vol./vol.
  • rat brain membranes are diluted out into this medium to a protein concentration of 0.5 mg/ml.
  • the samples are centrifuged for 30 minutes at 100,000 x g.
  • the 100,000 x g supernatants are aspirated out of the centrifuge tubes as far as possible without disturbing the pellets of insoluble material.
  • the remaining supernatant is poured out of the tubes and filtered through a 0.2 ⁇ cellu ⁇ lose acetate or nylon filter unit (Corning Inc. , Corning, NY) to remove particulate matter dislodged from the pellet. This filtered supernatant is then combined with the material removed by aspiration.
  • the final product (about 50% pure; see Figure IA) is further purified by reverse phase HPLC on a Brownlee "Aquapore" C8 column (l x 25 cm) . Elution is by a gradient of aceto- nitrile mixed in water/0.1% trifluoroacetic acid. Two closely spaced product peaks are eluted from the column ("Fl" and "F2" in Figure IB) . These two peptide fractions are lyophilized and solubilized in water at l mg/ml. Aliquots are stored frozen at -90°C.
  • the resin is washed with 20 bed volumes of solubilization buffer + 0.15% D:L + 1/500 volume of the 100X 4Pase protease inhibitor mixture. Methods of eluting the columns will be specified for individual experiments (see Results) .
  • the eluates from the SA-A columns are incubated overnight (12-15 hours) with 1/200 to 1/400 volumes of immobilized wheat germ agglutinin (WGA-agaros ⁇ or WGA-A; Vector Labs, Burlingame, CA) .
  • the WGA-A is pelleted by centrifugation, washed twice with 50-100 volumes of solubilization buffer + 0.15% D:L (after removing the supernatants containing material not bound to WGA) and then either: (A) eluted with 8 mM triacetylchitotriose (TAC; Sigma) in solubilization buffer + 0.15% D:L (3 sequential elutions where resin is mixed with 2 volumes of elution buffer at room temperature for 15-20 minutes, pelleted by centrifugation and super ⁇ natant removed and saved) or B. solubilized directly by addition of IX Laemmli sample buffer and heating at 90°C for 10-15 minutes.
  • TAC triacetylchitotriose
  • the assay for R:L complex exploits the well known glycoprotein nature of receptors which, like most cell surface proteins, contain covalently linked carbohydrate.
  • the ligand, ⁇ -endorphin is not glycosylated and will not bind to a carbohydrate-binding lectin, such as wheat germ agglutinin (WGA) .
  • WGA wheat germ agglutinin
  • Rat brain membranes are incubated with [ 125I] ⁇ -endorph ⁇ n as described above (experimental Procedures. 2.B.). The
  • total binding sample is incubated with only [ 125 I] ⁇ - endorphin.
  • nonspecific binding sample is incubated with [ 125I] ⁇ -endorphin plus 10—6 M nonlabelled ⁇ -endorphin.
  • [125I] -endorphin in the 100,000 x g supernatant are counted as described in "Experimental Procedures. 2.A.”. Some samples are held on ice as an internal standard for the ratio of total to nonspecific cpm in the starting material ("A. 100,000 x g supernatant"; see “A/B” ratio). other samples (“B. Supernatants + WGA-agarose”) are warmed to room temperature and then receive 100 ⁇ M GTP-g-8 (Sigma; diluted from a 2 M stock solution in H.O) , 500 M NaCl (diluted from a 5 M stock solution in H.O) or no treatment and are further incubated for 10 minutes at room tempera ⁇ ture.
  • WGA-agarose immobilized wheat germ agglutinin
  • Vector Labs, Burlingame, CA immobilized wheat germ agglutinin
  • the WGA-agarose is pelleted by centrifu ⁇ gation, the supernatants are removed and the WGA-agarose is washed once in solubilization buffer + 0.15% D:L and counted for radioactivity.
  • the membrane bound complex between [ 125I] ⁇ - endorphin and its receptor can be solubilized mostly in intact form. This is shown by the adsorption of a high proportion of the solubilized [ 125I] ⁇ -endorphin to immobil ⁇ ized WGA. Not only is a high proportion of the specifically bound radioligand adsorbed to WGA, as would be expected if it is bound to the receptor, but WGA selects for specifical ⁇ ly bound material. This is shown by the large increase in the ratio of total cpm/nonspecific cpm in the WGA-bound material. Also, the soluble R:L complex is stable enough to be separated from free ligand in a step taking 2-3 hours.
  • a biotinylated ⁇ -endorphin can be used to form a R:L complex that could be adsorbed in intact form to immobilized streptavidin.
  • binding to immobilized WGA can serve as an assay for the soluble R:L complex, and binding to WGA can serve as a purification step for the receptor.
  • the R:L complex can be easily dissociat ⁇ ed. This provides a means for eluting the receptor from an affinity column.
  • a soluble complex between the receptor and biotinyl ⁇ -endorphin could be bound to immobilized streptavidin and the receptor then eluted by GTP (partial elution) , GTP + NaCl or NaCl.
  • the IC 5Q s for reduction of radioligand binding by competition with cold ligand are: ⁇ -endorphin, 1 nM; biotinyl- ⁇ -endorphin (Fl) , l nM; and biotinyl- ⁇ -endorphin
  • the ligand specificity of the 66K glycoprotein is further tested by blocking binding of the biotinylated ⁇ -endorphin with a large molar excess of nonbiotinylated ligand.
  • 100 nM biotinyl- ⁇ -endorphin (1:1 Fl + F2) is competed with a combination of 50 ⁇ M ⁇ -endorphin + 50 ⁇ M met-enkephalin, the yield of the 66K glycoprotein is greatly diminished (Figure 5A) .
  • 40 ⁇ M naloxone effectively competes with 60 nM biotinyl- ⁇ - endorphin to nearly eliminate the recovery of 66K glyco ⁇ protein (Figure 5B) .
  • Two different peptides one known to exhibit mu-receptor selec- tive binding ([D-Ala 2, N-MePhe4, gly-ol5]enkephalin or DAGO; Bachem; 300 fold selectivity for mu over delta) and the other known to exhibit delta receptor selective binding ([D-Pen 2 ' 5 , pCL-Phe ]enkephalin or pCl-DPDPE; 500-fold selectivity for delta over mu) are used to block binding of biotinyl- ⁇ -endorphin to rat brain membranes. This pair of ligands is appropriate because their affinities for their respective receptors are very similar (approximately l mM K_) .
  • Each incubation contains 3 nM biotinyl- ⁇ -endorphin, and the blocking peptides are included at 50, 500, and 5000 nM.
  • the ligand mixtures are incubated with unsolu- bilized membranes for one hour at room temperature and then purification of the receptor proceeds as described herein. A summary of the conditions is provided in Table 3.
  • the ability of the respective peptides to block ⁇ -endorphin binding is determined by observing the relative recovery of biotinylated ⁇ -endorphin bound 66 kDa protein from each sample. Results are depicted in Figure 8. It can be seen that the 66 kDa protein is recovered in about the same amounts from. the control as when the pCl-DPDPE is used as a competitor (Lanes 1, 5, 6 and 7). In contrast, DAGO blocked recovery of receptor almost completely at 500 nM and completely at 5000 nM (lanes 3 and 4), thereby confirming the identity of the protein as a mu-subtype opioid receptor.
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO

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Abstract

L'invention se rapporte à un récepteur d'opioïdes du sous-type ν essentiellement pur, qui est capable de fixer la β-endorphine.
PCT/US1993/002539 1992-03-23 1993-03-22 Recepteur d'opioides purifie WO1993019086A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0612845A3 (en) * 1993-02-26 1994-09-21 American Cyanamid Co Purified opioid receptor.
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0612845A3 (en) * 1993-02-26 1994-09-21 American Cyanamid Co Purified opioid receptor.
US6258556B1 (en) 1993-02-26 2001-07-10 The United States Of America As Represented By The Department Of Health And Human Services cDNA and genomic clones encoding human μ opiate receptor and the purified gene product
WO1994028132A3 (fr) * 1993-05-20 1995-05-04 Arch Dev Corp Recepteurs d'opioides: compositions et procedes
US6319686B1 (en) 1993-05-20 2001-11-20 Arch Development Corporation Nucleic acids encoding kappa opioid receptors
US7235366B1 (en) 1993-05-20 2007-06-26 Arch Development Corporation Methods of identifying agonists and antagonists of opioid receptors

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