Biophysical characterization of the purified alpha 1-adrenergic receptor and identification of the hormone binding subunit

The alpha 1-adrenergic receptor has been solubilized in active form from rat hepatic membranes with the nonionic detergent, digitonin, and purified by affinity and gel filtration chromatography to homogeneity with a specific activity of 14,400 pmol/mg of protein. The affinity chromatographic steps of the purification procedure were achieved by the use of a newly synthesized analog (2-[4(2-succinoyl)piperazin-1-yl]-4-amino-6,7-dimethoxyquinazoline, CP-57,609) of the highly selective alpha 1-adrenergic antagonist, prazosin, immobilized via an amide linkage to agarose. The resulting purified receptor bound [3H]prazosin and a variety of adrenergic agents with the specificity, stereoselectivity, and affinities equivalent to those observed with membrane-bound and solubilized receptor preparations. The purified receptor.digitonin complex had a Stokes radius of 49 A and a sedimentation coefficient (s20w) of 7.1, as determined by AcA-34 gel filtration chromatography and sucrose gradient density centrifugation, respectively. Based on these hydrodynamic parameters, the calculated molecular weight of the receptor.digitonin complex was estimated at 147,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis following the final purification step revealed a single band of protein at 59,000 daltons from which [3H]prazosin binding activity could be recovered after renaturation of the receptor protein. These findings indicate that the protein purified from rat hepatic membranes is the hormone binding component of the alpha 1-adrenergic receptor and that the receptor molecule most likely contains more than one Mr = 59,000 subunit.     

Purification of the receptor for nerve growth factor from A875 melanoma cells by affinity chromatography

The receptor for nerve growth factor (NGF) has been purified to near homogeneity from octylglucoside extracts of A875 melanoma cell membranes by the use of repetitive affinity chromatography on NGF-Sepharose. Elution of purified receptor (NGF receptor) was accomplished with 0.15 M NaCl, pH 11.0, containing phosphatidylcholine and octylglucoside. Chromatography on two columns of NGF-Sepharose yielded a 1500-fold purification of the receptor, as assessed by 125I-NGF binding, and permitted recovery of 9% of the total binding activity in the soluble extract. Scatchard analysis of equilibrium binding of 125I-NGF provided similar Kd values for NGF receptors in soluble extracts of A875 membranes (2.2 nM) and with purified NGF receptor (3.1 nM). Examination of NGF receptor after electrophoresis on sodium dodecyl sulfate-polyacrylamide gels revealed the presence of two major peptides, of Mr = 85,000 and Mr = 200,000. Affinity labeling experiments, done with 125I-NGF and A875 cells, soluble extracts of A875 cell membranes, and purified receptor, show that both of these components of the NGF receptor can be specifically cross-linked to 125I-NGF.     

Studies on the interaction of steroid substrates with adrenal microsomal cytochrome P-450 (P-450C21) in liposome membranes

Cytochrome P-450 (P-450C21), purified from bovine adrenocortical microsomes, was incorporated into the single bilayer liposomes of egg yolk phosphatidylcholine by gel filtration, using a high pressure liquid chromatography system. Interaction of the steroid substrates, 17 alpha-hydroxyprogesterone and progesterone, with P-450C21 in the liposomes was studied in the equilibrium state by measuring substrate-induced spectral change. The apparent dissociation constant of the P-450C21-substrate complex increased with phosphatidylcholine concentration in the system, showing the substrate to be partitioned between the aqueous and lipid phases. Partition coefficients, determined by equilibrium dialysis and the Hummel-Dreyer method, were 3500 for progesterone and 2000 for 17 alpha-hydroxyprogesterone at 25 degrees C. The binding process of the substrates to P-450C21 in the liposomes and their dissociation were measured by a stopped flow method. The apparent rate of substrate binding to P-450C21 in the liposomes was not effected by substrate partitioning, indicating partitioning to occur much more quickly than substrate binding to P-450C21. Absorption changes observed in the stopped flow experiments were analyzed at a rapid equilibrium of partitioning. Based on these results, the substrate binding site of P-450C21 was concluded to face the lipid phase of the liposome membranes.     

A gamma-aminobutyric acid/benzodiazepine receptor complex of bovine cerebral cortex

The gamma-aminobutyric acid/benzodiazepine receptor from bovine cerebral cortex was solubilized with sodium deoxycholate and purified by affinity chromatography on benzodiazepine-agarose and ion exchange chromatography. The benzodiazepine binding protein was enriched 1800-fold. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol showed the presence of two major bands of Mr = 57,000 and 53,000. [3H]Flunitrazepam, after UV irradiation, was incorporated irreversibly into both bands of the isolated protein. A high affinity binding site for gamma-aminobutyric acid was co-purified with the benzodiazepine binding site and the two sites were shown to reside on the same physical structure. The dissociation constants were 10 +/- 4 nM for [3H] flunitrazepam and 12 +/- 3 nM for the gamma-aminobutyric acid agonist [3H]muscimol. The maximum specific activity for [3H] muscimol binding was 4.3 nmol/mg of protein. The ratio of [3H]muscimol to [3H]flunitrazepam binding sites was between 3 and 4. Gel filtration and sucrose density gradient sedimentation studies gave a Stokes radius of 7.3 +/- 0.5 nm and a sedimentation coefficient of 11.1 +/- 0.3 S, respectively. The purified complex had a pharmacological profile that corresponds to the receptor specificity found in membranes and crude soluble extracts.     

Solubilization of the 17 alpha-ethinyl estradiol-stimulated low density lipoprotein receptor of male rat liver

Pharmacological doses of 17 alpha-ethinyl estradiol induce a low density lipoprotein (LDL) receptor in the liver of male rats. Our aim was to solubilize this receptor. Isolated liver membranes (8,000-100,000 g fraction) from male rats treated with 17 alpha-ethinyl estradiol and from control rats were solubilized in 1% (w/v) Triton X-100. Using Amberlite XAD-2, more than 90% of the detergent was then removed. Liposomes were prepared by precipitating the solubilized proteins with acetone in the presence of phosphatidylcholine. The receptor activity of these liposomes was assayed using human 125I-labeled LDL. Filtration was used to separate bound from free 125I-labeled LDL. The assay was optimized; 0.25 mM CaCl2, 25 mM NaCl, pH 8.0, were chosen as the standard conditions. Binding of 125I-labeled LDL was dependent on Ca2+. Liposomes containing solubilized membrane proteins from treated rats displayed Ca2+-dependent binding which was 11 times higher than for control rats. The specific binding of 125I-labeled LDL was saturable with a Kd = 18 micrograms/ml. 125I-Labeled LDL was displaced by unlabeled lipoproteins containing apolipoproteins B and E and by dimyristoylphosphatidylcholine liposomes containing purified apolipoprotein E, but not by HDL3. The binding was abolished by pronase and was inhibited by suramin. Ligand blotting with 125I-labeled LDL revealed one band of protein with an apparent molecular weight of 133,000 daltons. These properties are characteristic of the low density lipoprotein receptor.     

Size characteristics of the solubilized saxitoxin receptor of the voltage-sensitive sodium channel from rat brain

The saxitoxin receptor of the voltage-sensitive sodium channel from rat brain was solubilized with Triton X-100 and stabilized with phosphatidylcholine. The size characteristics of the detergent . phospholipid . receptor complex were studied by gel filtration and sucrose gradient sedimentation in H2O and D2O. The complex has Stokes radius = 80 A, S20,W = 12 S, v = 0.82 ml/ g, and Mr = 601,000 +/- 48,000. Assuming v = 0.73 ml/g for the saxitoxin receptor protein, the mass of the complex consists of 47.4% detergent and phosphatidylcholine and 52.6% saxitoxin receptor protein with Mr = 316,000 +/- 63,000.     

Sedimentation behavior of solubilized gonadotropin receptor from plasma membranes of bovine corpus luteum

The gonadotropin receptors associated with plasma membrane fractions were solubilized by detergents, including Triton X-100, Lubrol WX, Lubrol PX and sodium deoxycholate before and after equilibration with ¹²⁵I-labelled human chorionic gonadotropin. The binding activity remained in solution even after centrifugation at 300 000 × g for 3 h. The solubilized gonadotropin receptor or gonadotropin receptor complex was characterized by gel filtration and sucrose density gradient centrifugation. Sucrose density gradient centrifugation of solubilized gonadotropin-receptor complex in the presence of Triton X-100 had a sedimentation coefficient of 6.5 S whereas the solubilized uncomplexed receptor had a sedimentation coefficient of 5.1 S. In the absence of the detergent, solubilized hormone receptor complex from plasma membrane fractions I and II sedimented with a apparent sedimentation coefficient of 6.6 S and 7.4 S, respectively. Similary, the free receptor also showed higher sedimentation profile with a apparent sedimentation coefficient of 6.7 S for fraction I and 7.2 S for fraction II. Treatment of plasma membranes with phospholipase A and C inhibited the binding of ¹²⁵I-labelled human chorionic gonadotropin in a dose dependent manner, whereas phospholipase D was without any effect. Doses of 1.4 mI.U. of phospholipase A or 0.6 mI.U. of phospholipase C were required to produce 50% inhibition of the binding activity. These phospholipases had no effect on the performed ¹²⁵I-labelled human chorionic gonadotropin-receptor complex nor on the sedimentation profile of solubilized gonadotropin receptor complex.     

Sedimentation behavior of solubilized gonadotropin receptor from plasma membranes of bovine corpus luteum.

The gonadotropin receptors associated with plasma membrane fractions were solubilized by detergents, including Triton X-100, Lubrol WX, Lubrol PX and sodium deoxycholate before and after equilibration with 125I-labelled human chorionic gonadotropin. The binding activity remained in solution even after centrifugation at 300 000 X g for 3 h. The solubilized gonadotropin receptor or gonadotropin receptor complex was characterized by gel filtration and sucrose density gradient centrifugation. Sucrose density gradient centrifugation of solubilized gonadotropin-receptor complex in the presence of Triton X-100 had a sedimentation coefficient of 6.5 S whereas the solubilized uncomplexed receptor had a sedimentation coefficient of 5.1 S. In the absence of the detergent, solubilized hormone receptor complex from plasma membrane fractions I and II sedimented with an apparent sedimentation coefficient of 6.6 S and 7.4 S, respectively. Similarly, the free receptor also showed higher sedimentation profile with an apparent sedimentation coefficient of 6.7 S for fraction I and 7.2 S for fraction II. Treatment of plasma membranes with phospholipase A and C inhibited the binding of 125I-labelled human chorionic gonadotropin in a dose dependent manner, whereas phospholipase D was without any effect. Doses of 1.4 mI. U. of phospholipase A or 0.6 mI.U. of phospholipase C were required to produce 50% inhibition of the binding activity. These phospholipases had no effect on the preformed 125I-labelled human chorionic gonadotropin-receptor complex nor on the sedimentation profile of solubilized gonadotropin receptor complex.     

Enrichment and initial characterization of the solubilized receptor for mouse gamma interferon

The work reported here constitutes a first step in characterizing the receptor for mouse gamma interferon at the biochemical level. The myelomonocytic cell line, WEHI-3, was the source of starting material. Iodinated recombinant mouse gamma interferon incubated with WEHI-3 cells, as well as membranes prepared from them, bound specifically to a single class of sites with a Kd of 7 x 10(-9)M. Membranes were solubilized with the non-ionic detergent octyl-beta-D-glucopyranoside. As solubilization proceeded, binding activity could be assayed by precipitating the receptor with acetone in the presence of egg phosphatidylcholine liposomes. The Kd of the receptor in association with liposomes was 13 nM. Again here, only a single class of binding activity was found, and specificity for gamma, compared to other interferons, was maintained. This is the first time that the receptor for mouse gamma interferon has been solubilized and recovered in functional form. Further characterization included at least a 200-fold enrichment of binding activity by ligand affinity chromatography, resulting in the identification of a 95 kDa protein as the most likely candidate for either the receptor or a binding subunit thereof.     

Catalytic properties of phospholipid exchange protein from bovine heart.

A protein which catalyzes the exchange of phosphatidylcholine between membranes has been purified from heart tissue homogenates up to 300-fold by acidic pH precipitation, (NH4)2SO4 precipitation, gel filtration, and ion-exchange chromatography. Binding of the protein to phosphatidylcholine liposomes as measured by Sepharose chromatography was nondetectable. However, isoelectric focusing experiments showed that individual molecules of phosphatidylcholine were transferred from liposomes to the soluble, partially purified protein. Exchange of phospholipid between liposomes and mitochondria was not affected by the presence of moderate amounts of cholesterol in liposomes. A search for competitive inhibitors among moieties similar to phosphatidylcholine failed to show strong binding sites in the hydrophilic part of the substrate. High concentrations of Na+, Ca2+ and Mg2+ impaired the exchange activity.     

Incorporation of partially purified cation channels from cardiac sarcolemmal membrane into planar lipid bilayers

Voltage-dependent calcium channels are vital to cardiac muscle contraction. Therefore it is very important to isolate physiologically active channel proteins, however there have been few reports on their solubilization and reconstitution. Highly purified sarcolemmal membranes from bovine cardiac muscle were solubilized with octylglucoside, partially purified by gel filtration, and reconstituted into planar lipid bilayer by the direct insertion method. At least, two cation channel activities were observed: one with about 4.2 pS and the other with about 28 pS in conductance. From the reversal potential, it was concluded that Ba2+ ions are the current carrier through these two channels.     

Co-purification of an atrial natriuretic factor receptor and particulate guanylate cyclase from rat lung

An atrial natriuretic factor (ANF) receptor from rat lung was solubilized with Lubrol-PX and purified by sequential chromatographic steps on GTP-agarose, DEAE-Sephacel, phenyl-agarose, and wheat germ agglutinin-agarose. The ANF receptor was enriched 19,000-fold. The purified receptor has a binding profile and properties that correspond to the affinity and specificity found in membranes and crude detergent extracts. Polyacrylamide gel electrophoresis of the purified preparation in the presence of sodium dodecyl sulfate and dithiothreitol showed the presence of one major protein band with a molecular mass of 120,000 daltons. When purified preparations were incubated with 125I-ANF, then cross-linked with disuccinimidyl suberate, the 120,000-dalton protein was specifically radiolabeled. This high affinity binding site for ANF co-purified with particulate guanylate cyclase. Particulate guanylate cyclase was purified to a specific activity of 19 mumol cyclic GMP produced/min/mg of protein utilizing Mn-GTP as substrate. This represented a 15,000-fold purification compared to the initial lung membrane preparation with Lubrol-PX. Gel permeation high performance liquid chromatography and glycerol density gradient sedimentation studies of the purified preparation also resulted in co-migration of specific ANF binding and guanylate cyclase activities. The co-purification of these activities suggests that both ANF binding and guanylate cyclase activities reside in the same macromolecular complex. Presumably ANF binding occurs at the external membrane surface and cyclic GMP synthesis at the internal membrane surface of this transmembrane glycoprotein.     

Purification of the thromboxane A2/prostaglandin H2 receptor from human blood platelets

S-145 (5Z-7-(3-endo-phenylsulfonylamino-(2.2.1.)-bicyclohept -2-exo-yl) heptenoic acid) is a potent and selective antagonist for thromboxane A2/prostaglandin H2 receptor. Using this compound as an immobilized ligand for affinity chromatography and [3H]S-145 as a radioligand, we have purified the thromboxane A2/prostaglandin H2 receptor from the membranes of human blood platelets. The purification procedures consisted of solubilization of the receptor with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), affinity chromatographies on columns of S-145 affinity gel, wheat germ agglutinin agarose and red agarose, and repeated gel filtration high performance liquid chromatography on a TSK gel G-3000SW column. On the second gel filtration high performance liquid chromatography, the [3H]S-145 binding activity was eluted as a symmetrical peak which overlapped exactly with a peak of ultraviolet absorption at 280 nm. By these procedures, the receptor was purified about 8700-fold from the solubilized extract with a recovery of 6%. The final preparation showed a broad protein band at Mr 57,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and maximally bound 19.2 nmol of [3H]S-145/mg protein with a Kd of 29.8 nM. The [3H]S-145 binding to the purified receptor was specifically displaced by several thromboxane A2/prostaglandin H2 analogues.     

Solubilization and reconstitution of the renal phosphate transporter

Proteins from brush-border membrane vesicles of rabbit kidney cortex were solubilized with 1% octylglucoside (protein to detergent ratio, 1:4 (w/w). The solubilized proteins (80.2 +/- 2.3% of the original brush-border proteins, n = 10, mean +/- S.E.) were reconstituted into artificial lipid vesicles or liposomes prepared from purified egg yolk phosphatidylcholine (80%) and cholesterol (20%). Transport of Pi into the proteoliposomes was measured by rapid filtration in the presence of a Na+ or a K+ gradient (out greater than in). In the presence of a Na+ gradient, the uptake of Pi was significantly faster than in the presence of a K+ gradient. Na+ dependency of Pi uptake was not observed when the liposomes were reconstituted with proteins extracted from brush-border membrane vesicles which had been previously treated with papain, a procedure that destroys Pi transport activity. Measurement of Pi uptake in media containing increasing amounts of sucrose indicated that Pi was transported into an intravesicular (osmotically sensitive) space, although about 70% of the Pi uptake appeared to be the result of adsorption or binding of Pi. However, this binding of Pi was not dependent upon the presence of Na+. Both Na+-dependent transport and the Na+-independent binding of Pi were inhibited by arsenate. The initial Na+-dependent Pi transport rate in control liposomes of 0.354 nmol Pi/mg protein per min was reduced to 0.108 and 0 nmol Pi/mg protein per min in the presence of 1 and 10 mM arsenate, respectively. Future studies on reconstitution of Pi transport systems must analyze and correct for the binding of Pi by the lipids used in the formation of the proteoliposomes.     

Fusion of Sendai virions with phosphatidylcholine-cholesterol liposomes reflects the viral activity required for fusion with biological membranes

Sendai virus envelopes were reconstituted after solubilization of intact virions with either Triton X-100 or octylglucoside. Envelopes obtained from Triton X-100, but not from octylglucoside solubilized virions, were hemolytic and promoted cell-cell fusion. Fluorescence dequenching studies [using N-4-nitrobenzo-2-oxa-1,3-diazole phosphatidylethanolamine-bearing viral envelopes] revealed that both preparations fused with negatively charged phospholipids. Fusion with phosphatidylcholine (PC)/cholesterol (chol) liposomes was promoted only by the hemolytic viral envelopes. Fluorescence dequenching studies, using intact virions bearing octadecylrhodamine B chloride, revealed that intact virions fused with PC/chol as well as with negatively charged phospholipids. Only fusion with PC/chol liposomes was inhibited by phenylmethylsulfonyl fluoride and dithiothreitol, reagents which are known to block the viral ability to fuse with biological membranes.     

Glycerol, sodium phosphate, and sodium chloride permit the solubilization and partial purification of rat hepatic alpha 1-receptors by 3-(3-cholamidylpropyl)-dimethylammonio-1-propanesulfonate

CHAPS [3-(3-cholamidylpropyl)-dimethylammonio-1-propanesulfonate], a zwitterionic detergent, has been used to solubilize the rat hepatic alpha 1-adrenergic receptor. Although the use of this detergent alone permitted a poor receptor solubilization, the inclusion of sodium phosphate, sodium chloride, and glycerol to the medium allowed 30% of the binding activity observed in plasma membranes to be recovered. Binding of the selective alpha 1-adrenergic antagonist, [3H]prazosin, by the solubilized preparation was saturable and of high affinity. In addition, binding of the radioligand was inhibited by a variety of adrenergic agents with affinity, specificity, and stereoselectivity comparable to that observed in plasma membranes. The use of glycerol in the solubilization medium permitted recovery of the solubilized receptor in a stable form (T1/2 = 72 h at 4 degrees C). Sequential affinity and size-exclusion gel chromatography allowed a 1000-fold purification of the solubilized receptor. The Stokes' radius and the apparent molecular mass of the purified receptor-Chaps complex (48.4 A and 160,000 Da, respectively), determined by gel filtration chromatography, were similar to those previously obtained for the rat hepatic alpha 1-receptor purified after solubilization with the nonionic detergent digitonin. These data indicate that the combination of Chaps, sodium phosphate, sodium chloride, and glycerol permitted the solubilization and partial purification of hepatic alpha 1-receptor in an active and stable form. The use of this technique might be useful for the solubilization of other membrane-bound proteins by Chaps whose biophysical characteristics make it an ideal detergent for reconstitution experiments.     

Solubilization and Partial Purification of α-Amino-3- Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Binding Sites from Rat Brain

alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) binding sites were solubilized from rat brain membranes using 1% Triton X-100 in 0.5 M potassium phosphate buffer containing 20% glycerol. The solubilized binding sites were stable, permitting biochemical and pharmacological characterization as well as partial purification. Pharmacological and binding analyses indicated that the solubilized binding sites were similar to the membrane-bound sites. Both the solubilized and the membrane-bound preparations contained high- and low-affinity AMPA binding sites in the presence of potassium thiocyanate. A similar rank order for inhibition of [3H]AMPA binding by several excitatory amino acid analogs was obtained for the soluble and membrane-bound preparations. [3H]AMPA binding to both soluble and membrane-bound preparations was increased in the presence of potassium thiocyanate. The solubilized AMPA binding sites migrated as a single peak with gel filtration chromatography, with an Mr of 425,000. Beginning with the solubilized preparation, AMPA binding sites were purified 54-fold with ion-exchange chromatography and gel filtration. The characterization and purification of these soluble binding sites is potentially useful for the molecular characterization of this putative excitatory amino acid receptor subtype.     

Isolation and characterization of membranes from bovine liver which are highly enriched in mannose 6-phosphate receptors

We have developed a method for the isolation of the subcellular organelles from bovine liver which are enriched in the cation- independent mannose 6-phosphate receptor (CI-MPR) and the cation- dependent mannose 6-phosphate receptor (CD-MPR). The purification scheme consists of sedimentation of a postnuclear supernatant fraction on a sucrose gradient followed by immunoisolation using specific anti- peptide antibodies conjugated to magnetic polystyrene beads. Antibodies that recognize the cytoplasmic domain of either the CI-MPR or the CD- MPR routinely give membrane preparations that are approximately 50-fold enriched in each of the respective receptors, as determined by quantitative Western blotting. The immunoisolated membranes are also enriched in the other MPR, as well as in the asialoglycoprotein receptor. They contain significantly lower levels of enzyme activities representative of the plasma membrane (5' nucleotidase) or the Golgi complex (galactosyltransferase and sialyltransferase). There is little or no enrichment for either the lysosomal enzymes beta-hexosaminidase and tartrate-resistant acid phosphatase, or the mitochondrial enzyme succinate-tetrazolium reductase. These data, together with electron microscopy of the immunoisolated material, suggest that the bulk of MPR- containing membranes we have isolated from bovine liver correspond to endosomes. Analysis by SDS-PAGE indicates that several proteins, including two with apparent molecular weights of 170 K and 400 K, are significantly enriched in the purified fractions and may represent potential markers for MPR-containing endosomes.     

Purification of the 5-hydroxytryptamine 5-HT3 receptor from NCB20 cells

A 5-hydroxytryptamine 5-HT3 receptor binding site has been purified from deoxycholate-solubilized NCB20 cell membranes. Purification (1,700-fold) was achieved in one step by affinity chromatography with L-685,603 immobilized on agarose. The 5-HT3 selective antagonist [3H]Q ICS 205-930 labeled a single population of receptors in the affinity-purified preparation with a Bmax of 3.1 +/- 0.9 nmol/mg protein and Kd of 0.40 +/- 0.05 nM (mean +/- S.E., n = 3). The rank order of potency for a series of competing compounds confirmed that [3H]Q ICS 205,930 was labeling a 5-HT3 receptor in the purified preparation, and the inhibition constants for all antagonists were unchanged after purification. The purified 5-HT3 binding site eluted from a Sepharose 6B gel filtration column in a similar manner to the crude solubilized preparation (Stokes radius of 4.9 nm, apparent molecular size 250,000). Polyacrylamide gel electrophoresis of the affinity-purified receptor showed two broad bands by silver staining, migrating with apparent molecular masses of 54,000 and 38,000. Gel filtration of the affinity purified material yielded a single peak labeled by [3H]Q ICS 205-930 with an apparent molecular size of 250,000, which was also composed of two bands of 54,000 and 38,000, consistent with these being the constituents of the 5-HT3 receptor.     

Purification and characterization of the glycine receptor of pig spinal cord

A large-scale purification procedure was developed to isolate the glycine receptor of pig spinal cord by affinity chromatography on aminostrychnine agarose. After an overall purification of about 10 000-fold, the glycine receptor preparations contained three major polypeptides of Mr 48 000, 58 000, and 93 000. Photoaffinity labeling with [3H]strychnine showed that the [3H]strychnine binding site is associated with the Mr 48 000 and, to a much lesser extent, the Mr 58 000 polypeptides. [3H]Strychnine binding to the purified receptor exhibited a dissociation constant KD of 13.8 nM and was inhibited by the agonists glycine, taurine, and beta-alanine. Gel filtration and sucrose gradient centrifugation gave a Stokes radius of 7.1 nm and an apparent sedimentation coefficient of 9.6 S. Peptide mapping of the [3H]strychnine-labeled Mr 48 000 polypeptides of purified pig and rat glycine receptor preparations showed that the strychnine binding region of this receptor subunit is highly conserved between these species. Also, three out of six monoclonal antibodies against the glycine receptor of rat spinal cord significantly cross-reacted with their corresponding polypeptides of the pig glycine receptor. These results show that the glycine receptor of pig spinal cord is very similar to the well-characterized rat receptor protein and can be purified in quantities sufficient for protein chemical analysis.