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.     

Solubilization and Characterization of D2-Dopamine Receptors in an Estrone-Induced, Prolactin-Secreting Rat Pituitary Adenoma

D2-dopamine (3,4-dihydroxyphenylethylamine) receptors were successfully solubilized with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate from an estrone-induced rat pituitary adenoma. Forty-five percent of initial protein and 48% of initial [3H]spiroperidol binding sites were solubilized. The high affinity as well as the stereoselectivity of the sites was preserved. The order of potency of dopaminergic agonists was found to be typical of D2 receptors. Target size analysis by radiation inactivation indicated a molecular weight of 143,000 +/- 3,000 and of 106,000 +/- 4,000 daltons for membrane-bound and solubilized receptors, respectively. This suggests the loss of a 37,000-dalton subunit during solubilization without significant modification of binding characteristics. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of receptor protein preparation photolabeled with N-(p-azido-m[125I]iodophenethyl)spiroperidol confirmed the existence of a 94,000-dalton peptide which probably constitutes the ligand binding site of the receptor. Thus, our data indicate that chronic estrogen treatment of rats, although inducing a pituitary adenoma, does not modify the pharmacological characteristics of D2 receptors. These data suggest therefore that these adenoma may represent an ideal source of material for further biochemical characterization of D2 receptors.     

Characterization of D2 Dopamine Receptors in Dopamine-Resistant Prolactin-Secreting Rat Pituitary Tumors 7315a and MtTW15

We have investigated the structure of D2 receptors present in two prolactin-secreting, dopamine-resistant, transplantable rat pituitary tumors, 7315a and MtTW15. These receptors specifically bind with high affinity the dopamine antagonist [3H]spiroperidol when membrane bound or solubilized by [3-(3-cholamidopropyl)-dimethyl-ammonio]-1-propane sulfonate 10 mM and are pharmacologically characterized as D2 type. Target-size analysis by radiation inactivation indicated a molecular mass of approximately 100,000 and 200,000 daltons for receptors present respectively in 7315a and MtTW15 tumors either membrane bound or solubilized. The minimal size of the D2 binding site was evaluated at 94,000 daltons by photoaffinity labeling with [125I]azido-N-(p-aminophenethyl)-spiperone followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A guanine nucleotide had no effect on the displacing potency of the agonist N-propylnorapomorphine evaluated with membrane-bound or solubilized receptors obtained from either tumor. These results suggest the absence or inactivation of a guanine nucleotide binding protein in the receptorial complex of these tumors. Thus, our data indicate that a structural anomaly is present in the D2 receptorial complex of these prolactin-secreting rat pituitary tumors, which may be responsible for their resistance to the inhibitory effects of dopamine.     

Solubilization and Characterization of Rat Brain α2-Adrenergic Receptor

alpha 2-Adrenergic receptors labelled by [3H]-clonidine (alpha 2-agonist) can be solubilized from the rat brain in a form sensitive to guanine nucleotides with a zwitterionic detergent, 3-[3-(cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS). About 40% of the original [3H]CLO binding sites in the membranes were solubilized with 6 mM CHAPS. Separation of the soluble [3H]CLO-bound complex was performed by the vacuum filtration method using polyethylenimine-treated GF/B filters. Solubilized [3H]CLO binding sites retained the same pharmacological characteristics of membrane-bound alpha 2-adrenergic receptors. Scatchard plots of [3H]CLO binding to solubilized alpha 2-receptors were curvilinear, indicating the existence of the two distinct binding components. Solubilized receptors were eluted as a single peak from Bio-Gel A-1.5 m column with a Stokes radius of 6.6 nm. The isoelectric point was 5.6-5.8. Regulations of the receptor binding by guanine nucleotides, monovalent cations, and sulfhydryl-reactive agents were maintained intact in the soluble state, whereas those by divalent cations were lost. The apparent retention of receptors and guanine nucleotide binding regulatory component(s) in the soluble state may allow a investigation of the regulation mechanisms of the brain alpha 2-adrenergic receptor system at the molecular level.     

Physical properties of the detergent-extracted nerve growth factor receptor of sympathetic ganglia.

The nerve growth factor (NGF) receptor from microsomes of adult rabbit superior cervical ganglia has been solubilized with Triton X-100 and sodium deoxycholate. The physical properties of the detergent-extracted NGF receptor were assessed by Sepharose 6B chromatography and sucrose density gradient ultracentrifugation studies in H2O and D2O. The predominant form of the NGF receptor has a Stokes radius of 71 A, a partial specific volume of 0.74 ml/g, a sedimentation coefficient of 4.3 S, and a frictional ratio of 1.8. From these parameters, it can be calculated that the NGF receptor in Triton X-100 is a minimally hydrophobic, highly asymmetric, intrinsic membrane protein with a molecular weight of approximately 135,000. A form of the receptor with a sedimentation coefficient of 10.4 S was occasionally seen which appears to represent an aggregated form of the 4.3 S moiety.     

Solubilization of D2 Dopamine Receptor Coupled to Guanine Nucleotide Regulatory Protein from Bovine Striatum

D2 dopamine receptor from bovine striatum was solubilized in a form sensitive to guanine nucleotides, by means of a zwitterionic detergent, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS). The presence of sodium ion markedly increased the solubilization yield. Treatment of the membranes with 10 mM CHAPS and 0.72 M NaCl solubilized 26% of the stereospecific [3H]spiperone binding sites in the original membrane preparations. The solubilized [3H]spiperone binding sites possessed characteristics of the D2 dopamine receptor: (a) localization of the site in the striatum but not in the cerebellum; (b) high affinity to nanomolar concentrations of [3H]spiperone; (c) displacement of [3H]spiperone binding by nanomolar concentrations of neuroleptics, but only by micromolar concentrations of dopamine and apomorphine; (d) equal activity of various dopamine agonists and antagonists in the soluble and membrane preparations. Guanine nucleotides decreased the affinity of the solubilized D2 dopamine receptor for dopamine agonists, but not for antagonists. The solubilized receptor complex was eluted in Sepharose CL-4B column chromatography as a large molecule, with a Stokes radius of approximately 90 A. These results indicate that the complex between the D2 dopamine receptor and GTP binding protein remains intact throughout the solubilization procedure.     

The Drosophila acetylcholine receptor subunit D alpha5 is part of an alpha-bungarotoxin binding acetylcholine receptor

The central nervous system of Drosophila melanogaster contains an alpha-bungarotoxin-binding protein with the properties expected of a nicotinic acetylcholine receptor. This protein was purified 5800-fold from membranes prepared from Drosophila heads. The protein was solubilized with 1% Triton X-100 and 0.5 M sodium chloride and then purified using an alpha-cobratoxin column followed by a lentil lectin affinity column. The purified protein had a specific activity of 3.9 micromol of 125I-alpha-bungarotoxin binding sites/g of protein. The subunit composition of the purified receptor was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This subunit profile was identical with that revealed by in situ labeling of the membrane-bound protein using the photolyzable methyl-4-azidobenzoimidate derivative of 125I-alpha-bungarotoxin. The purified receptor reveals two different protein bands with molecular masses of 42 and 57 kDa. From sedimentation analysis of the purified protein complex in H2O and D2O and gel filtration, a mass of 270 kDa was calculated. The receptor has a s(20,w) of 9.4 and a Stoke's radius of 7.4 nm. The frictional coefficient was calculated to be 1.7 indicating a highly asymmetric protein complex compatible with a transmembrane protein forming an ion channel. The sequence of a peptide obtained after tryptic digestion of the 42-kDa protein allowed the specific identification of the Drosophila D alpha5 subunit by sequence comparison. A peptide-specific antibody raised against the D alpha5 subunit provides further evidence that this subunit is a component of an alpha-bungarotoxin binding nicotinic acetylcholine receptor from the central nervous system of Drosophila.     

Purification by affinity chromatography of the glycine receptor of rat spinal cord

The glycine receptor of rat spinal cord was solubilized with the nonionic detergent Triton X-100 and subsequently purified by affinity chromatography on aminostrychnine-agarose and wheat germ agglutinin-Sepharose. An overall purification of 1950-fold was achieved. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and mercaptoethanol revealed three glycine receptor-associated polypeptides of Mr = 48,000, 58,000, and 93,000. [3H]Strychnine was incorporated irreversibly into the Mr = 48,000 polypeptide upon UV-illumination. The dissociation constant (KD) of [3H]strychnine binding to the purified glycine receptor was 9.3 +/- 0.6 nM. The glycine receptor agonists glycine, beta-alanine, and taurine inhibited the binding of [3H]strychnine to the purified receptor. Gel filtration and sedimentation in sucrose/H2O and sucrose/D2O gradients gave a Stokes radius of 7.7 nm, a partial specific volume of 0.780 +/- 0.005 ml/g and a sedimentation coefficient s20,w of 8.2 +/- 0.2 S for the purified glycine receptor. From these data, a molecular weight of 246,000 +/- 6,000 was calculated for the glycine receptor protein.     

Molecular weight of detergent-solubilized prostaglandin-F2 alpha receptor from bovine corpora lutea

A prostaglandin F2 alpha receptor localized in plasma membranes of bovine corpus luteum cells was solubilized by treatment with Triton X-100. Sepharose chromatographies of ([3H]prostaglandin F2 alpha)-receptor complex gave a Stokes' radius of 630 nm. In the absence of detergent, aggregated forms of the receptor appeared. Sedimentation experiments of solubilized receptor in sucrose/H2O and sucrose/2H2O density gradients gave the following values: sedimentation coefficient (S20, w) 4.6 S; partial specific volume (VB) 0.78 cm3/g and frictional ratio (f/fo) 1.6. Based on the sedimentation coefficient and the Stokes' radius and assuming that the receptor is a non-glycosylated protein the molar mass of the receptor-(Triton X-100) complex was 144000 g/mol. The VB value indicated that ca. 26% of the weight represented bound detergent and that the molecular weight of the prostaglandin F2 alpha receptor is approximately 107000.     

Size characteristics of the solubilized sodium channel saxitoxin binding site from mammalian sarcolemma

The sodium channel saxitoxin binding component from rat sarcolemma was solubilized with medium chain length non-ionic detergents including NP-40, Brij-96 and Lubrol-PX. Phospholipid was required for stability of the binding component. Specific saxitoxin binding was significantly temperature sensitive even with optimal levels of phospholipid present. The solubilized saxitoxin binding component chromatographed on Sepharose 6B at a position corresponding to that of a globular protein of 95--10 A Stokes radius, but had an apparent s20,w typical of a smaller molecule (s20,w = 9.2--10). Column behavior and s20,w were independent of the specific detergent used for solubilization. Anomalous column behavior may reflect molecular asymmetry, contribution from bound detergent or similar considerations.     

Solubilization and characterization of the beta-bungarotoxin-binding protein of chick brain membranes

The previously characterized ( Rehm , H., and Betz, H. (1982) J. Biol. Chem. 257, 10015-10022) neuronal binding protein for the presynaptic neurotoxin beta-bungarotoxin (beta-BuTx) was solubilized from synaptic membrane fractions of chick brain using the nonionic detergent Triton X-100. 125I-beta-BuTx bound to the solubilized protein with a dissociation constant (KD) of 1.9 +/- 0.1 nM. This binding of 125I-beta-BuTx was Ca2+-dependent and pharmacologically specific. From different basic proteins tested, only unlabeled beta-BuTx and its antagonist dendrotoxin inhibited 125I-beta-BuTx binding. Potassium ions were required during solubilization and binding in order to detect 125I-beta-BuTx-binding activity. Sedimentation in sucrose/H2O and sucrose/D2O gradients and gel exclusion chromatography on Sepharose 6B indicated a s20,w of 12.8 +/- 0.6 S and a Stokes radius of 8.6 +/- 0.2 nm for the solubilized beta-BuTx-binding component. From these data, the protein molecular weight of the beta-BuTx binding site was calculated to be 431,000 +/- 45,000.     

The Binding Properties of the Particulate and Solubilized Sulfonylurea Receptor from Cerebral Cortex Are Modulated by the Mg2+ Complex of ATP

Glibenclamide closes an ATP-sensitive K+ channel (K-ATP channel) by interaction with the sulfonylurea receptor in the plasma membrane of pancreatic B cells and thereby initiates insulin release. Previous studies demonstrated that the Mg2+ complex of ATP decreases glibenclamide binding to the sulfonylurea receptor from pancreatic islets. The aim of the present study was to examine the effect of adenine and guanine nucleotides on binding of sulfonyl-ureas to the cerebral sulfonylurea receptor. For this purpose, binding properties of the particulate and solubilized site from rat or pig cerebral cortex were analyzed. Maximum recovery of receptors in detergent extracts amounted to 40-50%. Specific binding of [3H]glibenclamide to the solubilized receptors corresponded well to specific binding to microsomes. In microsomes and detergent extracts, the Mg2+ complexes of ATP, ADP, GTP, and GDP inhibited binding of [3H]glibenclamide. These effects were not observed in the absence of Mg2+. In detergent extracts, Mg-ATP (300 microM) reduced the number of high-affinity sites for [3H]-glibenclamide by 52% and increased the dissociation constant for [3H]glibenclamide by eightfold; Mg-ATP was half-maximally effective at 41 microM. Alkaline phosphatase accelerated the reversal of Mg-ATP-induced inhibition of [3H]glibenclamide binding. The data suggest similar control of the sulfonylurea receptor from brain and pancreatic islets by protein phosphorylation.     

Characterization of Developmentally Regulated cAMP/Ca2+-Independent Protein Kinases from Dictyostelium discoideum

Cell-free extracts of the slime mold Dictyostelium discoideum were assayed for phosphorylating activity towards endogenous proteins and towards histone H1, casein and myelin basic protein (MBP). During development, protein kinase activity towards all of these substrates steadily increased and peaked between the aggregation and the pseudoplasmodial stages. Particulate-associated kinase activity was solubilized with 1% CHAPS, and separated into 300–400 kDa and ∼ 100 kDa components on Sephacryl S-300. The 300–400 kDa peak exhibited the most pronounced developmental increase in MBP phosphorylating activity. It was further fractionated on DEAE-Sephacel and heparin-Sepharose, and in each case, it coeluted with the histone H1 phosphorylating activity. The activity of this kinase was unaffected by cAMP and calmodulin, but it was reduced to 50% by ∼ 350 mM NaCl, 5 mM NaF and 40 μg polylysine/ml. The ∼ 100 kDa peak exhibited the most pronounced increase in casein kinase activity during development. Most of the casein phosphorylating activity did not bind to DEAE-Sephacel; it was distinct from casein kinase 2, which was not developmentally regulated. In parallel with these elevated kinase activities during development, there was increased in vitro phosphorylation of a number of Dictyostelium proteins, including two major phosphoproteins of 140 and 94 kDa.     

Characterization of High-Affinity Dopamine D2 Receptors and Modulation of Affinity States by Guanine Nucleotides in Cholate-Solubilized Bovine Striatal Preparations

3,4-Dihydroxyphenylethylamine (dopamine) D2 receptors, solubilized from bovine striatal membranes using a cholic acid-NaCl combination, exhibited the typical pharmacological characteristics of both agonist and antagonist binding. The rank order potency of the agonists and antagonists to displace [3H]spiroperidol binding was the same as that observed with membrane-bound receptors. Computer-assisted analysis of the [3H]spiroperidol/agonist competition curves revealed the retention of high- and low-affinity states of the D2 receptor in the solubilized preparations and the proportions of receptor subpopulations in the two affinity states were similar to those reported in membrane. Guanine nucleotide almost completely converted the high-affinity sites to low-affinity sites for the agonists. The binding of the high-affinity agonist [3H]N-n-propylnorapomorphine ([3H]NPA) was clearly demonstrated in the solubilized preparations for the first time. Addition of guanylyl-imidodiphosphate completely abolished the [3H]NPA binding. When the solubilized receptors were subjected to diethylaminoethyl-Sephacel chromatography, the dopaminergic binding sites eluted in two distinct peaks, showing six- to sevenfold purification of the receptors in the major peak. Binding studies performed on both peaks indicated that the receptor subpopulation present in the first peak may have a larger proportion of high-affinity binding sites than the second peak. The solubilized preparation also showed high-affinity binding of [35S]guanosine-5'-(gamma-thio)triphosphate, a result suggesting the presence of guanine nucleotide binding sites, which may interact with the solubilized D2 receptors. These data are consistent with the retention of the D2 receptor-guanine nucleotide regulatory protein complex in the solubilized preparations and should provide a suitable model system to study the receptor-effector interactions.     

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.     

The GTP-Insensitive Component of High-Affinity [3H]8-Hydroxy-2-(Di-n-Propylamino)tetralin Binding in the Rat Hippocampus Corresponds to an Oxidized State of the 5-Hydroxytryptamine1A Receptor

Previous studies on central 5-hydroxytryptamine1A (5-HT1A) receptors have consistently shown the existence of a GTP-insensitive component of agonist binding, i.e., binding of [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) that persists in the presence of 0.1 mM GTP or guanylylimidodiphosphate (GppNHp). The molecular basis for this apparent heterogeneity was investigated pharmacologically and biochemically in the present study. The GppNHp-insensitive component of [3H]8-OH-DPAT binding increased spontaneously by exposure of rat hippocampal membranes or their 3-[3-(cholamidopropyl)dimethylammonio]-1-propane sulfonate-soluble extracts to air; it was reduced by preincubation of solubilized 5-HT1A binding sites in the presence of dithiothreitol and, in contrast, reversibly increased by preincubation in the presence of various oxidizing reagents like sodium tetrathionate or hydrogen peroxide. In addition, exposure of hippocampal soluble extracts to short-cross-linking reagents specific for thiols produced an irreversible increase in the proportion of GppNHp-insensitive over total [3H]8-OH-DPAT binding. The pharmacological properties of this GppNHp-insensitive component of [3H]8-OH-DPAT binding were similar to those of 5-HT1A sites in the absence of nucleotide. Sucrose gradient sedimentation of solubilized 5-HT1A binding sites treated by dithiothreitol or sodium tetrathionate showed that oxidation prevented the dissociation by GTP of the complex formed by the 5-HT1A receptor binding subunit (R[5-HT1A]) and a guanine nucleotide-binding protein (G protein). Moreover, the oxidation of -SH groups by sodium tetrathionate did not prevent the inactivation of [3H]8-OH-DPAT specific binding by N-ethylmaleimide, in contrast to that expected from an interaction of both reagents with the same -SH groups on the R[5-HT1A]-G protein complex. These data suggest that the appearance of GTP-insensitive [3H]8-OH-DPAT specific binding occurs as a result of the (spontaneous) oxidation of essential -SH groups (different from those preferentially inactivated by N-ethylmaleimide) on the R[5-HT1A]-G protein complex.     

Solubilization and Characterization of Prostaglandin E2 Binding Protein from Porcine Cerebral Cortex

The specific binding protein for prostaglandin (PG) E2 was solubilized in an active form from the crude mitochondrial (P2) fraction of porcine cerebral cortex. After incubation with 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) at 4 degree C for 30 min, the PGE2 binding to the supernatant fraction (103,000 g, 60 min) was determined by the polyethylene glycol method. The maximum yield (approximately 30% of the binding activity to the P2 fraction) was obtained with 10 mM CHAPS. The specific [3H]PGE2 binding to the solubilized fraction was time-dependent and the equilibrium was reached at around 60 min at 37 degrees C. By dilution of the reaction mixture, the binding site-[3H]PGE2 complex formed after 5-min incubation slowly dissociated, whereas that formed after 60-min incubation did not dissociate to a significant extent. The binding was highly specific for PGE2 and inhibited by unlabeled PGs in the following order: PGE2 greater than PGE1 much greater than PGF2 alpha greater than PGE2 methyl ester greater than PGA2 greater than 13,14-dihydro-15-keto-PGE2 greater than PGD2. Scatchard analyses of the solubilized fraction suggested the presence of high- and low-affinity sites. Heat treatment and preincubation with trypsin or proteinase K markedly reduced the binding. The binding activity was eluted in a single peak both from gel filtration and from ion-exchange columns using HPLC. These results suggest that a specific protein solubilized may be responsible for the binding site.     

Isolation of a Ca2+-Dependent Actin-Fragmenting Protein from Brain, Spinal Cord, and Cultured Neurones

Extracts of ox spinal cord and chicken brain were fractionated by ion-exchange chromatography and assayed for their ability to reduce the viscosity of muscle F-actin solutions. Two distinct peaks of activity were obtained, one of which was further purified by affinity chromatography on a DNAase-actin Sepharose column. Following molecular exclusion chromatography, the actin component appeared as a complex of 1 molecule of a protein with molecular weight 90,000 and 2 molecules of actin (42,000). This tightly bound complex was resistant to most methods of protein separation, but was resolvable into its component proteins by sodium dodecyl sulphate acrylamide gel electrophoresis. The protein of molecular weight 90,000 could be eluted from such a gel in a fully active form. The activity of the protein from ox spinal cord was closely similar to that of gelsolin, an actin-fragmenting protein originally isolated from rabbit lung macrophages. Like gelsolin, the protein from ox spinal cord produced fragmentation of muscle F-actin filaments at Ca2+ concentrations greater than 10(-7) M, and had a nucleating effect on the polymerisation of muscle actin; the latter was measured most easily by the enhancement of fluorescence of muscle actin conjugated to N-(1-pyrenyl)iodoacetamide. Nucleation was more effective in the presence of Ca2+, but also occurred in its absence, and the same was true of complex formation between the 90,000 protein and muscle G-actin. On the basis of its actin-fragmenting activity, we estimate that the 90,000 molecular weight protein constitutes 0.2% of the protein initially extracted from ox spinal cord. A very similar protein, indistinguishable in its action on actin but containing variable amounts of a protein of molecular weight 85,000 as well as 90,000, was isolated from chicken brain. A similar protein was also detected in pure cultures of sympathetic neurones by enrichment on a DNAase-actin affinity column and by immune blotting and by immunofluorescence. We conclude that a protein similar, if not identical to macrophage gelsolin is present in neurones and that it probably plays a part in the actin-based movements of these cells.     

Characterization of the nicotinic acetylcholine receptor isolated from goldfish brain.

We have studied the binding of alpha-bungarotoxin to a particulate fraction of goldfish brain enriched in synaptosomes. The binding is specific and saturable and exhibits the pharmacological properties of a nicotinic cholinergic receptor. Equilibrium binding measurements yield a single dissociation constant (KD) of 0.92 nM. Kinetic analysis revealed one association rate constant and two dissociation rate constants. Dissociation constants calculated from kinetic measurements were 1.9 nM and 12.5 pM. The toxin . receptor complex is readily solubilized in nonionic detergent. The isoelectric point of the toxin . receptor complex was found to be 5.00 +/- 0.01. Sedimentation velocity analysis in sucrose/H2O and sucrose/D2O gradients in conjunction with Sepharose 4B chromatography and diffusion experiments yielded a sedimentation constant of 11.45, a partial specific volume of 0.79 cm3/g for the toxin . receptor . detergent complex, and a molecular weight of approximately 340,000 for the toxin . receptor complex.     

Hydrodynamic properties of the purified glutamate-binding protein subunit of the N-methyl-D-aspartate receptor.

The hydrodynamic properties of the previously purified glutamate-binding protein from rat synaptic membranes were determined in order to estimate the molecular size of the protein in its native state. This protein is apparently a subunit of a multisubunit complex that forms the N-methyl-D-aspartate subtype of glutamate receptor and has a molecular size of approximately 70 kDa based on electrophoretic migration under denaturing conditions. On the basis of results obtained from H2O/D2O sucrose density gradient sedimentation and gel filtration chromatography of the purified glutamate-binding protein we calculated the partial specific volume of the protein-detergent complex to be 0.766 cc3/g, the Stokes radius of the complex as 4.9 nm, the Mc of the complex as 203,000 +/- 22,000 and the Mr of the protein as 182,000 +/- 19,000. These results are indicative of stable self-association of the glutamate-binding protein and are in agreement with recent studies indicating that more than one molecule of glutamate may be required to activate the N-methyl-D-aspartate receptor-associated ion channel.