Molecular organization of glutamate-sensitive chemo-stimulated nerve cell membranes. Interaction of monoclonal antibodies with glutamate-binding membrane proteins in the rat brain, human neuroblastoma and molluscan neurons

Hybrid cells obtained by fusion of myeloma PX63-Ag8-653 with immune splenocytes of BALB/c mice were found to produce monoclonal antibodies with a high degree of specificity to rat and human brain. The kinetics of specific IgG binding to purified fractions of glutamate-binding membrane proteins from rat and human brain were analyzed in Scatchard plots. The presence of a single type of binding sites with Kd = 100 nM was demonstrated. The monoclonal antibodies were shown to inhibit the specific binding of tritium-labeled L-glutamate to different brain synaptic membranes. Addition of monoclonal antibodies to the incubation medium induced a modulating effect of physiological responses to L-glutamate in Planorbarius corneus neurons. The possible use of specific antibodies to glutamate-binding proteins as immunochemical markers for the study of glutamate receptor topography on membrane surface was demonstrated with the aid of neuroblastoma cells N18 Tg2a and rat brain tissue slices. An analysis of glutamate receptor binding sites with the use of monoclonal antibodies revealed that these antibodies specifically recognize the active center in the receptor molecules which have identical antigen determinant sites in different biological systems.     

Molecular organization of glutamate-sensitive chemoexcitable membranes of nerve cells. Function of glutamate-binding proteins of the central nervous system when incorporated into liposomes

The functioning of the glutamate-binding protein of rat brain cortex synaptic membranes was studied by its incorporation into liposomes. The optimal conditions for the receptor protein incorporation were established and the kinetics of 22Na+ and 86Rb+ incorporation into the liposomes in the presence of L-glutamate were analyzed. Modelling of the CNS glutamate receptor functions was found to be dependent on the lipid composition and amount of the incorporated membrane protein. The selective transport of 22Na+ into the liposomes was stimulated in the presence of 10(-4) M glutamate. Addition of monoclonal antibodies against glutamate-binding proteins blocked the incorporation of Na+ into the liposomes. The experimental results are suggestive of the nativity of the liposome-incorporated membrane protein, which is capable of binding glutamate and regulating selective transport of Na+. It was assumed that the glutamate receptor macromolecule represents an integral complex made up of several low molecular weight subunits of glucoprotein nature that form a selective ionic channel.     

Solubilization, partial purification, and reconstitution of glutamate- and N-methyl-D-aspartate-activated cation channels from brain synaptic membranes.

L-Glutamate-activated cation channel proteins from rat brain synaptic membranes were solubilized, partially purified, and reconstituted into liposomes. Optimal conditions for solubilization and reconstitution included treatment of the membranes with nonionic detergents in the presence of neutral phospholipids plus glycerol. The affinity batch chromatography procedure described previously [Chen et al. (1988) J. Biol. Chem. 263, 417-427] was used to obtain a fraction enriched in glutamate-binding proteins. Quench-flow procedures were developed to characterize the rapid kinetics of ion flux induced by receptor agonists. [14C]Methylamine, a cation that permeates through the open channel of both vertebrate and invertebrate glutamate receptors, was used to measure the activity of glutamate receptor-ion channel complexes in reconstituted liposomes. L-Glutamate caused an increase in the rate of [14C]methylamine influx into liposomes reconstituted with either solubilized membrane proteins or partially purified glutamate-binding proteins. The increase in methylamine influx was dependent on the concentration of L-glutamic acid with an estimated Kact for L-glutamate equal to 0.2 microM for synaptic membrane proteins and 0.32 microM for purified proteins. Of the major glutamate receptor agonists, only N-methyl-D-aspartate activated cation fluxes in liposomes reconstituted with glutamate-binding proteins. Glutamate-activated methylamine flux was completely inhibited by the N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonopentanoic acid. In liposomes reconstituted with glutamate-binding proteins, N-methyl-D-aspartate- or glutamate-induced influx of Na+ led to a transient increase in the influx of the lipid-permeable anion probe S14CN-. Electrophoretic analysis of partially purified proteins reconstituted in liposomes indicated enrichment of several bands, the most prominent being those of molecular size equal to approximately 69, 60, 35, and 25 kDa. Antibodies raised against the purified 71- and 63-kDa glutamate-binding proteins reacted strongly with the approximately 69-kDa band of reconstituted proteins and markedly decreased the initial rate of glutamate-activated cation flux. These results indicate the functional reconstitution of N-methyl-D-aspartate-sensitive glutamate receptors and the role of the approximately 69-kDa protein in the function of these ion channels.     

Immune labeling and purification of a 71-kDa glutamate-binding protein from brain synaptic membranes. Possible relationship of this protein to physiologic glutamate receptors

Immunoblot studies of synaptic membranes isolated from rat brain using antibodies raised against a previously purified glutamate-binding protein (GBP) indicated labeling of an approximately 70-kDa protein band. Since the antibodies used were raised against a 14-kDa GBP, the present studies were undertaken to explore the possibility that the 14-kDa protein may have been a proteolytic fragment of a larger Mr protein in synaptic membranes. Protease activity during protein purification was prevented by introducing five protease inhibitors, and a three-step purification procedure was developed that yielded a high degree of purification of glutamate-binding proteins. The major protein enriched in the most highly purified fractions was a 71-kDa glycoprotein, but a 63-kDa protein was co-purified during most steps of the isolation procedure. The glutamate-binding characteristics of these isolated protein fractions were very similar to those previously described for the 14-kDa GBP, including estimated dissociation constants for L-glutamate binding of 0.25 and 1 microM, inhibition of glutamate binding by azide and cyanide, and a selectivity of the ligand binding site for L-glutamate and L-aspartate. The neuroexcitatory analogs of L-glutamate and L-aspartate, ibotenate, quisqualate, and D-glutamate, inhibited L-[3H]glutamate binding to the isolated proteins, as did the antagonist of L-glutamate-induced neuronal excitation, L-glutamate diethylester. On the basis of the lack of any detectable glutamate-related enzyme activity associated with the isolated proteins and the presence of distinguishing sensitivities to analogs that inhibit glutamate transport carriers in synaptic membranes, it is proposed that the 71-kDa protein may be a component of a physiologic glutamate receptor complex in neuronal membranes.     

Immunochemical characterization of brain synaptic membrane glutamate-binding proteins

Two glutamate-binding proteins (71 and 63 kDa) were previously purified from synaptic plasma membranes (Chen, J.-W., Cunningham, M.D., Galton, V., and Michaelis, E. K. (1988) J. Biol. Chem. 263, 417-426). These proteins may play a role in glutamate neurotransmission in brain. Polyclonal antibodies were raised against the denatured glutamate-binding proteins in rabbits, including sets of antibodies against each of the binding proteins. The antibodies reacted specifically against both 71- and 63-kDa proteins. The antibodies recognized the denatured form of the proteins in Western blots and the native state of the proteins in enzyme-linked immunosorbent assays and in immunoaffinity chromatography and extraction procedures. All antibodies labeled most strongly the 71-kDa protein in Western blots, but extracted both proteins from solubilized synaptic membrane preparations. These findings indicate that the two proteins are closely related immunologically but the reactivity on Western blots differs between these two proteins. Immunoextraction of the 71- and 63-kDa proteins led to a approximately 60% decrease in L-[3H]glutamate-binding activity associated with synaptic membrane proteins. Of the brain subcellular fractions examined, the isolated synaptic plasma membranes had the strongest reaction in enzyme-linked immunosorbent assays toward the antiglutamate-binding protein antisera. Electron microscopy combined with gold particle immunohistochemistry revealed the sites labeled by the antibodies as entities present either on the surface or within the postsynaptic membranes and the associated densities of brain nerve ending particles (synaptosomes). Immunohistochemical procedures of gold labeling with silver enhancement of labeled sites revealed selective neuronal labeling in brain regions enriched in glutamate neurotransmitter pathways such as the hippocampus. Labeling was along dendrites and around cell bodies of pyramidal neurons. Based on the pattern of histochemical labeling, the distribution of immune reactivity in synaptic membranes, and the extractions of a major component of membrane glutamate-recognizing proteins by the antibodies, the glutamate-binding proteins must play a role in glutamate neurotransmission.     

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.     

A chloride- and calcium-dependent glutamate-binding protein from rat brain. Identification as a ubiquitous constituent of the inner mitochondrial membrane

We have recently solubilized and enriched a chloride- and calcium-dependent glutamate-binding protein from rat brain (Brose, N., Halpain, S., Suchanek, C., and Jahn, R. (1989) J. Biol. Chem. 264, 9619-9625). The partially purified protein fraction, containing two major protein components of 51,000 Da and 105,000 Da, was used to generate a rabbit antiserum. This serum quantitatively precipitated the binding activity from membrane extracts. Small amounts of the antiserum inhibited glutamate binding when chloride was absent from the incubation medium. Three protein bands were labeled by the serum on immunoblots. From the affinity purified antibody fractions contained in the serum, only the antibodies directed against a 51,000-Da protein were able to immunoprecipitate the binding activity, indicating that this protein is an essential component of the binding site. A survey of a variety of rat tissues by immunoblot analysis revealed a ubiquitous distribution of the protein. After subcellular fractionation of liver and brain, the 51,000-Da protein copurified with mitochondrial markers. Furthermore, exclusive labeling of mitochondria was observed by light and electron microscopy immunocytochemistry. Subfractionation of purified liver mitochondria resulted in a selective association of the protein with inner mitochondrial membranes. Pharmacological characterization of glutamate binding to liver mitochondrial membranes revealed a pattern almost identical to that of the chloride- and calcium-dependent glutamate-binding site in rat brain.     

Superoxide modification and inactivation of a neuronal receptor-like complex

Excessive superoxide (O(-)(2)) formation is toxic to cells and organisms. O(-)(2) reacts with either iron-sulfur centers or cysteines (Cys) of cytoplasmic proteins. Reactions with membrane proteins, however, have not been fully characterized. In the present studies, the reaction of O(-)(2) with a protein complex that has glutamate/N-methyl-D-aspartate (NMDA) receptor characteristics and with one of the subunits of this complex was examined. Exposure of the complex purified from neuronal membranes and the recombinant glutamate-binding protein (GBP) subunit of this complex to the O(-)(2)-generating system of xanthine (X) plus xanthine oxidase (XO) caused strong inhibition of L-[3H]glutamate binding. Inhibition of glutamate binding to the complex and GBP by O(-)(2) was greater than that produced by H(2)O(2), another product of the X plus XO reaction. Mutation of two cysteine (Cys) residues in recombinant GBP (Cys(190,191)) eliminated the effect of O(-)(2) on L-[3H]glutamate binding. Both S-thiolation reaction of GBP in synaptic membranes with [35S]cystine and reaction of Cys residues in GBP with [3H]NEM were significantly decreased after exposure of membranes to O(-)(2). Inhibition of cysteylation of membrane GBP by O(-)(2) was still observed after iron chelation by desferrioxamine, albeit diminished, and was not altered by the presence of catalase. Overall, the results indicated that GBP exposure to O(-)(2) modified Cys residues in this protein. The modification was not characterized but it was probably that of disulfide formation.     

Characterization and partial purification of a chloride- and calcium-dependent glutamate-binding protein from rat brain

A glutamate-binding protein was solubilized from rat brain synaptic plasma membranes using sodium cholate. Its properties were characterized after addition of exogenous phospholipids and formation of proteoliposomes. Glutamate binding was dependent on calcium and chloride ions with maximal binding at concentrations of 10(-5) M calcium and 10 mM chloride ions. The effects of the two ions were synergistic rather than additive. In addition, glutamate binding was not affected by inhibitors specific for N-methyl-D-aspartate and kainate receptor subtypes, but was inhibited by quisqualate (Ki = 50 microM) and DL-2-amino-4-phosphonobutyrate (Ki = 1.3 mM). Furthermore, binding was abolished by 100 microM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and 1 mM dithiothreitol. These properties resemble those of the chloride- and calcium-dependent binding site. Starting from the detergent extract, the glutamate-binding protein was purified 123-fold using fractionated ammonium sulfate precipitation, chromatography on hydroxyapatite and on DEAE-Sephacel as sequential purification steps. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protein fraction showed two major bands migrating with Mr values of 51,000 and 105,000. The properties of the partially purified binding protein were similar to those of the detergent extract. Glutamate binding to the partially purified protein is not due to a sequestration process or product binding to N-acetylated alpha-linked dipeptidase. Thus, the functional role of the binding protein remains to be established.     

Purification and Molecular Characterization of the Brain Synaptic Membrane Glutamate-Binding Protein

A glutamate-binding protein from rat brain synaptic plasma membranes has been purified to apparent homogeneity. This protein has a Mr of 14,300 based on amino acid and carbohydrate analyses. The protein is enriched with tryptophan residues, which contribute substantially to its hydrophobic nature. It also has a relatively high content of acidic amino acids, which determine is low isoelectric point (4.82). The protein exhibits either a single, high-affinity class of sites for L-[3H]glutamate binding (KD = 0.13 microM) when binding is measured at low protein concentrations, or two classes of sites with high (KD = 0.17 microM) and low affinities (KD = 0.8 microM) when binding is measured at high protein concentrations. These observations suggest preferential binding of L-glutamate to a self-associating form of the protein. The displacement of protein-bound L-[3H]glutamic acid by other neuroactive amino acids has characteristics similar to those observed for displacement of L-glutamate from membrane binding sites. Chemical modification of the cysteine and arginine residues results in an inhibition of glutamate binding activity. The possible function of this protein in the physiologic glutamate receptor complex of neuronal membranes is discussed.     

Glutamate-binding membrane proteins from human platelets]

Solubilization of the total membrane fraction of human platelets in a 2% solution of sodium deoxycholate and subsequent affinity chromatography on glutamate agarose resulted in two protein fractions possessing a glutamate-binding activity. As can be evidenced from radioligand binding data, the first fraction contains two types of binding sites (Kd1 = 1 microM, Bmax 1 = 100 pmol/mg of protein; Kd2 = 9.3 microMm Bmax2 = 395 pmol/mg of protein). The second fraction has only one type of binding sites (Kd = 1 microM, Bmax = = 110 pmol/mg of protein). SDS-PAAG electrophoresis revealed the presence in the first fraction of proteins with Mr of 14, 24, 56 and 155 kDa, whereas the second fraction was found to contain 14, 46, 71 and 155 kDa proteins. Solid phase immunoenzymatic analysis using poly- and monoclonal specific antibodies against mammalian brain glutamate-binding proteins revealed a marked immunochemical similarity of the isolated protein fractions with human brain synaptic membrane glutamate-binding proteins.     

Purification and characterization of a high-molecular-weight endogenous glutamate-binding inhibitor in porcine brain

A high-molecular-weight glutamate-binding inhibitor (HGBI) from porcine brain extract was purified to homogeneity. The results of this purification process show that glutamate receptor activity can be regulated by a high-molecular-weight protein, which inhibits [³H]L-glutamate binding to excitatory amino acid (EAA) receptors. The purified HGBI appears to be a protein with a molecular weight of approximately 70 kD. The purified HGBI is negatively charged, suggesting that it may contain acidic amino acids, and most likely,L-glutamate- andL-aspartate-enriched regions, responsible for its surface charge as well as for its binding to glutamate receptors. Inhibition of [³H]L-glutamate binding by the purified HGBI is reversible, and appears to change the binding kinetics. This endogenous ligand for glutamate receptors has unique characteristics separating it from all the other ligands found so far in the EAA receptor system. This HGBI represents a new class of modulator for the EAA receptor, thus further investigation of the function and structure of the HGBI should provide new understanding of the mechanisms of EAA-mediated neurotransmission.     

Molecular characteristics of glutamate receptors in the mammalian brain

Summary The strong excitatory activity of L-glutamic acid on central nervous system neurons is thought to be produced by interaction of this amino acid with specific neuronal plasma membrane receptors. The binding of L-glutamate to these surface receptors brings about an increase in membrane permeability to Na⁺ and Ca²⁺ ions presumably through direct activation of ion channels linked to the membrane receptors. The studies described in this paper represent attempts to define the subcellular distribution and pharmacological properties of the recognition site for L-glutamic acid in brain neuronal preparations, to isolate and explore the molecular characteristics of the receptor recognition site, and, finally, to demonstrate the activation of Na⁺ channels in synaptic membranes following the interaction of glutamate with its receptors.Radioligand binding assays with L-[³H] glutamic acid have been used to demonstrate a relative enrichment of these glutamate recognition sites in isolated synaptic plasma membranes. The specific binding of L-[³H] glutamate to these membrane sites exhibits rapid association and dissociation kinetics and rather complex equilibrium binding kinetics. The glutamate binding macromolecule from synaptic membranes has been solubilized and purified and was shown to be a small molecular weight glycoprotein (M_T 13 000). This protein tends to form aggregates which have higher specific activity at low concentrations of glutamate than the M_T 13 000 protein has. The overall affinity of the purified protein is lower than that of the high affinity sites in the membrane. Nevertheless, the purified protein exhibits pharmacological characteristics very similar to those of the membrane binding sites. On the basis of its pharmacological properties this protein belongs in the category of the physiologic glutamate preferring receptors.By means of differential solubilization of membrane proteins with Na-cholate, it was shown that this recognition site is an intrinsic synaptic membrane protein whose binding activity is enhanced rather than diminished by cholate extraction of the synaptic membranes. The role of membrane constituents in regulating the binding activity of this protein has been explored and a possible modulation of glutamate binding by membrane gangliosides has been demonstrated. Finally, this glutamate binding glycoprotein is a metalloprotein whose activity is dependent on the integrity of its metallic (Fe) center. This is a clear distinguishing characteristic of this protein vis-à-vis the glutamate transport carriers.The presence of functional glutamate receptors in synaptosomes and resealed synaptic plasma membranes has also been documented by the demonstration of glutamate-activated Na⁺ flux across the membrane of these preparations. The bidirectionality, temperature independence, and apparent desensitization of this stimulated flux following exposure to high concentrations of glutamate are properties indicative of a receptor-initiated ion channel activation. It would appear, then, that the synaptic membrane preparations provide a very useful system for the study of both recognition and effector function of the glutamate receptor complex.     

Effect of growth conditions on glutamate transport in the wild-type strain and glutamate-utilizing mutants of Escherichia coli.

The effects of growth conditions on the glutamate transport activity of intact cells and membrane vesicles and on the levels of glutamate-binding protein in wild-type Escherichia coli K-12 CS101 and in two glutamate-utilizing mutants, CS7 and CS2TC, were studied. Growth of CS101 on aspartate as the sole source of carbon or nitrogen resulted in a severalfold increase in glutamate transport activity of intact cells and membrane preparations to levels characteristic of the operator-constitutive mutant CS7. The high glutamate transport activity of mutant CS7 was not depressed further by growth on aspartate. Synthesis of glutamate-binding protein was not enhanced by aspartate in either strain. Mutant CS2TC produces a heat-labile repressor of glutamate permease synthesis and is therefore able to grow on glutamate at 42 C but not at 30 C. CS2TC cells grown in a glycerol-minimal medium at the restrictive temperature (30 C) exhibit low glutamate transport activity. Growth on aspartate at 30 C results in derepressed synthesis of glutamate permease. Cells grown on glycerol at 42 C have high glutamate transport activity. No further derepression is obtained upon growth on aspartate. Growth of CS101 and CS7 in "rich broth" greatly reduces the levels of glutamate-binding protein but does not appreciably affect glutamate transport by whole cells or membrane preparations. The identity of the carrier and the role of the binding protein in glutamate transport are discussed in the light of these findings.     

Molecular organization of glutamate-sensitive chemoexcitatory membranes of nerve cells. Comparative analysis of glutamate-binding membrane proteins from the cerebral cortex of rats and humans

The kinetics of 3H-L-glutamate binding to human brain synaptic membranes revealed the existence of one type of binding sites with Kd and Vmax comparable with those for freshly isolated rat brain membranes. The fraction of glutamate-binding proteins (GBP) was shown to contain three components with Mr of 14, 60 and 280 kD whose stoichiometry is specific for human and rat brain. All fractions were found to bind the radiolabeled neurotransmitter and to dissociate into subunits with Mr of 14 kD after treatment with-potent detergents (with the exception of the 56-60 kD component). Study of association-dissociation of GBP protein subunits by high performance liquid chromatography confirmed the hypothesis on the oligomeric structure of glutamate receptors which are made up of low molecular weight glycoprotein-lipid subunits and which form ionic channels by way of repeated association. Despite the similarity of antigen determinants in the active center of glutamate receptors from human and rat brain, it was assumed that the stoichiometry of structural organization of receptor subunits isolated from different sources is different. The functional role of structural complexity of human brain glutamate receptors is discussed.     

Ionotropic glutamate receptors.

The glutamate-binding sites of ionotropic glutamate receptors are formed from two extracellular domains of a single subunit. Conformational changes induced by agonist binding produce mechanical processes that are translated into ion gating and receptor desensitization. The interactions between macromolecular assemblies of synaptic proteins and ionotropic glutamate receptors, and their subsequent roles in receptor clustering and specificity are being elucidated. Kainate receptor pharmacology is finally revealing its secrets as a result of the availability of selective pharmacological agents.     

Molecular structure of the beta-adrenergic receptor

The beta-adrenergic receptor from several tissues has been purified to homogeneity or photoaffinity radiolabeled and its subunit molecular weight determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. In this study we have examined the oligomeric structure of nondenatured beta 1- and beta 2-adrenergic receptor proteins, as solubilized with the detergent digitonin. Model systems used were frog and turkey red blood cell as well as rat, rabbit, and bovine lung plasma membrane preparations. To correct for the effects of detergent binding, sedimentation equilibrium analysis in various solvents, as adapted for the air-driven ultracentrifuge, was used. With this approach an estimate of 6 g of digitonin/g of protein binding was determined, corresponding to a ratio of 180 mol of digitonin/mol of protein. Protein molecular weights estimated by this method were 43 500 for the turkey red blood cell beta 1 receptor and 54 000 for the frog red blood cell beta 2 receptor. Molecular weights of 60 000-65 000 were estimated for beta 1 and beta 2 receptors present in mammalian lungs. These values agree with estimates of subunit molecular weight obtained by SDS gel electrophoresis of purified or photoradiolabeled preparations and suggest beta-adrenergic receptors to be digitonin solubilized from the membrane as single polypeptide chains.     

Conformational changes at the agonist binding domain of the N-methyl-D-aspartic acid receptor

The conformational changes in the agonist binding domain of the glycine-binding GluN1 and glutamate-binding GluN2A subunits of the N-methyl D-aspartic acid receptor upon binding agonists of varying efficacy have been investigated by luminescence resonance energy transfer (LRET) measurements. The LRET-based distances indicate a cleft closure conformational change at the GluN1 subunit upon binding agonists; however, no significant changes in the cleft closure are observed between partial and full agonists. This is consistent with the previously reported crystal structures for the isolated agonist binding domain of this receptor. Additionally, the LRET-based distances show that the agonist binding domain of the glutamate-binding GluN2A subunit exhibits a graded cleft closure with the extent of cleft closure being proportional to the extent of activation, indicating that the mechanism of activation in this subunit is similar to that of the glutamate binding α-amino-5-methyl-3-hydroxy-4-isoxazole propionate and kainate subtypes of the ionotropic glutamate receptors.     

A membrane associated glutamate binding protein from Caenorhabditis elegans and Haemonchus contortus

1. A glutamate binding protein has been identified in membrane preparations from the free living nematode, Caenorhabditis elegans, and from the parasitic nematode, Haemonchus contortus. 2. This putative glutamate receptor was solubilized with 30 mM octyl-B-glucoside and partially purified by anion exchange and gel filtration chromatography. 3. An 80-fold purification with recovery of 75% of the glutamate binding activity was achieved. 4. The soluble C. elegans binding protein displayed a Kd for glutamate of 0.1 microM, in close agreement with the findings for the membrane associated binding protein. 5. Quisqualate was capable of displacing glutamate from the soluble C. elegans receptor, again in agreement with previous findings for the membrane bound receptor. 6. The fact that a parasitic nematode, Haemonchus contortus, also possesses this putative glutamate receptor, strengthens the case for using C. elegans as a model system for the study of parasitic nematode neuromuscular physiology.