The sites of high affinity binding of L-[3H]glutamic acid in human platelets. A new type of platelet receptor?

The total membrane fraction of human platelets was found to contain high affinity sites of L-[3H]glutamic acid binding (Kd = 100 nM, Bmax = 1.06 pmol/mg protein). The pH optimum for binding is at pH approximately 6.9 Na+ (1-150 mM) inhibit glutamate binding by platelet membranes (IC50 = 12 mM). Ca2+ (50-100 microM) stimulate the binding by 10-20% and inhibit it by 20-30% at concentrations of 1-5 mM. Monoclonal antibodies to the glutamate receptor strongly suppress the L-[3H]glutamate binding by platelet membranes (IC50 = 300 nm). The presence in human platelets of a glutamate-sensitive receptor complex similar to the central nervous system glutamate receptor is postulated.     

L-[3H]Glutamate Binding to a Membrane Preparation from Crayfish Muscle

The binding of L-[3H]glutamate to an isolated membrane preparation from crayfish tail muscle has been studied. The muscle homogenate was osmotically shocked, frozen and thawed, and thoroughly washed before incubation with L-[3H]glutamate. The preparation showed high specific binding of L-glutamate with a KD of 0.12 microM and Bmax of 4.7 pmol/mg protein measured in Tris/HCl pH 7.3 and at 4 degrees C. Nonspecific binding was 5-10% of total binding. The glutamate binding was highly stereospecific [K0.5 (D-glutamate), 270 microM] and showed a high degree of discrimination between L-glutamate and L-aspartate [K0.5 (L-aspartate), 54 microM]. In mammalian CNS preparations potent agonists of L-glutamate such as kainate and N-methyl-D-aspartate had no effect at 1 mM, and quisqualate was a weak inhibitor of L-glutamate binding [K0.5 (quisqualate), 162 microM]. Ibotenate was the most potent inhibitor [K0.5 (ibotenate), 0.27 microM], and various esters of L-glutamate were of intermediate potency as displacers of L-[3H]glutamate binding (K0.5 values from 6 to 60 microM). The glutamate binding site from crayfish muscle is clearly different from any of the subclasses of glutamate receptors in mammalian CNS. A possible physiological function of the binding site is a postsynaptic receptor for glutamate, either an extra-junctional or a junctional receptor.     

ACTH receptors in nervous tissue. High affinity binding-sequestration of [125I]Phe2,Nle4]ACTH 1-24 in homogenates and slices from rat brain

We have demonstrated specific, high affinity binding of a biologically active Tyr23-monoiodinated derivative of ACTH, [125I][Phe2,Nle4]ACTH 1-24, in rat brain homogenates. Similarly, in metabolically inhibited and noninhibited rat whole brain slices there is a specific "binding-sequestration" process that is dependent on time, protein concentration, and pH. In homogenates, binding curves were best described by a two-site model and provided the following parameters: Kd1 = 0.65 +/- 0.47 nM, Bmax1 = 21 +/- 41 fmol/mg protein; Kd2 = 97 +/- 48 nM, Bmax2 = 3.5 +/- 1.8 pmol/mg protein. In metabolically viable brain slices, concentration-competition curves of [125I][Phe2,Nle4]ACTH 1-24 binding-sequestration can be described by three components (Kd1 = 14 +/- 24 nM, Bmax1 = 50 +/- 95 fmol/mg protein; Kd2 = 2.4 +/- 1.9 microM, Bmax2 = 44 +/- 49 pmol/mg protein; Kd3 = 0.16 +/- 1.0 mM, Bmax3 = 5.3 +/- 54 nmol/mg protein). Metabolic inhibition, by removal of glucose and addition of 100 microM ouabain, abolishes the lowest affinity, highest capacity binding-sequestrian component only (Kd1 = 7.1 +/- 14 nM, Bmax1 = 8.7 +/- 16 fmol/mg protein; Kd2 = 7.4 +/- 4.49 microM, Bmax2 = 37 +/- 27 pmol/mg protein). The two binding-sequestration parameter estimates obtained from metabolically inhibited tissue slices are not significantly different from those of the two higher affinity components obtained with noninhibited tissue. Thus, metabolic inhibition permits demonstration of ACTH receptor binding only, unconfounded by sequestration or internalization of ligand:receptor complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenosine transporters in chromaffin cells: subcellular distribution and characterization

Adenosine transporters in freshly isolated and cultured chromaffin cells were quantified by the [3H]dipyridamole binding technique, showing a maximal bound capacity of 0.4 +/- 0.05 pmol/10(6) cells (240,000 +/- 20,000 transporters by cell). Scatchard analysis showed a similar affinity for [3H]dipyridamole in isolated cells and subcellular fractions (Kd = 5 +/- 0.6 nM). For enriched plasma membrane preparations and chromaffin granule membranes, the maximal binding capacities were also very similar, 2.3 +/- 0.3 and 1.8 +/- 0.4 pmol/mg protein, respectively. When [3H]nitrobenzylthioinosine was employed as a radioligand, the maximal bound capacity in cultured chromaffin cells was 0.053 +/- 0.004 pmol/10(6) cells (32,000 +/- 3000 transporters per cell) with a high affinity constant (Kd = 0.25 +/- 0.03 nM); similar values were obtained in all subcellular fractions (Kd = 0.1 +/- 0.01). Also, plasma and chromaffin granule membranes showed similar maximal binding values (0.4 +/- 0.06 pmol/mg protein). Photoincorporation studies with [3H]nitrobenzylthioinosine into plasma membrane polypeptides showed the presence of three molecular species of 115 +/- 10; 58 +/- 6 and 42 +/- 5 kDa. Chromaffin granule membranes showed only the 105 +/- 9 and 51 +/- 4 molecular species.     

Some properties of prostaglandin E2 receptors in rabbit alveolar bone cell membranes

[3H]prostaglandin E2 (PGE2) binding receptors exist in rabbit alveolar bone cell membranes. The presence of high (Kd = 3.9 X 10(-9) M) and low (Kd = 8.8 X 10(-8) M) affinity binding sites of [3H]PGE2 was demonstrated. The saturation values of [3H]PGE2 for high and low affinity binding sites were 0.13 pmol/mg protein and 1.22 pmol/mg protein, respectively. The digestion of the membranes with pronase, phospholipase C, D and neuraminidase led to a decrease of [3H]PGE2 binding but phospholipase A2 did not.     

Chemoreception in Hydra vulgaris (attenuata): initial characterization of two distinct binding sites for L-glutamic acid.

To elucidate the relationship between L-glutamic acid and the putative chemoreceptor for glutathione, binding of L-[3H]glutamate to a crude membrane fraction from Hydra vulgaris (attenuata) has been characterized. The binding of L-[3H]glutamate was rapid, reversible and saturable. A Scatchard analysis of the specific binding revealed values of 10 microM for the dissociation constant (Kd) and 170 pmol/mg for the maximal capacity of binding sites (Bmax). A maximum of 65% of the specific L-[3H]glutamate binding was inhibited by the chemostimulatory peptide, glutathione. This glutathione-sensitive glutamate binding presumably represents the association of glutamate with a putative chemoreceptor which modulates feeding behavior in hydra. The remaining 35% of the specific L-[3H]glutamate binding may be due to a second class of glutamate binding sites which is insensitive to glutathione. The identification of glutathione-insensitive glutamate binding is the first indication of a putative glutamate receptor, which may mediate an action independent of the glutathione-induced feeding response. The glutathione-insensitive and glutathione-sensitive sites must have similar affinities for glutamate since these sites were indistinguishable by Scatchard analysis. A preliminary characterization of the glutathione-insensitive site, performed in the presence of saturating levels of glutathione, revealed inhibition of glutathione-insensitive glutamate binding by kainate and quisqualate, but not by N-methyl-D-aspartate. A glutathione-insensitive L-[3H]glutamate binding suggests that kainate and alpha-aminoadipate may be selective ligands for the glutathione-insensitive and glutathione-sensitive glutamate binding sites, respectively.     

Comparison of bile acid binding to sinusoidal and bile canalicular membranes isolated from rat liver

Simon et al. (J. Clin. Invest., 70 (1982) 401) studied cholate binding to crude liver plasma membrane vesicles and suggested that the binding may represent mainly the binding to the receptor (carrier) on the canalicular membrane. This hypothesis was supported by finding a good correlation between the number of cholate binding sites on liver plasma membrane and the maximal rate of biliary secretion (Tm) for taurocholate. We studied bile acid binding to sinusoidal and canalicular membrane vesicles isolated from rat liver by a rapid filtration technique. Scatchard analysis of the saturation kinetics showed both [3H]cholate and [3H]chenodeoxycholate bind to two classes of binding site on each membrane. However, little difference was observed between the binding to sinusoidal and canalicular membrane vesicles for each bile acid (cholate, Kd1 = 10.4 and 19.8 microM, n1 = 31.0 23.6 pmol/mg protein, Kd2 = 1.32 and 1.73 mM, n2 = 13.1 and 23.4 nmol/mg protein; and chenodeoxycholate, Kd1 = 0.207 and 0.328 microM, n1 = 36.7 and 27.4 pmol/mg protein, Kd2 = 1.16 and 2.26 mM, and n2 = 20.6 and 24.2 nmol/mg protein; numbers show the mean values sinusoidal and canalicular membrane vesicles, respectively). Chenodeoxycholate binding to sinusoidal membrane vesicles was markedly inhibited by cholate but not by Rose bengal, an organic anion dye. These studies indicate that both membranes (sinusoidal and canalicular membrane vesicles) have two kinds of binding site for bile acids, although no clear difference in the binding properties was observed between the two membranes. Consequently, the cholate binding Simon detected may represent the binding not only to canalicular membrane vesicles but also to sinusoidal membrane vesicles.     

Identification and properties of phencyclidine-binding sites in nervous tissues

[3H]Phencyclidine (PCP) binds to a single class of noninteracting binding sites in rat brain membranes with an affinity Kd of 0.25 microM and a maximal binding capacity BM of 2.4 pmol/mg of membrane protein. PCP derivatives also interact with the muscarinic and mu-opiate receptors in rat brain membranes with affinities that are one or two orders of magnitude lower than those observed for the [3H]PCP-binding sites. Activities of 25 PCP derivatives in the rotarod assay are closely correlated to affinities of these molecules for the [3H]PCP-binding sites, but not for the muscarinic or mu-opiate receptors. Monohydroxylation of PCP generally decreases the affinity of PCP for the [3H]PCP- and muscarinic-binding sites and does not change the affinity for the mu-opiate receptor. The metaphenolic derivative of PCP does not follow these general rules; the affinities of this derivative for the [3H]PCP- and mu-opiate-binding sites are 8 and 430 times higher, respectively, than those of PCP itself. Voltage-clamp experiments with N1E 115 neuroblastoma cells show that PCP is an efficient blocker of both the K+ channel (EC50 = 2.6 microM) and the Na+ channel (EC50 = 9.2 microM).     

Interaction of non-steroidal antiestrogens with dopamine receptor binding

The ability of various estrogen antagonists and agonists to compete with [3H]spiroperidol, [3H]domperidone, [3H]dihydroalprenolol, [3H]dihydroergocryptine, [3H]dopamine or [3H]5-hydroxytryptamine for binding to membrane preparations from rat brain tissue was tested. The non-steroidal triphenylethylene-type antiestrogens with an amine side chain--enclomiphene, nitromifene, tamoxifen and zuclomiphene--were found to be competitive inhibitors of [3H]spiroperidol (Kd = 0.12 nM; Bmax = 101 fmol/mg protein) and [3H]domperidone (Kd = 0.62 nM; Bmax = 86 fmol/mg protein) binding to striatal membranes. The Ki values ranged from 4-12 microM. Estradiol-17 beta (Ki = 480 microM) or diethylstilbestrol (Ki = 63 microM) were much less effective inhibitors exhibiting noncompetitive interaction with the in vitro binding of [3H]spiroperidol. The pharmacological relevance of the antiestrogen interactions with dopamine receptor binding is discussed with respect to adverse effects of the in vivo administered compounds such as nausea and vomiting.     

Two types of inositol trisphosphate binding in cardiac microsomes.

Two distinct types of [3H]IP3 binding were found in canine cardiac microsomes with high (Kd = 21 nM, Bmax = 0.66 pmol/mg) and low affinity (Kd = 230 nM, Bmax = 2.9 pmol/mg). Also found were low affinity [3H]IP4 binding (Kd = 190 nM, Bmax = 4.5 pmol/mg) and high affinity [3H]IP6 binding (Kd = 10 nM, Bmax = 4.9 pmol/mg). The rank order of potency to displace these radioligands indicates that binding of IP3 and IP6 is ligand-specific. Sucrose gradient centrifugation of the detergent-solubilized cardiac microsomes indicates that the molecular size of the cardiac high affinity IP3 receptor is similar to that of the aortic smooth muscle IP3 receptor and smaller than that of the ryanodine receptor which migrates more rapidly. The IP4 and IP6 binding migrates more slowly than the IP3 receptor.     

The binding of [3H]adenosine to synaptosomal and other preparations from the mammalian brain.

1. A high-affinity adenosine-binding site with Kd(adenosine) 0.5-1.3 microM was demonstrated in particulate and synaptosomal fractions isolated from the cerebral cortex of guinea pig, rat and ox. 2. Binding of [3H]adenosine to this site was inhibited by theophylline and by 2-chloroadenosine, but not by four other adenosine analogues. 3. Endogenous adenosine, found to be present in some preparations at approx. 1 pmol/mg of protein, diminished the binding capacity of the preparations for [3H]adenosine. 4. Addition of the adenosine deaminase inhibitor erythro-9-[1-(1-hydroxyethyl)heptyl]-adenine revealed the presence of a second lower affinity binding site with Kd (adenosine) 5-9 microM and a higher maximal adenosine-binding capacity. The inhibitor partially blocked binding to the high-affinity site in preparations from which adenosine deaminase had been removed by washing. 5. To preparations of particulate fractions maintained under iso-osmotic conditions, adenosine attachment was non-saturable and temperature-dependent, indicating the existence of an active uptake process. 6. The location and binding constant of the high-affinity adenosine-binding site suggest that it corresponds to the receptor site for adenosine-activated adenylate cyclase.     

Sodium-dependent high-affinity binding of [3H]hemicholinium-3 in the rat brain: a potentially selective marker for presynaptic cholinergic sites

This report describes the membrane binding properties of [3H]hemicholinium-3 ([3H]HC-3), a selective inhibitor of sodium-dependent high-affinity choline uptake (SDHACU) in cholinergic nerve terminals. Under the described assay conditions, [3H]HC-3 bind with a saturable population of high-affinity (apparent Kd = 1.9 nM) CNS membrane sites having the regional distribution: striatum much greater than hippocampus greater than cerebral cortex greater than cerebellum. High-affinity [3H]HC-3 binding is entirely dependent upon the presence of sodium chloride (EC50 = 35-50 mM) and is markedly reduced when other salts of sodium or monovalent ions are substituted. [3H]HC-3 binding is inhibited by choline (Ki = 6 microM) and acetylcholine (Ki = 35 microM) but markedly less sensitive to other cholinergic agents and metabolic inhibitors. In light of the similar ionic dependencies, regional distributions and pharmacological specificities of [3H]HC-3 binding and SDHACU, closely associated sites may be involved in both processes.     

Characterization of the sulfonylurea receptor on beta cell membranes

Specific, high affinity sulfonylurea receptors were characterized on membranes of an insulin-secreting hamster beta cell line (HIT cells). Saturable binding of the sulfonylurea, [3H]glyburide, was linear up to 0.8 mg/ml membrane protein. Scatchard analysis of equilibrium binding data at room temperature indicated the presence of a single class of saturable, high affinity binding sites with a Kd of 0.76 +/- 0.04 nM and a Bmax of 1.09 +/- 0.13 pmol/mg protein, n = 9. The insulin secretory potency of glyburide, glipizide, tolbutamide, tolazamide, and carboxytolbutamide was compared to the ability of these ligands to displace [3H]glyburide from the sulfonylurea receptor. Tolbutamide, tolazamide, and glipizide demonstrated reasonable agreement with ED50 values of 15 microM, 3 microM, and 30 nM and Ki values of 25.3 microM, 7.2 microM, and 45 nM, respectively. The inactive tolbutamide metabolite, carboxytolbutamide, at the highest concentration tested, only partially displaced [3H]glyburide from the receptor and was a very poor secretagogue. At 37 degrees C the affinity of [3H]glyburide binding, Kd = 2.0 nM, was similar to the ED50 of 5.5 nM when the free glyburide concentrations were corrected for binding of the drug to albumin. These studies suggest that sulfonylureas initiate their biologic effect through a high affinity, specific interaction with sulfonylurea receptors on the beta cell membrane.     

Stimulatory and protective effects of benzodiazepines on GABA receptors labeled with [3H]muscimol

We investigated the effects of benzodiazepines on [³H]muscimol binding to rat brain membranes and on heat inactivation of GABA receptors. Scatchard analysis of [³H]muscimol binding to frozen and 0.05% Triton X-100 treated membranes revealed two components; a higher affinity (Kd=2.2 nM, Bmax=1.2 pmol/mg protein) and a lower affinity component (Kd=15.9 nM, Bmax=4.4 pmol/mg protein). Diazepam and flurazepam (3 μM) increased significantly the specific binding of 40 nM but not of 2 nM [³H]muscimol. This stimulation was attributed to an increase in the affinity of the lower affinity component for GABA receptors. The time course of heat inactivation of GABA receptors revealed rapidly and then slowly denaturating Phases. These observations would suggest that there are multiple GABA receptors with different sensitivities to the heat treatment. Diazepam depressed remarkably the slowly denaturating phase(s). After heat treatment for 50 min, the single component of GABA receptors with Kd of 14.3 nM and Bmax of 0.6 pmol/mg protein survived, whereas in the membranes preincubated with 3 μM diazepam, the Kd and Bmax of the still viable GABA receptors were 14.8 nM and 1.14 pmol/mg protein, respectively. In light of these findings, the stimulation of the lower affinity component of GABA receptors may be related to the protective effect of these drugs against heat inactivation.     

Solubilization of phencyclidine receptors from rat cerebral cortex in an active ligand binding state

A phencyclidine (PCP) receptor binding site has been solubilized in an active ligand-binding state from rat cerebral cortical membranes with sodium deoxycholate. Optimal receptor solubilization occurs at a detergent/protein ratio of 0.5 (w/w); for 5 mg protein/ml solubilized with 0.25% sodium deoxycholate, about 60% of the protein and 25% of the receptor is solubilized. Specific binding of either [3H]-N-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) or [3H]MK-801 is measurable by filtration through Sephadex G-50 columns or glass fiber filters; more than 60% of the binding activity is stable after 48 h at 4 degrees C. In the presence of detergent, [3H]TCP binding exhibits a Kd of 250 nM, a Bmax of 0.56 pmol/mg protein, and a pharmacological profile consistent with that of the membrane-bound PCP receptor, although most drugs bind with affinities 2 to 8 fold lower than in membranes. Upon reduction of detergent concentration, binding parameters approximate those for the membrane-bound receptor ([3H]TCP binding: Kd = 48 nM, Bmax = 1.13 pmol/mg protein).     

Glucose transporters in chromaffin cells: subcellular distribution and characterization

The glucose transporter was identified and characterized by cytochalasin B binding in subcellular membrane fractions of chromaffin tissue. The binding was saturable with Kd of about 0.3 microM for each subcellular fraction. The Bmax capacity was 12-16 pmol/mg protein for enriched plasma membrane fractions, 6.3 pmol/mg protein for microsomal membrane preparations and 5.4 pmol/mg protein for chromaffin granule membranes. Irreversible photoaffinity labelling of the glucose-protectable binding sites with [3H]cytochalasin B followed by solubilization and polyacrylamide gel electrophoresis from enriched plasma membrane preparations demonstrated the presence of three molecular species: 97 +/- 10, 51.5 +/- 6 and 30 +/- 4 kDa. The chromaffin granule membranes showed only a molecular species of 80 +/- 10 kDa.     

Autoradiographic Characterization and Localization of Quisqualate Binding Sites in Rat Brain Using the Antagonist [3H]6-Cyano-7-Nitroquinoxaline-2,3-Dione: Comparison with (R,S)-[3H]α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Binding Sites

Using quantitative autoradiography, we have investigated the binding sites for the potent competitive non-N-methyl-D-aspartate (non-NMDA) glutamate receptor antagonist [3H]6-cyano-7-nitro-quinoxaline-2,3-dione ([3H]-CNQX) in rat brain sections. [3H]CNQX binding was regionally distributed, with the highest levels of binding present in hippocampus in the stratum radiatum of CA1, stratum lucidum of CA3, and molecular layer of dentate gyrus. Scatchard analysis of [3H]CNQX binding in the cerebellar molecular layer revealed an apparent single binding site with a KD = 67 +/- 9.0 nM and Bmax = 3.56 +/- 0.34 pmol/mg protein. In displacement studies, quisqualate, L-glutamate, and kainate also appeared to bind to a single class of sites. However, (R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) displacement of [3H]CNQX binding revealed two binding sites in the cerebellar molecular layer. Binding of [3H]AMPA to quisqualate receptors in the presence of potassium thiocyanate produced curvilinear Scatchard plots. The curves could be resolved into two binding sites with KD1 = 9.0 +/- 3.5 nM, Bmax = 0.15 +/- 0.05 pmol/mg protein, KD2 = 278 +/- 50 nM, and Bmax = 1.54 +/- 0.20 pmol/mg protein. The heterogeneous anatomical distribution of [3H]CNQX binding sites correlated to the binding of L-[3H]glutamate to quisqualate receptors and to sites labeled with [3H]AMPA. These results suggest that the non-NMDA glutamate receptor antagonist [3H]CNQX binds with equal affinity to two states of quisqualate receptors which have different affinities for the agonist [3H]AMPA.     

Estimation of platelet-activating factor receptors in the endometrium of the pregnant rabbit: regulation of ligand availability and catabolism by bovine serum albumin.

High affinity receptors have been demonstrated for the potent phospholipid autacoid, platelet-activating factor (PAF C18:0; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) in a variety of tissues, including the endometrium. Because of the relative instability of PAF and our previous demonstration that lyso-PAF (1-O-alkyl-2-lyso-sn-glycero-3-phosphorylcholine), the major metabolite of PAF, displaced [3H]PAF from endometrial PAF receptor sites, we have examined the ability of bovine serum albumin (BSA) to prevent degradation of PAF and have characterized PAF and lyso-PAF binding sites in purified rabbit endometrial membranes isolated on Day 6 of pregnancy. In buffer containing the phospholipase A2 inhibitors, quinacrine (10 microM) and dibromoacetophenone (2 microM), and 0.25% BSA, 87.4 +/- 3.2% of added [3H]PAF C18:0 remained intact after incubation at 25 degrees C for 150 min. The metabolic products, lyso-PAF and 1-O-alkyl-2-acyl-sn-glycero-3-phosphorylcholine (alkylacyl-GPC), only amounted to 5.2 +/- 3.2 and 3.3 +/- 1.1, respectively. At the same concentration, rabbit serum albumin (RSA) also significantly protected [3H]PAF C18:0 from metabolism, but bovine gamma globulin (BGG) was ineffective. The presence of 0.25% BSA, however, did not protect [3H]lyso-PAF C18:0 from extensive catabolism: the major product formed was [3H]alkylacyl-GPC. Insignificant amounts of [3H]PAF were formed. Under the same conditions (25 degrees C, 150 min) in the presence of 0.25% BSA, saturation analysis revealed the presence of two types of PAF C18:0 receptors in the endometrial membranes. Type 1 sites had a Kd of 0.42 +/- 0.03 nM (mean +/- SD; n = 3) and binding capacity of 0.11 +/- 0.01 pmol/mg protein. Type 2 receptor sites had a Kd of 5.96 +/- 0.35 nM and a binding capacity of 1.59 +/- 0.22 pmol/mg protein. Thus, in the presence of BSA, the binding capacities of the two classes of receptors were markedly reduced compared to values generated previously in its absence. The Kd of the Type 1 sites was not significantly changed by the presence of BSA. A single class of saturable high-affinity binding sites was demonstrable for lyso-PAF C18:0: Kds ranged from 0.76 +/- 0.58 to 11.1 +/- 0.62 nM, depending on which method of analysis was used (Eadie-Hofstee, Scatchard-Rosenthal, or the Lundon nonlinear method). The binding capacities were equally varied, ranging from 0.15 +/- 0.08 to 15.17 +/- 4.95 pmol/mg protein.(ABSTRACT TRUNCATED AT 400 WORDS)     

HIGH AFFINITY l-[3H]GLUTAMATE BINDING TO POSTSYNAPTIC RECEPTOR SITES ON RAT CEREBELLAR MEMBRANES

l [³H]glutamate binding was investigated in membrane preparations derived from rat cerebellum, an area of the brain where it is likely that a high density of postsynaptic glutamate receptors occurs. Glutamate was hound specifically and, in freshly prepared membranes, was optimal under physiological conditions of pH and temperature and was associated with the synaptic membrane fraction of the cell. Specific binding occurred through a single, high-affinity process with a K_D, of 744 nM and a capacity of 73 pmol/mg protein. Unlike the findings reported for GABA, the specific binding of glutamate to fresh membranes did not involve an uptake site. Comparison of the potencies of a wide range of compounds with known pharmacological activities, demonstrated that their ability to displace specific glutamate binding was consistent with specific interactions with glutamate receptors.