The Specific Interaction of S-100 Protein with Synaptosomal Particulate Fractions. Evidence for the Formation of a Tight Complex Between S-100 and Its Binding Sites

Abstract: The dissociation of the ¹²⁵I-labelled S-100 specifically bound to synaptosomal particulate fractions (SYN) has been studied under a variety of conditions after different association times. The results indicate that after a critical association time of about 20 min at 37°C, the bound protein becomes progressively less accessible to the dissociating agents or conditions employed. These findings support the view that the partial irreversibility of the ¹²⁵I-labelled S-100 binding to SYN could be due to the formation of a tight complex between the protein and its synaptosomal sites. These data are discussed mainly in relation to the particulate-bound fraction of native S-100.     

Subnuclear Distribution of the S-100 Protein Specific Binding Sites in Rat Brain

Abstract: Fractionation of isolated brain nuclei previously reacted with ¹²⁵I-labelled S-100 showed that most of the specifically bound radioactivity associated with the nuclear membranes and the nucleoli. Labelling of nucleoli, which indicates the entrance of ¹²⁵I-labelled S-100 into the nucleus, was observed at 37°C, but not at 0–4°C. When tested separately for ¹²⁵I-labelled S-100 specific binding, both the nuclear membranes and the nucleoli were found to bind ¹²⁵I-labelled S-100 in a biphasic manner, the binding displaying a high affinity and a low affinity component, as observed with intact nuclei. However, the binding to nuclear membranes was largely irreversible, while that to nucleoli was fully reversible after any association time.     

Specific Binding Sites for S-100 Protein in Isolated Brain Nuclei

Abstract: Isolated brain nuclei possess binding sites for S-100 protein. The interaction of S-100 with these sites is specific and time-, temperature-, and Ca⁺-dependent. The profile of the ¹²⁵I-labelled S-100 binding inhibition is biphasic, displaying a high-affinity component and a low-affinity component. The S-100 binding to brain nuclei is largely irreversible, probably owing to the formation of a tight complex between the protein and its nuclear binding sites. The S-100 binding to brain nuclei is in most aspects similar to that to synaptosomal membranes. Several lines of evidence indicate, however, that the S-100 binding to nuclei is not due to contamination of these structures with plasma membranes. Isolated liver nuclei do not possess the high-affinity component of S-100 binding.     

Binding protein for Escherichia coli heat-stable enterotoxin II in mouse intestinal membrane

Abstract The protein binding Escherichia coli heat-stable enterotoxin II (STII) was isolated from cell membranes of mouse intestine. The binding of ¹²⁵I-labeled STII to the proteins was inhibited by unlabeled STII, showing that it is specific. Proteins cross-linked with ¹²⁵I-STII were purified by column chromatography on hydroxyapatite and TSK gel. Analyses of the purified protein by SDS-polyacrylamide gel electrophorosis and gel filtration showed that the molecular mass was 25 kDa.     

Compared Binding Properties of 125I-Labeled Analogues of Neurotensin and Neuromedin N in Rat and Mouse Brain

Abstract: Neurotensin and neuromedin N are two structurally related peptides that are synthesized by a common precursor. The purpose of the present work was to characterize neuromedin N receptors in rat and mouse brain and to compare these receptors with those of neurotensin. A radiolabeled analogue of neuromedin N has been prepared by acylation of the N-terminal amino group of the peptide with the ¹²⁵I-labeled Bolton-Hunter reagent. This ¹²⁵I-labeled derivative of neuromedin N bound to newborn mouse brain homogenate with high affinity (K _d = 0.5 n M ). Cross-competition experiments between radiolabeled and unlabeled neurotensin and neuromedin N indicated that each peptide was able to displace completely and specifically the other peptide from its interaction with its receptor. Independently of the radioligand used, the affinity of neurotensin was always better than that of neuromedin N. Quantitative radioautographic studies demonstrated that the ratio of labeling intensities obtained with ¹²⁵I-labeled analogues of neurotensin and neuromedin N remained constant in all the brain areas. Our results do not support the existence of a specific neuromedin N receptor in rat and mouse brain and can be explained by the presence of a common receptor for both peptides.     

Heterogeneity of the S-100 Protein Specific Binding Sites in Synaptosomal Particulate Fractions and Subfractions

The specific interaction of S-100 protein with synaptosomal particulate fractions (SYN) was further investigated with special reference to the number of binding components and their localization in synaptosomal subfractions. Binding studies were conducted on SYN from various CNS regions, on synaptosomal subfractions from the cerebral cortex, and on cerebral cortex SYN under various conditions. The results suggest that S-100 binds to two populations of membrane sites: high -affinity sites, which seem to be confined to neuronal membranes (synaptosomal plasma membranes and synaptic vesicles), and low-affinity sites, which are also detected in other membranes. The data are consistent with the view that the biphasic profile of S-100 binding to SYN does not result from heterogeneity of the S-100 molecule, and that the Ca2+ conformation of the protein is as important as the proper conformation of the binding site for full expression of high-affinity binding.     

Preparation and Binding Properties of Radioiodinated Analogues of Dermorphin and Deltorphin with High Specificity for the μ- and δ-Opioid Receptors

Abstract: The synthesis, purification, chemical characterization, and binding properties of two ¹²⁵I-labeled analogues of dermorphin and deltorphin-I are described. Native deltorphin-I and [Lys⁷]dermorphin sequences were elongated by an aminopentyl chain on their C-terminal amide function and alkylated with the ¹²⁵I-labeled monoiodinated derivative of Bolton-Hunter reagent (BH^*). The resulting radiolabeled peptides, ε-BH^* [Lys⁷]dermorphin 5-aminopentylamide and ω-BH^* deltorphin-I 5-aminopentylamide, have kept most of the original properties of the parent peptides. They bind with high selectivity and specificity to the μ- (dermorphin analogue) or δ- (deltorphin-I analogue) opioid receptors from rat brain or from cells transfected with cDNAs encoding the μ and δ receptors. The autoradiographic distribution of specific binding sites for the ¹²⁵I-labeled dermorphin and deltorphin-I analogues in rat brain is in complete agreement with previously reported localizations of μ- and δ-opioid receptors. The two radiolabeled peptides are the best ligands of μ- and δ-opioid receptors currently available in terms of sensitivity, specificity, and selectivity.     

Transferrin Receptor on Chick Fibroblast Cell Surface and the Binding Affinity in Relevance to the Growth Promoting Activity of Transferrin

We examined the transferrin (Tf) receptor of chick skin fibroblasts using chick ¹²⁵I-Tf. When the cells were incubated with ¹²⁵I-Tf on ice, most of the cell-associated ¹²⁵I-Tf was found on the cell surface; on the other hand, a large part of it was located inside the cells when incubated at 37°C. By equilibrium binding assay, the number of Tf receptors per cell was determined as 6.7 × 10³. Dissociation constant was estimated to be 2.6 × 10⁻⁸ M.
The binding of ¹²⁵I-Tf was competitively inhibited by the addition of chick unlabeled Tf. Weaker inhibition was observed when bovine Tf was used as a competitor. Horse Tf had no effect on the binding of chick Tf. This agrees well qualitatively with chick cell growth-promoting activites of these Tfs.
Removal of Fe from Tf affected the affinity for its receptors. About 5- to 10-fold higher concentrations of chick apo–Tf was needed to achieve the same degree of inhibition of ¹²⁵I-Tf binding as that made by chick Fe-Tf.     

THE SPECIFIC INTERACTION OF S-100 PROTEIN WITH SYNAPTOSOMAL PARTICULATE FRACTIONS. SITE-SITE INTERACTIONS AMONG S-100 BINDING SITES1

Abstract— The Scatchard plot of the specific binding of the brain-specific S-100 protein to synaptosomal particulate fractions (SYN) is curvilinear, concave upwards. This could indicate the existence either of multiple classes of sites with different but fixed affinities, or of site-site interactions of the type defined as negative cooperativity among a single class of sites. To discriminate between these possibilities, the dissociation test described by De Meyts et al. (1976) for demonstrating negative cooperativity among insulin binding sites of human lymphocytes or liver membranes, was applied to the interaction of S-100 with SYN. The results show that the dissociation of the ¹²⁵I-labelled S-100-site complex is faster due to an ‘infinite’(100-fold) dilution of the complex plus an excess of unlabelled S-100 than due to dilution only, the effect of unlabelled S-100 being specific and dose-dependent. ¹²⁵I-IabeIIed S-100 dissociation is time, temperature, and Ca^{2 +}-dependent. The effect of unlabelled S-100 is more evident at a low site occupancy than at a high one, suggesting that at high site occupancies ¹²⁵I-labelled S-100 binding sites could be already negatively cooperating. It can be reasonably excluded that the effect of unlabelled S-100 is due to inhibition of rebinding of the dissociated tracer. Na⁺ and K⁺ stimulate the dissociation even at physiological concentrations. At low pH ¹²⁵I-labelled S-100 dissociates very little, while at high pH dissociation is greatly stimulated. Finally, the protein denaturating reagent urea accelerates the dissociation even at concentrations as low as 1m. These data suggest that negative cooperativity occurs among S-100 binding sites, but do not exclude other possibilities. Together with previously reported findings, they further support the view that S-100 binds to highly specific sites in nervous membranes.     

Methyl 3β-(4-[125I]Iodophenyl)Tropane-2β-Carboxylate In Vitro Binding to Dopamine and Serotonin Transporters Under "Physiological" Conditions

Abstract: Methyl 3β-(4-[¹²⁵I]iodophenyl)tropane-2β-carboxylate ([¹²³I]β-CIT) is a single photon emission computed tomographic radiotracer for in vivo labeling of dopamine (DA) and serotonin (5-HT) transporters. Single photon emission computed tomographic experiments in nonhuman primates showed that [¹²³I]β-CIT in vivo binding to DA transporters had a much slower washout than binding to 5-HT transporters. This observation was not predicted from previously published in vitro studies. These studies, performed at 22°C in nonphysiological buffer, reported similar affinity of [¹²⁵I]β-CIT for DA and 5-HT transporters. We now report [¹²⁵I]β-CIT binding parameters to fresh rat membranes at 22°C and 37°C, in a buffer mimicking the composition of cerebrospinal fluid. At both temperatures, binding to DA transporters was best fit by a twosite model, whereas binding to 5-HT transporters was compatible with one population of sites. At 22°C, [¹²⁵I]β-CIT showed similar affinity to high-affinity DA (0.39 n M ) and 5-HT transporter sites (0.47 n M ). Increasing the incubation temperature from 22°C to 37°C reduced binding to DA transporters by 60%, whereas binding to 5-HT transporters was only marginally affected. In vitro kinetic experiments failed to detect significant differences in on or off rates that could explain the observed in vivo kinetics. These experiments thus failed to explain [¹²³ I]β-CIT in vivo uptake kinetics, suggesting the existence of specific factors affecting the in vivo situation.     

Characterization of 5,7-Dichlorokynurenate-Insensitive d-[3H]Serine Binding to Synaptosomal Fraction Isolated from Rat Brain Tissues

Abstract: To explore target sites for endogenous d -serine that are different from the glycine site of the N -methyl- d -aspartate (NMDA) type glutamate receptor, we have studied the binding of d -[³H]serine to the synaptosomal P2 fraction prepared from the rat brain and peripheral tissues in the presence of an excess concentration (100 µ M ) of the glycine site antagonist 5,7-dichlorokynurenate (DCK). Nonspecific binding was defined in the presence of 1 m M unlabeled d -serine. Association, dissociation, and saturation experiments indicated that d -[³H]serine bound rapidly and reversibly to a single population of recognition sites in the cerebellar P2 fraction in the presence of DCK, with a K _D of 614 n M and a B _max of 2.07 pmol/mg of protein. d -Serine, l -serine, and glycine produced a total inhibition of the specific DCK-insensitive d -[³H]serine binding to the cerebellum with similar K _i values. Strychnine and 7-chlorokynurenate failed to inhibit the binding at 10 µ M . The profiles of displacement of the DCK-insensitive d -[³H]serine binding by various amino acids and glutamate and glycine receptor-related compounds differ from those of any other defined recognition sites. DCK-insensitive d -[³H]serine binding was at high levels in the cerebral cortex and cerebellum but very low in the kidney and liver. The present findings indicate that the DCK-insensitive d -[³H]serine binding site could be a novel candidate for a target for endogenous d -serine in mammalian brains.     

Human Y-79 Retinoblastoma Cells Exhibit Specific Corticotropin-Releasing Hormone Binding Sites

Abstract: In this study we have identified specific binding sites for corticotropin-releasing hormone (CRH) in human Y-79 retinoblastoma cell membranes by using ¹²⁵I-Tyrovine CRH (¹²⁵I-oCRH) as radioligand. Binding at 19°C was rapid with steady state being reached within 20 min, reversible and linear with membrane protein concentration. The ¹²⁵I-oCRH binding was enhanced by Mg²⁺ and inhibited by the GTP analogue guanosine 5'- O -(3'-thiotriphosphate). Y-79 cell membranes exhibited two populations of binding sites, a high-affinity site with an apparent dissociation constant ( K _D) of 1 n M and a low-affinity site with an apparent K _D of 500 n M . ¹²⁵I-oCRH binding was completely antagonized by human/rat CRH, [Met(O)²¹]oCRH, α-helical CRH_9–41, urotensin I, and sauvagine with a rank order of potency similar to that displayed by CRH receptors of other tissues. These data describe for the first time the presence of specific CRH-binding sites in retinal cells. The Y-79 cell line may therefore constitute a valuable model in which to study CRH action on retinal cells.     

The relationship between cytotoxic effect and binding to mammalian cultured cells of Clostridium perfringens enterotoxin

Abstract The relationship between the cytotoxic effect and binding to different cell lines of Clostridium perfringens enterotoxin was investigated. The enterotoxin released ⁵¹Cr from Vero and MDCK cells labeled with Na₂-⁵¹CrO₄. The effect varied depending upon the dose of enterotoxin and the duration and temperature of the interaction. The enterotoxin gave no effect on FL, KB, or L-929 cells. [¹²⁵I]Enterotoxin bound specifically to Vero and MDCK cells via a binding site of distinct nature, but not to FL, KB, or L-929 cells. The number of the binding sites located on one MDCK cell (1.98 × 10⁶ sites/cell) was three times that on one Vero cell (5.64 × 10⁵ sites/cell), although the binding affinity of MDCK cell ( K _a/ 3.76 × 10⁷ M⁻¹) was 0.1 that of Vero cells ( K _a/ 3.23 × 10⁸ M⁻¹). Binding of the enterotoxin to susceptible cells was temperature-independent.     

[1251]2α-BUNGAROTOXIN AND [3H]QUINUCLIDINYLBENZILATE BINDING IN CENTRAL NERVOUS SYSTEMS OF DIFFERENT SPECIES

Abstract— [¹²⁵I]Diiodo α-bungarotoxin ([¹²⁵I]₂BuTx) and [³H]quinuclidinylbenzilate ([³H]QNB) binding sites were measured in post-nuclear membrane fractions prepared from whole brains or brain regions of several species. Species studied included Drosophila melanogaster (fruit fly), Torpedo californiea (electric ray), Carassius auratus (goldfish), Ram pipiens (grass frog), Kana cutesheiana (bullfrog), Rattus norvegicus (rat, Sprague-Dawley), Mus muscalus (mouse, Swiss random, C58/J, LG/J), Oryctolagus cuniculus (rabbit, New Zealand Whitc), and Bos (cow). Acetyl-CoA: choline O -acetyltransferase (EC 2.3.1.6) levels were also determined in the post nuclear supernatants and correlated with the number of binding sites.
All species and regions except Drosophila had 16–150 fold more [³H]QNB binding sites than [¹²⁵I]₂BuTx binding sites. Brain regions with the highest levels of [¹²⁵I]₂BuTx binding were Drosophila heads (300 fmol/mg), goldfish optic tectum (80fmol/mg), and rat and mouse hippocampus (3040 fmol/mg). The highest levels of [³H]QNB binding were seen in rat and mouse caudate (1.3–1.6 pmol/mg). Lowest levels of [³H]QNB and [¹²⁵I]₂BuTx binding were seen in cerebellum. The utility of [¹²⁵I]₂BuTx and [³H]QNB binding as quantitative measures of nicotinic and muscarinic acetylcholine receptors in CNS is discussed.     

CHOLINERGIC SITES IN SKELETAL MUSCLE: INTERACTION WITH CONCANAVALIN A

Abstract– The interaction of normal and denervated skeletal muscle cholinergic sites with the lectin concanavalin A and concanavalin A-Sepharose are detailed. Concanavalin A blocks the binding of ¹²⁵I-α-bungarotoxin to both the high and low affinity sets of cholinergic sites described previously. The characteristics of the block of ¹²⁵I-α-bungarotoxin binding to the high affinity set (acetylcholine receptor) is not competitive. The data suggest that the concanavalin binds multivalently to the macro-molecular complex containing the ACh receptor site and sterically prevents the α-bungarotoxin binding. The interaction of both sets of cholinergic sites with concanavalin A-Sepharose was also studied. The macromolecule(s) containing both the high and low affinity sets of sites bind to the concanavalin A-Sepharose. The data indicate a multivalent association with the affinity resin. Following the affinity procedure, a partial purification in both sets of sites is effected. The equilibrium binding of ¹²⁵I-diiodo-α-bungarotoxin to the preparations from the affinity procedure (both normal and denervated muscle) was examined. The K_D of the α-bungarotoxin binding to the high affinity sets of sites (acetylcholine receptor) in both normal and denervated preparations changes from ∼10⁻⁹mol/l to ∼ 10⁻¹⁰ mol/l following purification. No change in the K_D of the α-bungarotoxin binding to the low affinity set of sites was observed following purification. The ¹²⁵l-α-bungarotoxin binding to the partially purified acetylcholine receptor was blocked by unlabelled α-bungarotoxin, concanavalin A, d-tubocurarine and carbamylcholine.     

Evidence for the Existence of Imidazoline-Specific Binding Sites in Synaptosomal Plasma Membranes of the Bovine Brainstem

Abstract: Nonadrenergic imidazoline-specific binding sites were characterized pharmacologically in crude cerebral membrane preparations, but little is known about their subcellular localization in neurons. As in the brain-stem these sites are involved in cardiovascular regulation and peripherally imidazolines modulate neurotransmitter release, we tried to determine a possible (pre)synaptic localization in brainstem. We found a specific enrichment in (entire) synaptosome, purified synaptosomal plasma membrane (37 fmol/mg), and mitochondrial (83 fmol/mg) fractions as compared with other membrane fractions (3–8 fmol/mg). Synaptosomes appeared to be free of postsynaptic structures, and purified synaptosomal plasma membranes were devoid of mitochondrial material, as determined by electron microscopy and by comparison with the distribution of marker enzymes such as monoamine oxidase. These results show for the first time that these extramitochondrial imidazoline-specific sites are neuronal and are located on presynaptic terminals. We found high affinities for unlabeled p -iodoclonidine (subnanomolar), clonidine (0.2 n M ), and efaroxan (11 n M ), but idazoxan did not compete significantly for the p -[¹²⁵I]iodoclonidine binding in these membranes. Therefore, these sites can be classified as I₁ imidazoline receptors. In summary, we describe for the first time that high-affinity I₁ receptors of the bovine brainstem are located on (pre)synaptic membranes.     

Taurine Interactions with Chick Retinal Membranes

Abstract: Binding of [³H]taurine to whole retinal membranes and to membranes obtained from retinal subcellular fractions was studied. [³H]Taurine bound to chick retinal membranes with high affinity and specificity. Two types of [³H]taurine binding associated to retinal membranes were observed, one with a K_D= 0.68 μM and the other one with a K_D,= 9.32 μM. Both types of binding were highly Na⁻-dependent. The Na⁺-dependent taurine binding was antagonized by strychnine. Bound [³H]taurine was effectively displaced by β-alanine but not by GABA or glycine. Taurine binding was preferentially localized in membranes obtained from the crude synaptosomal fraction, although it is also present in substantial amounts in all retinal membranes. A Na⁺-independent [³H]taurine binding exhibiting properties which might represent interaction with postsynaptic receptor sites could not be demonstrated in the chick retina.     

Characterisation and Molecular Identification of Adrenomedullin Binding Sites in the Rat Spinal Cord: A Comparison with Calcitonin Gene-Related Peptide Receptors

Abstract: Calcitonin gene-related peptide (CGRP) and its receptors are found in mammalian spinal cord. We show, for the first time, binding sites for the novel related peptide adrenomedullin in rat spinal cord microsomes. ¹²⁵I-Adrenomedullin binding showed high affinity ( K _D = 0.45 ± 0.06 n M ) and sites were abundant ( B _max = 723 ± 71 fmol/mg of protein). CGRP, amylin, and calcitonin did not compete at these sites ( K _i > 10 µ M ). High-affinity CGRP binding sites ( K _D = 0.18 ± 0.01 n M ) were much less numerous ( B _max = 17.7 ± 2.4 fmol/mg of protein) and showed competition by unlabeled adrenomedullin ( K _i = 34.6 ± 2.4 n M ). Chemical cross-linking revealed a major band for ¹²⁵I-adrenomedullin of M_r = 84,400 ± 1,200 and a minor band of M_r = 122,000 ± 8,700. ¹²⁵I-CGRP cross-linking showed bands of lower molecular weight (M_r = 74,500 ± 5,000 and 61,000 ± 2,200). Enzymic deglycosylation of the adrenomedullin binding site showed a considerable carbohydrate content. Neither adrenomedullin nor CGRP was able to increase cyclic AMP in spinal cord. Adrenomedullin mRNA was present in spinal cord, at one-third of its level in lung, and adrenomedullin immunoreactivity was present, at a low concentration (40 fmol/g of tissue). Thus, the presence of abundant binding sites and adrenomedullin mRNA and immunoreactivity anticipate an as yet undefined function for this peptide in spinal cord.     

Effects of Chronic Nicotine Treatment on the Accumulation of [3H]Tetraphenylphosphonium by Cerebral Cortical Synaptosomes

Abstract: Chronic exposure of rats to nicotine increases the number of [³H]nicotine binding sites in the brain; however, it is not clear whether nicotinic cholinergic receptor function is altered as well. In this study, we have used [³H]tetraphenylphosphonium as a probe of synaptosomal membrane potential to investigate whether exposure to nicotine in vivo alters the ability of cerebral cortical synaptosomes to maintain a potential difference and to depolarize in response to in vitro nicotine. Treatment of rats for 14 days with 0.475 mg of nicotine base/day via subcutaneously implanted minipumps resulted in a decrease in the synaptosomal accumulation of [³H]tetraphenylphosphonium in physiological buffer, corresponding to a decrease in estimated membrane potential from –55 mV to –50 mV. The onset of the decrease in membrane potential occurred after 7 days of in vivo nicotine treatment and was significantly correlated with an increase in [³H]nicotine binding to cerebral cortical synaptosomal (P₂) membranes. Nicotine, at in vitro concentrations of 3–1,000 μ M , decreased [³H]tetraphenylphosphonium accumulation in cerebral cortical synaptosomes from control animals. When compared to accumulation in buffer alone, in vitro nicotine and other nicotinic agonists did not significantly decrease [³H]tetraphenylphosphonium accumulation in cerebral cortical synaptosomes prepared from rats treated with nicotine in vivo. These studies provide evidence that chronic treatment with nicotine results in an average lower membrane potential in cerebral cortical synaptosomes and in functional down-regulation of the depolarization response to nicotinic cholinergic receptor stimulation.     

Identification and isolation of the binding substance for Clostridium perfringens enterotoxin on Vero cells

Abstract To identify the binding substance for Clostridium perfringens enterotoxin (CPE), the CPE-binding substances metabolically labelled with [³H]leucine on CPE-susceptible (Vero) and resistant (L-929) cells were analyzed by solubilization, immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. The CPE-binding substance was found on Vero cells, but not on L-929 cells. The molecular weight of the CPE-binding substance was found to be 60 000 on SDS-PAGE. The CPE-binding substances were isolated from Vero cells and Balb/c mouse intestinal brush border membranes by affinity chromatography on CPE-coupled Sepharose 4B. They were homogeneous substances with molecular weights of 60 000 on SDS-PAGE and inhibited to the same extent the binding reaction of ¹²⁵I-labeled CPE with Vero cells. These results suggests that the CPE-binding substances are the receptors of CPE on these cells.