A rapid method for the preparation of (125I)alpha-bungarotoxin

Electrofocusing followed by chromatography on Sephadex G-50 proved to be a fast method for the purification of the neurotoxin, α-bungarotoxin (α-BGT) from the venom of the krait (Bungarus multicinctus). The purity of the isolated α-BGT was checked by electrofocusing and ion-exchange chromatography. Blockade of the binding of [³H]ACh to the acetylcholine receptor in a membrane preparation of Torpedo electroplax was a convenient biochemical assay to identify the α-neurotoxin polypeptide. Iodination, accomplished by lactoperoxidase attached to Sepharose 4B, was simple and rapid, with a 60% recovery of [¹²⁵I]α-BGT. Biological activity of the α-BGT was determined by its toxicity to mice as well as by autoradiography of [¹²⁵I]α-BGT which proved its localization in the endplates of the mouse diaphragms.     

Evidence for heterogeneous distribution of α1, α2- and β-adrenergic binding sites on rat-liver cell surface

Fractionation of preparations of rat-liver membranes on linear sucrose gradients revealed different profiles for the binding of α₁-, α₂- and β-adrenergic radioligands. The peaks of binding activities of [³H]prazosin and [³H]epinephrine were clearly separated from those of [³H]yohimbine and [¹²⁵I]iodocyanopindolol which appeared at lower sucrose densities. Enzyme marker activities in the sucrose subfractions indicated the presence of plasma membranes in all of the subfractions. Furthermore, the binding peaks of the various adrenergic radioligands cannot be correlated with the presence of membranes derived from microsomes, lysosomes or Golgi apparatus. Pretreatment of rat livers with concanavalin A, in order to prevent the fragmentation of the plasma membranes during isolation, resulted in the shift of the binding of [³H]yohimbine and [¹²⁵I]iodocyanopindolol to sucrose-gradient subfractions of higher densities, clearly separate from fractions containing microsomes and Golgi apparatus. There was no distinct separation of the binding peaks of prazosin, yohimbine, and cyanopindolol in sucrose-gradient subfractions from concanavalin A-pretreated livers. These results are consistent with the hypothesis that α₁-, α₂-, and β-adrenergic binding sites are associated with plasma membranes, and are heterogeneously distributed on the rat-liver cell surface.     

The use of iodinated lectins for determining the degree of deglycosylation of high-mannose glycoproteins by endo-beta-N-acetylglucosaminidase H

A method which demonstrates that the removal of polymannosyl chains from glycoproteins by endo-β-N-acetylglucosaminidase H can be monitored reliably using only submicrogram quantities of glycoprotein is described. Glycoproteins and their endoglycosidase-treated forms are subjected to electrophoresis on SDS-polyacrylamide gels, which are then overlaid with [¹²⁵I]concanavalin A or [¹²⁵I]wheat germ agglutinin. The degree to which these lectins bind is measured by autoradiography. The complete loss of [¹²⁵I]concanavalin A binding by glycoproteins such as deoxyribonuclease I, ovalbumin, carboxypeptidase Y, and invertase is associated with the removal of their oligosaccharide chains. Invertase, unlike the above mannose-containing glycoproteins, acquires the capacity to bind [¹²⁵I]wheat germ agglutinin only upon partial or complete deglycosylation, a finding substantiated by wheat germ agglutinin-Sepharose column chromatography. In addition to providing a procedure for monitoring the enzymatic deglycosylation of mannose-containing glycoproteins, the lectin-gel binding technique is shown to provide an estimate of the mannose content of neutral glycoproteins at levels which cannot be detected by conventional methods. In some cases, this method may be useful in distinguishing between N- and O-glycosidic linkages where the oligosaccharide is predominantly mannosyl.     

Effects of Lectins on the Hemolysis of Rabbit Erythrocytes by Staphylococcal Alpha Toxin

When concanavalin A (1 μg/ml) or wheat germ agglutinin (2 μg/ml) was preincubated with a suspension of 2% rabbit erythrocytes for 5 min at 20 C, the binding of [¹²⁵I]-labeled staphylococcal alpha toxin to these erythrocytes was greatly inhibited and the hemolytic action of alpha toxin was decreased. The inhibitory effect of concanavalin A on hemolysis by alpha toxin was completely reversed in the presence of 0.1 M α-methyl-D -glucoside or α-methyl-D -mannoside. Phytohemagglutinin-P from Phaseolus vulgaris and soybean agglutinin inhibited hemolysis by the toxin at concentrations exceeding 20 μg/ml. The effect of concanavalin A on alpha-toxin hemolysis was studied further to ascertain the nature of the inhibition. Double reciprocal plots were made of hemolysis against alpha toxin concentrations, and the data suggested that inhibition of the initial rate of the hemolysis by concanavalin A is competitive in nature. This was probably due to an interaction with the alpha toxin binding sites on the cell membrane surface.     

Interactions of Phospholipid Vesicles with Murine Lymphocytes

The effect of unilamellar lipid vesicles composed of dioleoyl lecithin (DOL), egg yolk lecithin (EYL), 1:1 EYL:cholesterol (Chol), dipalmitoyl lecithin (DPL), and dimyristoyl lecithin (DML) on the mitogenic response in mouse lymphocytes was tested. Cortisone-resistant thymocytes were briefly treated with lipid vesicles and subsequently stimulated with concanavalin A (con A). All of the lipid vesicles induced an enhanced mitogenic response on day 3 as tested by [³H]TdR incorporation and by counting total cells. The order of enhanced [³H]TdR incorporation (?5.3 times the control) was DML>DPL>1:1 EYL:Chol>EYL?DOL> untreated control cells. These increases were paralleled by increased numbers of total cells. The response of spleen cells to a B-cell mitogen, bacterial lipopolysaccharide, was similarly enhanced by vesicle pretreatments in the same order. Vesicle treatments alone were not mitogenic.

Pretreatment of cells with lipid vesicles modified lectin binding: DML and DPL increased the binding of [¹²⁵I]con A by three to four times the control, whereas 1:1 EYL:Chol, EYL, or DOL had little or no effect. The binding of [125I]phytohemagglutinin-P (PHA-P) to vesicle-treated cells was indistinguishable from untreated cells. The lectin (con A; PHA-P)-induced agglutination of vesicle-treated cells was also modified by different lipid vesicles in the same order as the mitogenic response.

Based on the results presented in the accompanying report [6], we find that the cell surface adsorption properties of the applied lipid vesicles correlate with their ability to enhance the mitogenic response, and that they modify agglutinability and lectin binding. These results are further discussed in terms of the possible alteration of membrane properties and subsequent cellular activity.     

A study on concanavalin a binding to human erythrocytes

Interactions of concanavalin A with human erythrocytes were studied using ¹²⁵I-labelled concanavalin A and a centrifugal technique with dibutyl phthalate which permitted complete separation of bound and free concanavalin A. Binding of ¹²⁵I-labelled concanavalin A to human erythrocytes was dependent on cell concentration, pH and temperature. Specificity of binding was confirmed by inhibition and dissociation studies with sugars and native concanavalin A. Positive cooperative binding of concanavalin A to human erythrocytes was observed at low concanavalin A concentrations (less than 1 μ/ml) in both buffers studied. Positive cooperativity at higher concanavalin A concentrations (more than 100 μ/ml) was seen in Tris-Hepes buffer but not in phosphate-buffered saline. Consistent with this cooperative effect was the observation that although dissociation of ¹²⁵I-labelled concanavalin A from the erythrocytes was complete in the presence of 1 mg/ml of the native lectin, release was inhibited by low concentrations (1 μ/ml). A comparison of concanavalin A binding with hemagglutination studies suggest that the amount of concanavalin A bound determines the rate of erythrocyte agglutination and the size of the aggregates formed.     

Photoaffinity labeling the agonist binding domain of α4β4 and α4β2 neuronal nicotinic acetylcholine receptors with [I]epibatidine and 5[I]A-85380

The development of nicotinic acetylcholine receptor (nAChR) agonists, particularly those that discriminate between neuronal nAChR subtypes, holds promise as potential therapeutic agents for many neurological diseases and disorders. To this end, we photoaffinity labeled human α4β2 and rat α4β4 nAChRs affinity-purified from stably transfected HEK-293 cells, with the agonists [¹²⁵I]epibatidine and 5[¹²⁵I]A-85380. Our results show that both agonists photoincorporated into the β4 subunit with little or no labeling of the β2 and α4 subunits respectively. [¹²⁵I]epibatidine labeling in the β4 subunit was mapped to two overlapping proteolytic fragments that begin at β4V102 and contain Loop E (β4I109-P120) of the agonist binding site. We were unable to identify labeled amino acid(s) in Loop E by protein sequencing, but we were able to demonstrate that β4Q117 in Loop E is the principal site of [¹²⁵I]epibatidine labeling. This was accomplished by substituting residues in the β2 subunit with the β4 homologs and finding [¹²⁵I]epibatidine labeling in β4 and β2F119Q subunits with little, if any, labeling in α4, β2, or β2S113R subunits. Finally, functional studies established that the β2F119/β4Q117 position is an important determinant of the receptor subtype-selectivity of the agonist 5I-A-85380, affecting both binding affinity and channel activation.     

Relationship of prolactin receptors to concanavalin A binding

Concanavalin A, which binds to specific carbohydrate determinants on the cell surface, was used to investigate the binding of prolactin to its receptors in liver membranes from female rats. The binding of ¹²⁵I-labeled ovine prolactin to receptors was sharply inhibited by concanavalin A. This effect was reversed by the competitive sugar α-methyl-D-mannopyranoside and thus required the presence of specifically bound lectin. Concentrations of concanavalin A of up to 50 μg/ml caused a progressive decrease in the apparent affinity of the prolactin receptor for hormone. When higher concentrations were used, the number of available binding sites decreased. Concanavalin A-resistant receptors, about 30% of the total, had the same dissociation constant (K_d) as the controls. The binding of ¹²⁵I-labeled concanavalin A in the same membrane preparations showed the presence of two distinct types of concanavalin A binding. At low concentrations, the lectin bound with high affinity (K_d ≈ 6.6 · 10^?8 M). At high lectin concentrations, low affinity (K_d ≈ 6.7 · 10^?5 M) binding predominated. Since high affinity concanavalin A binding was saturated at 50 μg/ml, this class of binding most likely alters the affinity of the prolactin receptor for hormone; low affinity concanavalin A binding may mask prolactin receptors, making them inaccessible to the hormone.Binding sites for concanavalin A and prolactin appear to be independent but closely related since (i) concanavalin A did not displace bound prolactin from its receptor, and (ii) detergent-solubilized ¹²⁵I-labeled prolactin-receptor complexes bound to concanavalin A-Sepharose and were eluted by α-methyl-D-mannopyranoside.     

The effects of tunicamycin and 20-hydroxyecdysone on the cell surface properties of two insect cell lines

The radiolabeled lectins, concanavalin A* and wheat germ agglutinin, were used to study surface properties of two insect cell lines. We also looked at the effects of tunicamycin and 20-hydroxyecdysone on the binding of these lectins to one of the cell lines. Both UMBGE-2 and CH-MRRL cells bound both lectins, specifically. The CH-MRRL cells showed an overall higher binding for the lectins than the UMBGE-2 cells. This difference may account for some of the striking morphological difference seen between these cells. Tunicamycin and 20-hydroxyecdysone decreased the binding of both [¹²⁵I]-Con A and [¹²⁵I]-WGA to CH-MRRL cells. These results suggest that cell surface glycoproteins play a role in the modification of cellular morphology and in other hormone-mediated physiological functions.     

Comparative lectin-binding and agglutination properties of the strain-specific,TA3-St,and the non-strain-specific,TA3-Ha,murine mammary carcinoma ascites sublines. Further studies of receptors in epiglycanin

The complex carbohydrates at the cell surfaces of two TA3, murine mammary carcinoma ascites sublines (the strain-specific, TA3-St subline and the nonstrain-specific, TA3-Ha line) were compared by binding studies with ¹²⁵I-labelled concanavalin A (con A), Ricinis communis agglutinin (RCA), and eel-serum agglutinin (ESA). The TA3-Ha cell bound equal amounts of con A, 1.5-fold more RCA, and 4-fold more ESA than the TA3-St cell. Binding-inhibition studies by these lectins and two others [wheat-germ agglutinin (WGA) and potato lectin (STA)] suggest complementary binding-sites between con A and both RCA and ESA. Quantitative agglutination studies with the five lectins, and inhibition determinations by both neuraminidase-treated and untreated epiglycanin revealed that TA3-St, but not TA3-Ha, cells were agglutinated by con A, and that epiglycanin inhibited this agglutination, as well as the agglutination of rabbit erythrocytes by con A. The presence of a con A receptor on epiglycanin was also suggested by the binding of epiglycanin to con A-Sepharose, and its specific elution with methyl α-d-manno-pyranoside. TA3-St cells were agglutinated at a 10-15-fold lower concentration of either STA or RCA than TA3-Ha cells, but both cells were agglutinated by the same concentration of WGA and ESA. Inhibition by epiglycanin of agglutination of TA3-St cells by either STA or ESA occurred at a concentration lower than that of TA3-Ha cells, but epiglycanin inhibited RCA agglutination of TA3-Ha cells at a concentration     

Studies on phytohemagglutinins: XXII. Isolation and characterization of a lymphocyte receptor for concanavalin A

A receptor glycopeptide for concanavalin A was isolated from calf thymocytes by a method originally devised for the isolation of a lectin receptor from human erythrocytes (Kubánek, J., Entlicher, G.; and Kocourek, J. [1973] Biochim, Biophys. Acta 304, 93–102). The method consisted of pronase digestion of the lipid-depleted thymocyte membrane material followed by ethanol fractionation, separation on Sephadex and preparative paper electrophoresis. The isolated glycopeptide contains 10.4% of neutral sugar. The molar ratios of the sugar components mannose, galactose, glucosamine, glucose, fucose and sialic acid are 3 : 2 : 2 : 1 : 1 : 1. The minimum molecular weight calculated from the sugar composition is about 12 000.Concanavalin A receptor activity of the glycopeptide was demonstrated in three different ways: (i) Reduction of the ¹²⁵I-labeled concanavalin A binding to thymocytes. (ii) Prevention of concanavalin A induced agglutination of calf thymocytes. (iii) Inhibition of concanavalin A stimulated DNA synthesis in calf and rabbit thymocytes and rabbit lymph node lymphocytes cultivated in vitro.The isolated glycopeptide seems to be involved in the interaction of lymphocytes with concanavalin A and the subsequent stimulation.     

Antibodies to nicotinic acetylcholine receptors used to probe the structural and functional relationships between brain α-bungarotoxin binding sites and nicotinic receptors

Antibodies against peripheral nicotinic acetylcholine receptors (nAChR) were used to determine the proportion of brain α-bungarotoxin binding sites that are immunologically related to the peripheral nAChR. The α-bungarotoxin binding component partially purified from rat brain was labelled with [¹²⁵I]α-bungarotoxin and reacted with increasing concentrations of rabbit anti(nAChR) antisera. At least 75% of the brain protein could be immunoprecipitated by rabbit anti(rat muscle junctional nAChR) antiserum (M) whereas an antiserum against Torpedo nAChR (J) was without effect and clearly failed to cross-react with the brain component. Both antisera precipitated 100% of [¹²⁵I]α-bungarotoxin-labelled nAChR from Torpedo marmorata. The lower precipitation of the brain protein was not a consequence of [¹²⁵I]α-bungarotoxin dissociating during the precipitation. We conclude that the majority of α-bungarotoxin binding sites in brain are clearly recognised by the crossreacting antiserum.Release of [³H]dopamine from striatal synaptosomes could be elicited by nicotine in a dose-dependent manner and the response was prevented by the ganglionic blocker mecamylamine, although antagonism by α-bungarotoxin was less clearcut. Preincubation of the synaptosomes with antiserum M resulted in a statistically significant decrease in the [³H]dopamine response to nicotine at all agonist concentrations tested. Antiserum J, however, had no consistent effect on the response. Thus the actions of the antisera parallel their ability to recognise the brain α-bungarotoxin binding component. We conclude that the cholinergic regulation of dopamine release is in part mediated through a nAChR that is immunologically related to the nAChR of the neuromuscular junction and to the α-bungarotoxin binding component that can be isolated from rat brain.     

Synthesis and evaluation of novel benzothiazole derivatives based on the bithiophene structure as potential radiotracers for β-amyloid plaques in Alzheimer’s disease

In this study, six novel benzothiazole derivatives based on the bithiophene structure were developed as potential β-amyloid probes. In vitro binding studies using Aβ aggregates showed that all of them demonstrated high binding affinities with K_i values ranged from 0.11 to 4.64 nM. In vitro fluorescent staining results showed that these compounds can intensely stained Aβ plaques within brain sections of APP/PS1 transgenic mice, animal model for AD. Two radioiodinated compounds [¹²⁵I]-2-(5′-iodo-2,2′-bithiophen-5-yl)-6-methoxybenzo[d]thiazole [¹²⁵I]10 and [¹²⁵I]-2-(2,2′-bithiophen-5-yl)-6-iodobenzo[d]thiazole [¹²⁵I]13 were successfully prepared through an iododestannylation reaction. Furthermore, in vitro autoradiography of the AD model mice brain sections showed that both [¹²⁵I]10 and [¹²⁵I]13 labeled the Aβ plaques specifically with low background. In vivo biodistribution studies in normal mice indicated that [¹²⁵I]13 exhibited high brain uptake (3.42% ID/g at 2 min) and rapid clearance from the brain (0.53% ID/g at 60 min), while [¹²⁵I]10 showed lower brain uptake (0.87% ID/g at 2 min). In conclusion, these preliminary results of this study suggest that the novel radioiodinated benzothiazole derivative [¹²⁵I]13 may be a candidate as an in vivo imaging agent for detecting β-amyloid plaques in the brain of AD patients.     

Synthesis and characterization of radioiodinated 3-phenethyl-2-indolinone derivatives for SPECT imaging of survivin in tumors

Survivin, overexpressed in most cancers, is associated with poor prognosis and resistance to radiation therapy and chemotherapy. Herein, we report the synthesis of three 3-phenethyl-2-indolinone derivatives and their application as in vivo imaging agents for survivin. Of these, 3-(2-(benzo[d][1,3]dioxol-5-yl)-2-oxoethyl)-3-hydroxy-5- iodoindolin-2-one (IPI-1) showed the highest binding affinity (K_d?=?68.3?nM) to recombinant human survivin, as determined by quartz crystal microbalance (QCM). In vitro studies demonstrated that the [¹²⁵I]IPI-1 binding in survivin-positive MDA-MB-231 cells was significantly higher than that in survivin-negative MCF-10A cells. In addition, uptake of [¹²⁵I]IPI-1 by MDA-MB-231 cells decreased in a dose-dependent manner in the presence of the high-affinity survivin ligand S12; this is indicative of specific binding of [¹²⁵I]IPI-1 to cellular survivin protein in vitro. Biodistribution studies in MDA-MB-231 tumor-bearing mice demonstrated the moderate uptake of [¹²⁵I]IPI-1 in the tumor tissue (1.37%?ID/g) at 30?min that decreased to 0.32%?ID/g at 180?min. Co-injection of S12 (2.5?mg/kg) slightly reduced tumor uptake and the tumor/muscle ratio of [¹²⁵I]IPI-1. Although further structural modifications are necessary to improve pharmacokinetic properties, our results indicate that PI derivatives may be useful as tumor-imaging probes targeting survivin.     

[125I]LSD binding to serotonin and dopamine receptors in bovine caudate membranes

[¹²⁵I]LSD (labeled at the 2 position) has been introduced as the first ¹²⁵I-labeled ligand for serotonin 5-HT₂ (S2) receptors. In the present study we examined the binding of [¹²⁵I]LSD and its non-radioactive homologue, 2I-LSD, to bovine caudate homogenates. The binding of [¹²⁵I]LSD is saturable, reversible, stereospecific and is destroyed by boiling the membranes. The specific to total binding ratio in this tissue is 75–80% and Scatchard plots of the binding data reveal K_d = 1.1 nM, B_max = 9.6 fmol/mg wet weight tissue. The association and dissociation rate constants are highly temperature dependent. At 0°C the net dissociation is less than 5% after 1 h and the association rate is proportionately slow. IC₅₀ values for a variety of compounds show a clear 5-HT₂ (S2) serotonergic pattern at this [¹²⁵I]LSD site. Blockage of this primary 5-HT₂ (S2) caudate binding site by 0.3 μM mianserin reveals the presence of a weaker [¹²⁵I]LSD binding site with a K_d = 9.1 nM, B_max = 7.6 fmol/mg tissue. This secondary site is a D3 dopaminergic receptor site, as shown by the relative abilities of various displacers to inhibit this binding. Binding studies with nonradioactive 2I-LSD reveal a clear preference for D2 over D3 dopamine receptor sites. [¹²⁵I]LSD is a sensitive and selective label for 5-HT₂ (S2) serotonin receptor sites in both rat frontal cortex and bovine caudate membranes. Blockage of the primary bovine caudate [¹²⁵I]LSD binding site with mianserin allows the high sensitivity of [¹²⁵I]LSD to be applied to D2 dopamine receptor studies as well.     

Interaction between Alzheimer's disease βA4 precursor protein (APP) and the extracellular matrix: Evidence for the participation of heparan sulfate proteoglycans

The interaction between the Alzheimer amyloid precursor protein (APP) and an intact extracellular matrix (ECM), matrigel, obtained from Engelbreth-Holm-Swarm tumors was evaluated. Based on quantitative analyses of the binding data obtained from solid phase binding assays, two binding sites on the ECM were identified for [¹²⁵I]-APP (with apparent Kd₁ of 1.0 × 10 ⁻¹¹ M and Kd₂ of 1.6 × 10 ⁻⁹ M respectively). Over 70% of [¹²⁵I]-APP was displaced by heparin and N-desulfated heparin but not by chondroitin sulfate. Pretreatment of matrigel with heparitinase decreased the binding of [¹²⁵I]-APP by 80%. β-amyloid peptides (residues 1–40, 1–28, and 1–16) containing a heparin binding domain also displaced 80% of bound [¹²⁵I]-APP, which was totally displaced by intact APP. The binding of [¹²⁵I]-APP to matrigel increased by 210% with a decrease in the pH. These observations suggest that [¹²⁵I]-APP interacts mainly with heparan sulfate proteoglycan present in the ECM. The binding of [¹²⁵I]-APP to individual ECM components was also analyzed. [¹²⁵I]-APP was found to bind laminin and collagen type IV but not fibronectin. However, when these ECM constituents were combined, the extent of APP-binding decreased significantly, to levels comparable to those obtained with intact matrigel, suggesting that multiple interactions may occur between ECM constituents and [¹²⁵I]-APP. The results are discussed in terms of APP function and amyloidogenesis. J. Cell. Biochem. 65:145–158. © 1997 Wiley-Liss, Inc.     

Detection of nicotinic cholinergic transmission in malapteruruselectricus electrop lax

Biochemical and electrophysiological studies were conducted on the electric organ of the electric fish of the Nile, $\text{Malapterurus}$$\text{electricus}$, in order to determine if transmission was chemically mediated. There was no binding of [³H] acetylcholine, [³H] quinuclidinyl benzilate or [³H]-perhydrohistrionicotoxin; but low acetylcholinesterase activity was observed, as was binding of [¹²⁵I] α-bungarotoxin. The latter binding was detectable at 0.85 ± 0.07 pmol/g tissue, and was totally inhibited by 1 μM α-bungarotoxin or 100 μM d-tubocurarine. A tetrodotoxin-sensitive action potential was measured which was Na⁺- dependent. Depolarization (30–40 mV) was caused by carbamylcholine, and this was blocked by d-tubocurarine or α-bungarotoxin. The data suggest that this electric organ which may be a rich source for electrically excitable channels, is innervated by nicotonic cholinergic motoneurons, but the concentrations of acetylcholine receptors and acetylcholinesterase are very low.     

Appearance and distribution of carbohydrate-rich macromolecules on the epithelial surface of the developing rat palatal shelf.

The synthesis and appearance of carbohydrate-rich macromolecules by epithelial cells of the developing secondary palate was examined with concanavalin A (CON A) binding and [³H]glucosamine labeling. The amount of [¹²⁵I]CON A bound to the epithelial surface of the rat palatal shelf in vitro increased from day 15 of gestation to day 16 when initial adhesion to the opposite shelf occurs in vivo. Visualization of CON A binding by electron microscopy using the peroxidase method revealed a dramatic increase in binding between days 15 and 16 of gestation, most apparent on the medial-edge epithelial surface. The incorporation in vivo of [³H]glucosamine during this period into the medial-edge epithelial cells was detected with autoradiography. These results show that a glycoprotein-rich surface material appears on the superficial cells of the medial-edge epithelium prior to adhesion of the apposing shelves.     

Nicotinic cholinergic receptor in brain detected by binding of α-[3H]bungarotoxin

α-[³H]Bungarotoxin was prepared by catalytic reduction of iodinated α-bungarotoxin with tritium gas. Crude mitochondrial fraction from rat cerebral cortex bound 40 · 10^?15 ?60 · 10^?15 moles of α-[³H]bungarotoxin per mg of protein. This binding was reduced by 50% in the presence of approx. 10^?6 M d-tubocurarine or nicotine, 10^?5 M acetylcholine, 10^?4 M carbamylcholine or decamethonium or 10^?3 M atropine. Hexamethonium and eserine were the least effective of the drugs tested. Crude mitochondrial fraction was separated into myelin, nerve endings, and mitochondria. The highest binding of toxin per mg of protein was found in nerve endings, as well as the greatest nhibition of toxin binding of d-tubocurarine. Binding of α-[³H]bungarotoxin to membranes obtained by osmotic shock of the crude mitochondrial fraction indicates that the receptor for the toxin is membrane bound. ¹²⁵I-Labeled α-bungarotoxin, prepared with Na ¹²⁵I and chloramine T, was highly specific for the acetylcholine receptor in diaphragm, however, it was less specific and less reliable than α-[³H]bungarotoxin in brain. We conclude that a nicotinic cholinergic receptor exists in brain, and that α-[³H]bungarotoxin is a suitable probe for this receptor.     

Decrease of Hormone Binding Capacity of Estrogen Receptor by Calcium

Abstract

We have adressed the question as to whether calcium may modify the [³H]estradiol ([³H]E₂) binding properties of the estrogen receptor (ER). A human recombinant full length ER (yER) expressed in yeast was used to limit the potential interference of ER-associated proteins and proteases present in the target tissues.

Ca⁺⁺ (0.1–10 mM) always produced an important loss of [³H]E₂ binding capacity without any effect on the hormone binding affinity of residual receptors. This loss was reflected in a decrease of immunoreactivity for monoclonal antibodies raised against the hormone binding domain. An ER recombinant expressing solely this domain confirmed that the ion operated at this level. Binding of [¹²⁵I]Z-17 α-(2-iodovinyl)-11β-chloromethyl estradiol-17β (an compound with very high selectivity for ER) as well as [¹²⁵I]tamoxifen aziridine were similarly affected. Size-exclusion chromatography failed to reveal the emergence of any ER isoforms of low molecular weight rejecting the hypothesis of a Ca⁺⁺- induced proteolysis. In agreement with this conclusion, EDTA reversed the loss of [³H]E₂ binding capacity. Phosphoamino acids (PY, PT and PS) partly antagonized the effect of Ca⁺⁺ suggesting its interaction with phosphoamino acid residues. Worthy of note, the effect of Ca⁺⁺ appeared more marked when assessed by DCC than HAP assay. The phosphocalcic nature of the HAP matrix may explain this phenomenon which was observed with cytosolic ER from various origins.