Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Affinity Labels”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,135 records · Page 63Linked to original sources
PubMed1990

Characterization of vasoactive intestinal peptide receptors in retina.

Vasoactive intestinal peptide (VIP) is a neuropeptide having a wide range of effects on a large number of tissues. To gain insight into the role VIP plays in retinal function, VIP receptors in bovine retinal membranes were analyzed in competition binding assays and by affinity labeling studies and compared to VIP receptors in rat liver membranes. In both membrane preparations, high affinity VIP binding sites (KD approximately 1 nM) were detected. Secretin and glucagon, each having close structural homology to VIP, were found to have negligible effects on [125I]VIP binding in retina. In contrast, secretin (KD = 70 nM) was modestly effective in inhibiting [125I]VIP binding to rat liver membranes. Affinity labeling analysis revealed a VIP binding site of 59 kDa in both bovine retinal and rat liver membranes. Digestion of affinity-labeled receptor proteins with endoglycosidase F generated final cleavage products of approx. 45 kDa for both receptors. These results indicate that the retina expresses a high affinity, highly selective VIP receptor thereby supporting a specific function for VIP in this tissue.

Affinity Labels↗
PubMed1996

Reversible and irreversible inhibition of sheep liver sorbitol dehydrogenase with Cibacron Blue 3GA and Eriochrome Black T.

Due to the central role of sorbitol dehydrogenase in diabetic cataract, it is important to examine this enzyme's interaction with different inhibitory compounds such as dyes. The aim of the study was to investigate the binding of Cibacron Blue and Eriochrome Black T to the active site in sorbitol dehydrogenase. These dyes' effect on the enzyme was studied by steady state and affinity labelling kinetics. Both dyes were coenzyme competitive inhibitors with KEI values around 0.5 microM. Essentially the same KEI values were obtained using the dyes as protecting ligands against the affinity label D,L-alpha-Bromo-beta-(5-imidazolyl)-propionic acid. Both dyes were also able to inhibit the enzyme irreversibly through an affinity labelling mechanism, with KEI' values for Cibacron Blue and Eriochrome Black T of 2.2 and 3.1 mM, respectively. Dithiothreitol and NADH were competitive protecting ligands against both dyes. The rate of inactivation was fastest for Cibacron Blue at acid pH values, while the opposite was the case with EBT. Both Cibacron Blue and Eriochrome Black T bind to sorbitol dehydrogenase in two different ways. In both cases the complex formed prior to irreversible inhibition is the weakest. The tighter reversible complexes are suggested to share a common epitope in the coenzyme binding region. Both irreversible complexes involve binding close to the zinc ion at the active site and the sugar binding site. Due to different pH dependences it can be concluded that the affinity labelling mechanism is different for the two dyes and in neither case is the inactivation due to removal of the active site zinc ion.

Animals↗
PubMed1993

Localization and functional role of the calmodulin-binding domain of phospholamban in cardiac sarcoplasmic reticulum vesicles.

Limited proteolysis and affinity-labeling techniques have been used to localize the calmodulin-binding domain of phospholamban, the major substrate for both cAMP- and calmodulin-dependent protein kinases in cardiac sarcoplasmic reticulum (SR). SR vesicles, treated with increasing concentrations of trypsin (likely hydrolyzing at Arg-25 in the cytoplasmic region of phospholamban), exhibited a subsequent loss of both cAMP- and calmodulin-dependent phosphorylation, as well as calmodulin affinity-labeling of phospholamban. When SR vesicles were treated with increasing concentrations of chymotrypsin (which likely cleaves at Tyr-6 of phospholamban) there was no effect on the cAMP-dependent phosphorylation of phospholamban. However, similar concentrations of chymotrypsin resulted in a loss of both calmodulin affinity-labeling and calmodulin-dependent phosphorylation of phospholamban (at Thr-17). When SR vesicles were treated with increasing concentrations of Endoproteinase Lys-C (which hydrolyzes phospholamban at Lys-3) both the calmodulin affinity-labeling and the calmodulin-dependent, but not the cAMP-dependent, phosphorylation of phospholamban were inhibited. These data were complemented by 1H-NMR studies on the complex formed by calmodulin and a phospholamban peptide. These data suggest that binding of calmodulin to phospholamban may be an essential intermediate step in the calmodulin-dependent phosphorylation of phospholamban.

Affinity Labels↗
PubMed2000

Involvement of activated caspase-3-like proteases in N-methyl-D-aspartate-induced apoptosis in cerebrocortical neurons.

Excessive activation of glutamate receptors mediates neuronal death in a number of neurodegenerative diseases. The intracellular signaling pathways that mediate this type of neuronal death are only partly understood. Following mild insults via NMDA receptor activation, central neurons undergo apoptosis, but with more fulminant insults, necrosis intervenes. Caspases are important in several forms of apoptosis in vivo and in vitro. Previously, we have demonstrated that caspases are important in excitotoxicity-mediated apoptosis of cerebrocortical neurons. To determine the possible activation of caspase-3 in NMDA-induced neuronal apoptosis, we used an affinity-labeling technique: Biotinylated N-acetyl-Asp-Glu-Val-Asp-aldehyde (DEVD.CHO) preferentially labels conformationally active caspase-3-like proteases, allowing us to visualize affinity-labeled caspases with streptavidin-fluorescein isothiocyanate under confocal microscopy. NMDA-induced apoptosis of cerebrocortical neurons was associated with a time-dependent increase in conformationally active caspase-3-like proteases. The activation of caspases was apparent within 20 min of NMDA stimulation and was localized primarily in the cytosol. However, following incubation of neurons for 18-24 h, conformationally active caspase-3-like proteases were also detectable in nuclei. Double labeling with propidium iodide to detect chromatin condensation indicated that affinity-labeled caspase-3-like proteases were specifically expressed in apoptotic cells. To further confirm this, we used an antibody specific for the conformationally active fragment of caspase-3 and found largely concordant results. Moreover, preincubation with DEVD.CHO prevented NMDA-induced apoptosis. Our results suggest that caspase-3-like proteases play a major role in excitotoxin-induced neuronal apoptosis.

Animals↗
PubMed1984

Analysis of cholecystokinin-binding proteins using endo-beta-N-acetylglucosaminidase F.

We have previously shown that the cholecystokinin (CCK)-binding proteins in rat pancreatic plasma membranes consist of a major Mr 85,000 and minor Mr 55,000 and Mr 130,000 species as revealed by affinity labeling with 125I-CCK-33 using the cross-linker, disuccinimidyl suberate. The glycoprotein nature of these species was investigated using endoglycosidase F (endo F) and neuraminidase treatment and wheat germ agglutinin-agarose chromatography. Treatment of affinity-labeled membranes with endo F resulted in increased electrophoretic mobilities of all three binding proteins, indicating removal of N-linked oligosaccharide side chains. Endo F treatment of each protein in gel slices indicated the following cleavage relationships: Mr 85,000----65,000; Mr 55,000----45,000; Mr 130,000----110,000. Using limiting enzyme conditions to digest each protein contained in excised SDS gel slices, three and four products, respectively, were identified for the Mr 85,000 and 55,000 proteins. Similar treatment of the Mr 130,000 protein revealed only the Mr 110,000 product. These results indicated that the Mr 85,000 protein has at least three, the Mr 55,000 protein has at least four, and the Mr 130,000 protein has at least one, N-linked oligosaccharide side chain(s) on their polypeptide backbone. Neuraminidase treatment of affinity-labeled membranes caused slight increases in the electrophoretic mobilities of all three proteins, indicating the presence of sialic acid residues. Solubilization of affinity-labeled membranes in Nonidet P-40 followed by affinity chromatography on wheat germ agglutinin-agarose revealed that all three CCK-binding proteins specifically interact with this lectin and can be eluted with N-acetyl-D-glucosamine. Analysis of the proteins present in the eluted fractions by silver staining indicated a significant enrichment for proteins having molecular weights corresponding to the major CCK-binding proteins in comparison to the pattern of native membranes. Taken together, these studies provide definitive evidence that the CCK-binding proteins in rat pancreas are (sialo)glycoproteins.

Acetylglucosaminidase↗
PubMed1984

Characterization of nerve growth factor receptor in neural crest tumors using monoclonal antibodies.

The nerve growth factor (NGF) receptor was characterized by using a new series of anti-receptor monoclonal antibodies (MAbs). These MAbs (i) showed significantly greater reactivity with a melanoma cell line expressing higher levels of NGF receptor, (ii) inhibited the binding of 125I-labeled NGF to its receptor, and (iii) immunoprecipitated both metabolically labeled and 125I-labeled NGF affinity-labeled receptor. These experiments defined the receptor as a 75-kDa cell-surface protein. The NGF receptor was visualized by immunoperoxidase staining in tissue sections of human nevi, melanomas, neurofibromas, a pheochromocytoma, and peripheral nerves. Uniform staining of the cytoplasm suggests that, in addition to cell-surface NGF receptors, there is a population of intracellular receptors.

Affinity Labels↗
PubMed2004

Identification of cellular target proteins for signaling cyclic phosphates.

Cyclic glycerophosphates and their deoxy analogs were previously found to induce intracellular tyrosine and threonine phosphorylation in Chinese hamster ovary (CHO) cells. Further studies have indicated that these compounds induce neuronal outgrowth in PC-12 cells, as well as elevation of the state of cellular differentiation in human breast cancer cell lines. The mechanism by which these cyclic phosphates operate is not yet fully delineated. Using an affinity labeling approach we probed for possible cyclic phosphate target proteins in CHO cells. A 170 kDa protein that was labeled by an affinity cyclic phosphate reagent was identified by mass spectrometry as the largest subunit of the eukaryotic initiation factor 3 (eIF3). Using In-Gel kinase assays allowed the detection of a approximately 70 kDa target kinase directly activated by cyclic phosphates. Identification of these proteins may provide a basis for deciphering the mechanisms, by which cyclic phosphates exert their effects.

Affinity Labels↗
PubMed1993

Homodimers of the 60 kDa phosphatidylinositol-anchored transforming growth factor-beta 2 binding proteins in FBHEC and MG-63 cells.

Phophatidylinositol (PI) anchored TGF-beta 2 binding proteins of approximately 60 and 140 kDa were recently identified in MG-63 human osteogenic sarcoma and fetal bovine heart endothelial cells (FBHEC) (Cheifetz, S., Massagué, J. (1991) J. Biol. Chem. 266, 20767-20772). The relationship between these two PI-anchored TGF-beta 2 binding proteins was investigated. Specifically labeled bands of approximately 60 and 110 kDa were observed when 125I-TGF-beta 2 labeled FBHEC and MG-63 cells were separated by SDS-PAGE under non-reducing conditions. Partial proteolysis of the affinity labeled 60 and 110 kDa species yielded similar peptides. The integrity of the 110 kDa species under denaturing conditions is dependent on both disulfide bonds and chemical cross-linking: reduction of the 110 kDa species yielded an affinity labeled species co-electrophoresing with the 60 kDa band; cleavage of chemical cross-links in the 110 kDa complex yielded a labeled 60 kDa component. These results indicate that the 110 kDa affinity labeled species in FBHEC and MG-63 cells is a homodimer. Within this complex the binding protein monomers can be chemically cross-linked to opposite arms of the disulfide-linked TGF-beta 2 homodimer.

Affinity Labels↗
PubMed1993

The developmental pattern of rabbit brain insulin and insulin-like growth factor receptor expression.

To examine the effect of development on rabbit brain insulin and insulin-like growth factor (IGF) receptor expression, we characterized and quantitated receptor mRNAs by Northern blot analysis and affinity-labeled ligand bound receptors by SDS-PAGE and autoradiography. At various stages of development ranging from 23 to 30 day gestational (term approximately 31 days), 1 to 10 day postnatal ages and the adult, no change in the whole brain insulin receptor mRNA (7.0, 6.0 and 5.5 kb) and affinity-labeled receptor protein (approximately 125 kDa) levels was observed. The IGF-I receptor mRNA (11.5, 6.5 and 4.5 kb) and affinity-labeled receptor (approximately 125 kDa) protein levels declined during the neonatal stages of development. In the case of the IGF-II receptor, while the mRNA levels (9.0 and 4.5 kb) remained constant, the corresponding affinity-labeled receptor protein (approximately 230 kDa) declined with maturation. We conclude that a differential regulation of brain insulin, IGF-I and IGF-II receptor expression occurs during development.

Animals↗
PubMed1978

Analysis of RNA initiated in isolated mouse myeloma nuclei using purine nucleoside 5'[gamma-S]triphosphates as affinity probes.

The purine ribonucleoside triphosphate analogues adenosine 5'[gamma-S]triphosphate and guanosine 5'[gamma-T]triphosphate were used as affinity probes for studying RNA chain initiation in isolated nuclei from the mouse myeloma 66.2 cell line. Transcripts initiated with either nucleotide analogue were isolated by affinity chromatography on a mercury-agarose affinity column. The binding was specific and dependent upon the inclusion of the sulfur nucleotide analogues in the in vitro synthetic reaction. Several lines of evidence indicate that the affinity-labeled RNA is initiated in vitro. First, the sulfur nucleotide is recovered in high yield as a single nucleoside 5'[gamma-S]triphosphate 2',3'-monophosphate product following alkaline hydrolysis of RNA bound to the affinity column. Second, authentic ribosomal 5S RNA is known to initiate with GTP; in vitro 5S RNA is bound to mercury-agarose only if [gamma-S]GTP is used as the affinity label in the synthesis, and not if [gamma-S]ATP is used. Under the conditions studied, nuclei incorporated 1.2--2.4 pmole of UMP per 10(6) nuclei per min, and the rate of synthesis was unaffected by substitution of the nucleotide analogues for the normal nucleotides. The percentage of the total RNA synthesized that was incorporated into sequences initiated in vitro was 7.8 +/- 1.5% with [gamma-S]ATP and 9.6 +/- 6.4% with [gamma-S]GTP. The size of the total RNA synthesized, determined by sedimentation on sucrose density gradients containing dimethylsulfoxide, ranged from less than 5S to 45S, and the size of the affinity-labeled sequences ranged from less than 5S to 28S. Approximately half of the incorporation into RNA initiated in vitro was sensitive to a concentration of alpha-amanitin which selectively inhibits polymerase II activity. Most of the remaining incorporation into initiated sequences can be abolished by concentrations of alpha-amanitin that are inhibitory for polymerase III activity. Over 70% of the total incorporation into ribosomal 5S RNA transcripts was into sequences initiated in vitro. This initiation was catalyzed by polymerase III and was specific for GTP as the initiating nucleotide. The RNAase T1 fingerprint of the newly initiated 5S RNA indicates that this gene is accurately initiated and faithfully elongated in vitro. The use of these affinity label probes provides much greater sensitivity for studying the initiation of RNA in vitro.

Amanitins↗
PubMed2000

Ligands presumed to label high affinity and low affinity ATP binding sites do not interact in an (alpha beta)2 diprotomer in duck nasal gland Na+,K+-ATPase, nor Do the sites coexist in native enzyme.

The interaction of ligands deemed to be ATP analogues with renal Na(+),K(+)-ATPase suggests that two ATP binding sites coexist on each functional unit. Previous studies in which fluorescein 5-isothiocyanate (FITC) was used to label the high affinity ATP site and 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) was used to probe the low affinity site suggested that the two sites coexist on the same alphabeta protomer. Other studies in which FITC labeled the high affinity site and erythrosin-5-isothiocyanate (ErITC) labeled the low affinity site led to the conclusion that the high and low affinity sites exist on separate interacting protomers in a functional diprotomer. We report here that at 100% inhibition of ATPase activity by FITC, each alphabeta protomer of duck nasal gland enzyme has a single bound FITC. Both TNP-ADP and ErITC interact with FITC-bound protomers, which unambiguously demonstrates that putative high and low affinity ATP sites coexist on the same protomer. In unlabeled nasal gland enzyme, TNP-ADP and ErITC inhibit both ATPase activity and p-nitrophenyl phosphatase activity, functions attributed to the putative high and low affinity ATP site, respectively, by interacting with a single site with characteristics of the high affinity ATP binding site. In FITC-labeled enzyme, TNP-ADP and ErITC inhibit p- nitrophenyl phosphatase activity but at much higher concentrations than with the unmodified enzyme. Low affinity sites do not exist on the unmodified enzyme but can be detected only after the high affinity site is modified by FITC.

Adenosine Diphosphate↗
PubMed1986

Photoaffinity labeling of extracellular and intracellular androgen binding proteins.

A method is described for the photoaffinity labeling of human and rabbit serum sex hormone binding globulin (SHBG) in ammonium sulphate precipitates utilizing 3H-delta 6-testosterone as affinity label. The precipitation step diminished albumin contamination and at the same time concentrated the binding globulin. Photolysis was conveniently carried out with an electronic flash. Unbound and non-covalently bound steroids were adsorbed by prolonged dextran-coated charcoal treatment. Sephadex G-200 gel filtration column chromatography showed a single peak of covalently bound radioactivity with the elution volume of SHBG. With some modifications, the method was applied to the affinity labeling of the prostate androgen receptor utilizing 3H-methyltrienolone (R 1881) as affinity label. The receptor was also precipitated from prostate cytosol with ammonium sulphate. After labeling, photolysis and heating at 50 degrees C, non covalently bound 3H-R 1881 was removed by dextran coated charcoal treatment. Sephadex G-25 microcolumn chromatography after heating showed a peak of radioactivity eluting with macromolecules if photolysis had been carried out, while it disappeared in the absence of previous photolysis. However, the photolabeled receptor had a sedimentation coefficient different from the non-irradiated receptor, suggesting that photolysis induced a change in the configuration of the complex.

Affinity Labels↗
PubMed1992

Identification of glutamic acid 105 at the active site of Bacillus amyloliquefaciens 1,3-1,4-beta-D-glucan 4-glucanohydrolase using epoxide-based inhibitors.

Bacillus amyloliquefaciens 1,3-1,4-beta-D-glucan 4-glucanohydrolase (EC 3.2.1.73) was modified by the mechanism-based, affinity-labeling reagent [14C](3,4)-epoxybutyl beta-D-cellobioside. Following partial inactivation a completely inactivated enzyme preparation containing 1.1 mol of covalently bound inhibitor/mol of protein was obtained by chromatography on a cellulosic matrix. The inactivated enzyme was digested with endoproteinase Glu-C and radioactive peptides purified by reversed-phase high performance liquid chromatography (HPLC). The affinity label was esterified exclusively to the gamma-carboxylate of Glu105 in the sequence Gly-Thr-Pro-Trp-Asp-Glu-Ile-Asp-Ile-Glu109. The sequence motif Glu-(Ile/Leu)-Asp-Ile is found in many glucanases and xylanases and may therefore serve to identify the catalytic nucleophile in beta-glycanases, which otherwise exhibit a low degree of sequence identity. The esterification of Glu105 by the affinity label abolished endoproteinase Glu-C-mediated hydrolysis of the Glu-Ile106 peptide bond. Identification of phenylthiohydantoin-Glu105 during automated sequence analysis was not possible unless the affinity label was liberated by prior base hydrolysis. These observations formed the basis for the development of a highly sensitive approach for the identification of catalytic carboxylates in polysaccharide hydrolases employing non-radioactive inhibitors, comparative HPLC mapping, electrospray mass spectrometry, and Edman degradation.

Amino Acid Sequence↗
PubMed1987

[Affinity-histochemical labeling methods using colloidal gold. Overview of developments in the past 15 years].

A survey is given of the development of affinity-histochemical labelling methods using colloidal gold for electron and light microscopy. Historical aspects and trends are discussed and principle methodologic variations are illustrated. A comprehensive compilation of previously published data provides a concise overview of technical variations and applications of the colloidal gold method.

Animals↗
PubMed1977

Structure of the steroid-binding site of human placental estradiol-17beta-dehydrogenase.

We recently demonstrated that human placental estradiol-17beta-dehydrogenase possesses a histidyl residue in the catalytic region of the active site by affinity-labeling studies with 16alpha-bromoacetoxyestradiol-3-methyl ether. We now report the synthesis of 12beta-bromoacetoxy-4-estrene-3,17-dione and its use in affinity labeling of the enzyme. The steroid was synthesized by incubation of 4-estrene-3,17-dione with Colletotrichum gloesporioides. The product was recrystallized from ethanol and structure assured by IR and NMR spectroscopy. The steroid is a substrate, which indicates that it binds at the active site. When the enzyme is incubated with a 150-fold molar excess of 12beta-bromoacetoxy-4-estrene-3,17-dione in potassium phosphate buffer at pH 7.0, the enzyme is inactivated in a time-dependent, irreversible manner. Inactivation follows pseudo-first-order kinetics with a half life of 18 hours. Analysis of a hydrolysate of the enzyme after inactivation with 12beta-bromo[2'-3H]acetoxy-4-estrene-3,17-dione reveals tritiated 1-, 3-, and 1,3-dicarboxymethylhistidine. The affinity labeling of a histidyl enzyme residue by both 16alpha- and 12beta-bromoacetoxy steroids localizes that residue near the point of catalysis and suggests that it may participate in the catalytic event.

17-Hydroxysteroid Dehydrogenases↗
PubMed1984

Binding of ligands to the catalytic zinc ion in horse liver alcohol dehydrogenase.

The affinity of nitrogen and sulfur ligands for the catalytic zinc ion in horse liver alcohol dehydrogenase has been investigated by their influence on the affinity labeling reaction with iodoacetate. All the nitrogen compounds including ammonia, a primary and a secondary amine, and heterocycles containing a pyridine-type nitrogen with the exception of 2,2-dipyridyl were found to activate the affinity labeling reaction. Activation results from inner-sphere ligand coordination to the catalytic zinc ion. Closely related pyridine compounds gave a regular increase in affinity for the enzyme with increasing basicity, as expected for coordination to a metal ion. The sulfur compounds penicillamine and mercaptoethanol also activated the affinity labeling reaction, but dimercaptopropanol bound very tightly as a bidentate inhibited the reaction. The anions hydrosulfide, diethyldithiocarbamate, and cyanide coordinated to the catalytic zinc ion, whereas azide, thiocyanate, tetrazole, and iodide complexed the anion-binding site. The anionic metal ligands increased the rate of inactivation of the enzyme with iodoacetamide by binding to the catalytic zinc ion, while the binding of iodoacetate to the anion-binding site was prevented.

2,2'-Dipyridyl↗
PubMed1992

Nonsteroidal estrogens bearing acyl azide functions: potential electrophilic and photoaffinity labeling agents for the estrogen receptor.

In an effort to develop novel affinity labeling agents for the estrogen receptor, we have synthesized two nonsteroidal ligands, a 1-aroyl-2-aryl tetralin system (1) and a 2-aryl-3-aroylbenzo[b]thiophene system (2). These agents, patterned after the Lilly antiestrogens trioxifene and LY 117018, respectively, embody acyl azide functions as part of a benzoyl chromophore. The acyl azide group has weak acylating activity, suitable for electrophilic affinity labeling, but this function is also photoreactive and, in its particular embodiment within these ligands, it could provide an efficient photochemical route to the highly reactive singlet acyl nitrene. The tetralin system (1) was prepared in nine steps from 6-methoxy-1-tetralone, and the benzothiophene system (2) was prepared in four steps from a known substituted benzo[b]thiophene precursor. In competitive binding assays, both compounds show reasonable binding affinity for the rat and lamb uterine estrogen receptor: estradiol = 100%, 1 = 3%, and 2 = 12%. When assayed by indirect receptor consumption assays, both compounds appear to have substantial capacity for irreversible binding (electrophilic reaction) with the receptor. This reactivity, which suggests that acylation of the receptor has occurred, is photoreversible. The nature of this ligand-receptor interaction is being investigated further.

Affinity Labels↗
PubMed1980

Independent activation of the acetylcholine receptor from Torpedo californica at two sites.

Membrane vesicles enriched in acetylcholine receptor were prepared from the electroplax tissue of Torpedo californica. The receptor was reduced with dithiothreitol to expose a sulfhydryl group near the ligand binidng site and then treated in one of the following ways: (1) affinity alkylated treated in one of the following ways: (1) affinity alkylated with bromoacetylcholine, a receptor activator, (2) affinity alkylated with maleimidobenzyltrimethylammonium, a receptor inhibitor, or (3) reoxidized to the native state with dithiobis(2-nitrobenzoate). The affinity labels blocked half of the binding sites for alpha-bungarotoxin. The toxin sites not protected by the affinity labels were protected by carbamylcholine based on studies of toxin binding kinetics. The functional response of native and affinity-alkylated receptors was measured by a sodium ion flux procedure. In the absence of added cholinergic activators, only slow ion flux was observed. In the presence of carbamylcholine, a receptor activator, both native and modified membranes showed the increased sodium flux associated with functional receptors. The concentration of carbamylcholine required for a 50% maximal response was higher in the affinity-labeled membranes. Preincubation of the membranes with carbamylcholine blocked the increased ion flux, indicating that desensitization could be induced. The results provide evidence for the existence of two functional sites on the acetylcholine receptor. Each site corresponds to a bungarotoxin binding site and can be independently activated and desensitized.

Acetylcholine↗