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PubMed1993

Structural characterization of the trypsinized estrogen receptor.

Structural differences between the unoccupied and ligand-occupied rat uterine estrogen receptors (ERs) were investigated using partial proteolysis followed by immunoblotting, affinity labeling, and gel filtration chromatography. Trypsin digestion of the unoccupied ER at 4 degrees C resulted in retention of 70-80% of high-affinity [3H]estradiol binding. Only two fragments of the rat ER were detected after prolonged trypsin treatment of the unoccupied ER followed by affinity labeling with [3H]tamoxifen aziridine. One fragment represents the intact steroid binding domain (28 kDa), and the other fragment is about 10 kDa. The small 10-kDa fragment of the ER detected by denaturing gel electrophoresis is shown to be held in a large oligomeric complex in solution using gel filtration chromatography. This oligomeric complex probably represents the steroid binding domain, which has its tertiary structure maintained predominantly by noncovalent interactions between the trypsin-generated fragments. The estrogen, anti-estrogen, and unoccupied trypsinized ERs all result in similar patterns of fragments after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detection by immunoblotting. Although no new trypsin cleavage sites were exposed, the sensitivity of the available trypsin sites was altered by heating the ER and, to a lesser extent, by hormone treatment. Gel filtration chromatography of the trypsinized estradiol- and 4-hydroxytamoxifen-occupied ERs demonstrates similar, diffuse peaks centered at about the correct size for the intact steroid binding domain (28 kDa), whereas the trypsinized unoccupied ER results in a sharp, discrete peak centered at about 80 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Heterodimeric transforming growth factor beta. Biological properties and interaction with three types of cell surface receptors.

Type beta transforming growth factors (TGF) are disulfide-linked homo- and heterodimers of two related polypeptide chains, beta 1 and beta 2. The homodimers TGF-beta 1 and TGF-beta 2 are widely distributed, but the heterodimer TGF-beta 1.2 has been found only in porcine platelets (Cheifetz, S., Weatherbee, J.A., Tsang, M.L.-S., Anderson, J.K., Mole, J.E., Lucas, R., and Massagué, J. (1987) Cell 48, 409-415). Here we characterize the receptor binding and biological properties of TGF-beta 1.2 and compare them with those of TGF-beta 1 and TGF-beta 2. Three types of cell surface receptors previously identified by affinity labeling with 125I-TGF-beta 1 are available for binding to TGF-beta 1.2. These three types of receptors are detected as 65-kDa (type I), 85-95-kDa (type II), and 250-350-kDa (type III) affinity-labeled receptor complexes on electrophoresis gels. They co-exist in many cell types, have high affinity for TGF-beta 1, and varying degrees of affinity for TGF-beta 2. Of the 11 cell lines screened in the present study none showed evidence for additional receptor types that would bind TGF-beta 2 but not TGF-beta 1. In receptor competition studies, TGF-beta 1, TGF-beta 1.2, and TGF-beta 2 competed for binding to type I and type II receptors with a relative order of potencies of 16:5:1 and 12:3:1, respectively, whereas all three forms of TGF-beta were equipotent as ligands for the type III receptors. The three forms of TGF-beta were equally potent at stimulating the biosynthesis of extracellular sulfated proteoglycan in BRL-3A rat liver epithelial cells, a response that presumably involves the type III receptor present in these cells. In contrast, the ability of the three ligands to inhibit the growth of B6SUt-A multipotential hematopoietic progenitor cells which display only type I receptors decreased in the order TGF-beta 1, TGF-beta 1.2, and TGF-beta 2 with a relative potency of 100:30:1. The results indicate that the presence of one beta 1 chain in TGF-beta 1.2 increases (with respect to TGF-beta 2) the biological potency and binding affinity toward receptor types I and II, but the presence of a second beta 1 chain in the dimer is required for full potency.

Binding, Competitive↗

PubMed2002

Identification of ceramide binding proteins in neuronal cells: a critical point of view.

Much discussion has centered on the biochemical mechanism by which ceramide is produced and functions as a signalling molecule in cells. To identify proteins involved in ceramide signalling, we synthesized a radioactively labelled ceramide analogue equipped with a photosensitive group: N-(p-trifluoromethyl-diazirinyl)phenyl-ethyl-2-[35S]-2-thioacetyl-D-erythro-C18-sphingosine ([35S]-TDS-ceramide). This compound was then employed in photo-affinity labelling experiments in primary cultured cerebellar neurons. Due to the hydrophobic nature of the compound, most of the cell-associated radioactivity was recovered in the lipid fraction while only about 0.1% of radioactivity was photocoupled to proteins. In order to improve protein labelling the cytosolic fraction of rapidly growing human neuroblastoma cells (SH-SY5Y) was isolated and subjected to ceramide affinity chromatography prior to photo-affinity labelling. Following electrophoresis proteins photocoupled to ceramide were identified by MALDI mass spectrometry in combination with tryptic digestion and turned out to be either cytoskeletal or stress proteins that are highly abundant in cytosol and contain at least one hydrophobic domain.

Animals↗

PubMed1986

Characterization of the receptor for heat-stable enterotoxin from Escherichia coli in rat intestine.

The receptor for the heat-stable enterotoxin (ST) from Escherichia coli was solubilized with Lubrol-PX from rat intestinal brush-border membranes and characterized. The binding kinetics and analog specificity of the solubilized receptor were virtually identical to those obtained with the membrane-bound receptor. Furthermore, the regulation of the receptor's affinity by cations was also maintained after solubilization, indicating a conservation of the toxin-binding site after removal of the receptor from its membrane environment. Gel filtration and sucrose density gradient sedimentation studies gave a Stokes radius of 5.5 nm and a sedimentation coefficient of 7.0 S for the solubilized receptor. The isoelectric point of the receptor was determined as 5.5 using Sephadex isoelectric focusing electrophoresis. In all of these separation techniques, the ST receptor showed a single peak of activity that was clearly separated from that of guanylate cyclase. When 125I-ST was cross-linked to brush-border membranes with disuccinimidyl suberate, the affinity-labeled receptor solubilized with 0.1% Lubrol-PX eluted at a similar position as the native receptor on gel filtration chromatography. Analysis of the affinity-labeled receptor by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of reducing agent and by autoradiography revealed the presence of three specifically labeled polypeptides with apparent molecular weights of 80,000, 68,000, and 60,000. These results suggest that the ST receptor is solubilized by Lubrol-PX in an active form with preservation of its regulation by cations. Also, the ST receptor is separable from particulate guanylate cyclase indicating that the receptor is coupled to the activation of guanylate cyclase by an as yet undefined mechanism. Three subunit peptides may constitute a binding region of the receptor.

Animals↗

PubMed1985

Transforming growth factor-beta modulates the high-affinity receptors for epidermal growth factor and transforming growth factor-alpha.

The epidermal growth factor (EGF) receptor mediates the induction of a transformed phenotype in normal rat kidney (NRK) cells by transforming growth factors (TGFs). The ability of EGF and its analogue TGF-alpha to induce the transformed phenotype in NRK cells is greatly potentiated by TGF-beta, a polypeptide that does not interact directly with binding sites for EGF or TGF-alpha. Our evidence indicates that TGF-beta purified from retrovirally transformed rat embryo cells and human platelets induces a rapid (t 1/2 = 0.3 h) decrease in the binding of EGF and TGF-alpha to high-affinity cell surface receptors in NRK cells. No change due to TGF-beta was observed in the binding of EGF or TGF-alpha to lower affinity sites also present in NRK cells. The effect of TGF-beta on EGF/TGF-alpha receptors was observed at concentrations (0.5-20 pM) similar to those at which TGF-beta is active in promoting proliferation of NRK cells in monolayer culture and semisolid medium. Affinity labeling of NRK cells and membranes by cross-linking with receptor-bound 125I-TGF-alpha and 125I-EGF indicated that both factors interact with a common 170-kD receptor structure. Treatment of cells with TGF-beta decreased the intensity of affinity-labeling of this receptor structure. These data suggest that the 170 kD high-affinity receptors for EGF and TGF-alpha in NRK cells are a target for rapid modulation by TGF-beta.

Animals↗

PubMed2002

Comparative proteomics based on stable isotope labeling and affinity selection.

Disease, external stimuli (such as drugs and toxins), and mutations cause changes in the rate of protein synthesis, post-translational modification, inter-compartmental transport, and degradation of proteins in living systems. Recognizing and identifying the small number of proteins involved is complicated by the complexity of biological extracts and the fact that post-translational alterations of proteins can occur at many sites in multiple ways. It is shown here that a variety of new tools and methods based on internal standard technology are now being developed to code globally all peptides in control and experimental samples for quantification. The great advantage of these stable isotope-labeling strategies is that mass spectrometers can rapidly target those proteins that have changed in concentration for further analysis. When coupled to stable isotope quantification, targeting can be further focused through chromatographic selection of peptide classes on the basis of specific structural features. Targeting structural features is particularly useful when they are unique to types of regulation or disease. Differential displays of targeted peptides show that stimulus-specific markers are relatively easy to identify and will probably be diagnostically valuable tools.

Animals↗

PubMed1987

Structure and proteolysis of the growth hormone receptor on rat hepatocytes.

125I-Labeled human growth hormone is isolated in high molecular weight (Mr) (300,000, 220,000, and 130,000) and low molecular weight complexes on rat hepatocytes after affinity labeling. The time-dependent formation of low molecular weight complexes occurred at the expense of the higher molecular weight species and was inhibited by low temperature or inhibitors of serine proteinases. Exposure to reducing conditions induced loss of Mr 300,000 and 220,000 species and augmented the amount of Mr 130,000 complexes. The molecular weight of growth hormone (22,000) suggests that binding had occurred with species of Mr 280,000, 200,000, and 100,000. Two-dimensional gel electrophoresis demonstrated that the 100,000-dalton receptor subunit is contained in both the 280,000- and 200,000-dalton species. Reduction of interchain disulfide bonds in the growth hormone receptor did not alter its elution from gel filtration columns, but intact, high molecular weight receptor constituents were separated from lower molecular weight degradation products. Digestion of affinity-labeled growth hormone-receptor complexes with neuraminidase increased the mobility of receptor constituents on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These observations show that the growth hormone receptor is degraded by hepatic serine proteinases to low molecular weight degradation products which can be separated from intact receptor by gel filtration. Intact hormone-receptor complexes are aggregates of 100,000-dalton sialoglycoprotein subunits held together by interchain disulfide bonds and by noncovalent forces.

Animals↗

PubMed1987

Purification of prolactin receptors from sow mammary gland and polyclonal antibody production.

After solubilization with Triton X-100 or 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS), prolactin receptors from mammary crude membranes of primiparous lactating sows (pretreated with bromocriptine) have been purified by affinity chromatography using ovine prolactin or a monoclonal antibody against rabbit prolactin receptor. Comparative analysis of these two methods of purification demonstrated that use of an immunoaffinity step allowed a great improvement of receptor yield (40%) compared to the hormone affinity method (10%). In addition, partially purified fractions obtained by immunoaffinity appeared more homogeneous and had much higher specific activity. Affinity labelling of prolactin receptors from crude membranes or solubilized extracts with iodinated ovine prolactin, followed by electrophoretic analysis (SDS-PAGE) and autoradiography, revealed one binding unit of approximately 45 kDa. When partially purified receptor preparations were labelled with 125I, submitted to an additional affinity chromatography and analyzed by SDS-PAGE, prolactin receptors appeared as a single form having a molecular weight of 42-45 kDa, which is not associated with itself or other subunits by disulfide linkages. Partially purified fractions were used to produce anti-prolactin receptor serum from goats. These polyclonal antibodies were able to completely inhibit the binding of lactogenic hormones in sow and rabbit mammary membranes. They were also able to recognize hormone-receptor complexes, but more specifically in sow mammary gland. These antisera could inhibit prolactin binding to its receptors in several organs of various species, suggesting that prolactin receptors shared numerous antigenic similarities between species and particularly between sow and rabbit. These similarities appeared to be located essentially on the part of the molecule more specifically involved in the recognition of the hormone.

Affinity Labels↗

PubMed2003

The voltage-dependent anion channel is the target for a new class of inhibitors of the mitochondrial permeability transition pore.

The relevance of the mitochondrial permeability transition pore (PTP) in Ca2+ homeostasis and cell death has gained wide attention. Yet, despite detailed functional characterization, the structure of this channel remains elusive. Here we report on a new class of inhibitors of the PTP and on the identification of their molecular target. The most potent among the compounds prepared, Ro 68-3400, inhibited PTP with a potency comparable to that of cyclosporin A. Since Ro 68-3400 has a reactive moiety capable of covalent modification of proteins, [3H]Ro 68-3400 was used as an affinity label for the identification of its protein target. In intact mitochondria isolated from rodent brain and liver and in SH-SY5Y human neuroblastoma cells, [3H]Ro 68-3400 predominantly labeled a protein of approximately 32 kDa. This protein was identified as the isoform 1 of the voltage-dependent anion channel (VDAC). Both functional and affinity labeling experiments indicated that VDAC might correspond to the site for the PTP inhibitor ubiquinone0, whereas other known PTP modulators acted at distinct sites. While Ro 68-3400 represents a new useful tool for the study of the structure and function of VDAC and the PTP, the results obtained provide direct evidence that VDAC1 is a component of this mitochondrial pore.

Animals↗

PubMed1984

Alpha 1-adrenergic receptor structure.

The structure of the alpha 1-adrenergic receptor was investigated by comparing polypeptides identified by sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel electrophoresis with the size of the intact receptor in cell membranes as determined by target size analysis. The alpha 1-adrenergic receptor from rat liver membranes affinity-labeled with [3H]phenoxybenzamine, a covalent affinity reagent, appeared as a single polypeptide with a molecular mass of 85,000 daltons (Da) on NaDodSO4-polyacrylamide gels. In the absence of protease inhibitors, smaller peptides of 58-62 kDa and 40-45 kDa, specifically labeled with [3H]phenoxybenzamine, were also apparent on NaDodSO4 gels. In order to determine whether the 85-kDa protein represented all or only a portion of the alpha 1-receptor, radiation inactivation (target size analysis) was undertaken. Radiation-induced receptor inactivation was measured by the loss of specific [3H]phenoxybenzamine and [3H]prazosin binding and by the loss of affinity-labeled alpha 1-adrenergic receptors on NaDodSO4 gels. Target size analysis of rat liver alpha 1-receptors indicated that the intact membrane-bound receptor has an average molecular mass of 160,000 Da. These data suggest that the intact alpha-receptor may exist in the membrane as a dimer of two 85,000-Da subunits. The structure of the alpha 1-receptor was further studied by limited proteolysis of the 85-kDa protein isolated from NaDodSO4 gels. Trypsin, chymotrypsin, and papain produce smaller peptides similar to those produced during membrane isolation in the absence of protease inhibition. Limited proteolysis of the membrane-bound receptor produces water-soluble peptides, the largest of which is 45,000 Da. This peptide contains the ligand-binding domain and protrudes from the membrane into the extracellular space.

Animals↗

PubMed1983

Epidermal growth factor-like transforming growth factor. II. Interaction with epidermal growth factor receptors in human placenta membranes and A431 cells.

A 125I-labeled derivative of rat epidermal growth factor-like transforming growth factor (125I-eTGF) that retains the chemical and biological properties of native eTGF has been prepared to characterize the interaction of eTGF with epidermal growth factor (EGF) membrane receptors. Binding of 125I-eTGF and 125I-labeled mouse EGF (125I-EGF) to membrane preparations from human placenta and the A431 human epidermoid carcinoma cell line was competed for to the same extent and with the same potency by either unlabeled eTGF or EGF. Components of placenta membranes and A431 membranes (145-170 kDa) were the major species affinity-labeled by cross-linking with membrane-bound 125I-eTGF or 125I-EGF. Affinity labeling of these receptor species was fully inhibited by both native eTGF and native EGF when present in excess during incubation of membranes with 125I-eTGF or 125I-EGF. The binding affinity and capacity of receptors in A431 membranes and placenta membranes were strikingly similar for eTGF and EGF. However, binding of 125I-eTGF to membrane receptors was highly sensitive to pH, being optimal only within the pH 8.0-8.5 range whereas EGF binding was optimal at pH 7.0-9.0. eTGF binding and EGF binding were modified in parallel by exposure of placental membranes to phytohemagglutinin, concanavalin A, or wheat germ agglutinin. Prolonged exposure of A431 cells to eTGF or EGF induced the same extent of down-regulation of both eTGF binding and EGF binding. eTGF-induced down-regulation of receptors in A431 cells followed the same kinetics as EGF-induced receptor down-regulation. The data indicate that despite the limited sequence homology between eTGF and EGF, these two ligands exhibit a remarkably similar mode of interaction with a common eTGF/EGF membrane receptor type.

Carcinoma, Squamous Cell↗

PubMed1992

A model for the catalytic site of F1-ATPase based on analogies to nucleotide-binding domains of known structure.

An updated topological model is constructed for the catalytic nucleotide-binding site of the F1-ATPase. The model is based on analogies to the known structures of the MgATP site on adenylate kinase and the guanine nucleotide sites on elongation factor Tu (Ef-Tu) and the ras p21 protein. Recent studies of these known nucleotide-binding domains have revealed several common functional features and similar alignment of nucleotide in their binding folds, and these are used as a framework for evaluating results of affinity labeling and mutagenesis studies of the beta subunit of F1. Several potentially important residues on beta are noted that have not yet been studied by mutagenesis or affinity labeling.

Adenylate Kinase↗

PubMed1979

Synthesis, characterization and fluorescence studies on N-alpha-dansylalanyllysyl trypsino (HIS-46)-methane.

The 1-dimethylamino-5-naphthalene sulfonyl (dansyl) derivative of alanyllsyl chloromethane was synthesized and employed to introduce the fluorescent dansyl moiety specifically into the active site of trypsin via affinity labelling. The potential of dansylalanyllysyl chloromethane lies in its high degree of selectivity and markedly faster rate of enzyme inactivation when compared to previously synthesized, single residue affinity label chromophores. This permits the practical utilization of stoichiometric amounts of the inhibitor to achieve 100% inactivation of trypsin, even at high dilutions. The transfer of energy between the four tryptophan residues of trypsin and the bound dansyl group has been investigated in the fluorescent inhibitor-enzyme conjugate. From transfer efficiency measurements mean distances of 19.0 A and 19.3 A between the point of attachment of the dansyl group and the four tryptophan residues of trypsin have been calculated. These compare well with the mean value of 18.8 A derived from calculations based on crystallographic model studies.

Amino Acids↗

PubMed1989

Two distinct types of endothelin receptors are present on chick cardiac membranes.

Competitive displacement experiments of 125I-endothelin (ET)-1, -2, or -3 binding to chick cardiac membranes were performed with unlabeled ET-1, -2, -3, and sarafotoxin S6b (STX) as competitors. 125I-ET-1 and -2 binding was competitively inhibited by increasing concentrations of these unlabeled peptides in the same order; i.e. ET-2 greater than or equal to ET-1 greater than ET-3 greater than STX. In contrast, the order of potency in displacing 125I-ET-3 binding was ET-3 greater than ET-2 greater than or equal to ET-1 greater than STX. Affinity labeling of the membranes by cross-linking with 125I-ET-1 and -2 via disuccinimidyl tartarate yielded one major specific band with an apparent Mr = 53,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. On the other hand, affinity labeling with 125I-ET-3 showed that two major and one minor bands of Mr = 34,000, 46,000, and 53,000, respectively, were specifically labeled. These results indicate the presence of two distinct types of ET receptors, one of which has higher affinity for ET-1 and -2 than ET-3 and the other is conversely ET-3-preferring.

Animals↗

PubMed1999

Enzyme amplification as detection tool in continuous-flow systems. I. Development of an enzyme-amplified biochemical detection system coupled on-line to flow-injection analysis.

The on-line coupling of flow-injection analysis (FIA) to an enzyme-amplified biochemical detection (EA-BCD) system is described. The aim of this study is the development of a detection system able to detect biotin-containing compounds at low concentration levels. The detection system is based on the interaction of biotin with enzyme-labeled affinity proteins. Biotin possesses a high affinity to both streptavidin and anti-biotin Fab fragments, which are both tested. Several biotin derivatives are available with different reactive probes, which can be used to label analytes of interest. Therefore, biotin acts as a universal probe for the enzyme-amplified biochemical detection. Alkaline phosphatase (AP) was used as enzyme label. Several parameters, such as substrate type and concentration, concentration of enzyme-labeled affinity protein, reaction time and reaction temperature were examined. Biotin aminocaproic acid was used as a model compound. In addition to biotin aminocaproic hydrazide, other biotinylation reagents were also examined. With fluorescence detection of the enzyme-generated product, a mass detection limit of 1 fmol was achieved.

Biotin↗

PubMed1988

Absence of TGF-beta receptors and growth inhibitory responses in retinoblastoma cells.

The responses of retinoblastoma tumor cells and normal retinal cells to various growth inhibitory factors were examined. Whereas fetal retinal cells were highly sensitive to the antimitogenic effects of transforming growth factor beta 1 (TGF-beta 1), retinoblastoma tumor cell lines were all resistant to this factor. Binding assays and affinity labeling of these cells with radioiodinated TGF-beta 1 revealed that the cells did not have TGF-beta receptors. The retinoblastoma cells lacked the three affinity-labeled proteins of 65, 95, and 300 kilodaltons typically seen in human cell lines and thus differed from normal retinal cells and from other types of neuroectodermal tumors that display the normal pattern of receptors. Loss of TGF-beta receptors, which is a rare event among tumor cells, may represent one mechanism through which these cells escape from negative control and form retinoblastomas.

Cell Division↗

PubMed1992

Subtypes of betaglycan and of type I and type II transforming growth factor-beta (TGF-beta) receptors with different affinities for TGF-beta 1 and TGF-beta 2 are exhibited by human placental trophoblast cells.

Transforming growth factor-beta is likely to be an important factor controlling placental activities, including growth, differentiation, invasiveness, hormone production, and immunosuppression. We have used a chemical cross-linking technique with either 125I-TGF-beta 1 or 125I-TGF-beta 2 and bis(sulfosuccinimidyl) suberate (BS3) to characterize TGF-beta binding components on human placental cells in primary culture. Trophoblast-enriched primary cultures exhibited a predominant affinity-labelled complex characteristic of membrane-anchored betaglycan (formerly termed the Type III TGF-beta receptor) and relatively low levels of the Type I and Type II TGF-beta receptor complexes. The results from affinity labelling saturation and competition experiments with TGF-beta 1 and TGF-beta 2 suggest the existence of two distinct subtypes of betaglycan: one subtype has a lower capacity and higher affinity, binds both TGF-beta 1 and TGF-beta 2, yet has a preferential affinity for TGF-beta 2; the second subtype has a higher capacity and lower affinity and binds TGF-beta 1 exclusively. In contrast, mesenchymal cell-enriched placental primary cultures possessed only one subtype of the betaglycan component that binds the two TGF-beta isoforms with similar affinities and capacities as observed on most cell lines. These experiments demonstrate that the betaglycan component which exhibits a higher affinity for TGF-beta 2 than for TGF-beta 1, that we had observed previously on term placental membranes, is actually present on trophoblast cells. In addition to the two distinctive betaglycan subtypes, subtypes of the Type I and II TGF-beta receptors were detected on the trophoblast-enriched cultures. In competition experiments, when 125I-TGF-beta 1 was used as the radiotracer, the Type I and II TGF-beta receptors show a much higher affinity for TGF-beta 1 than for TGF-beta 2, as observed with other cell types. However, when 125I-TGF-beta 2 was used, low abundance subtypes of both the Type I and II receptors that show similar affinities for TGF-beta 1 and TGF-beta 2 were also revealed.

Cell Separation↗

PubMed1986

Pharmacological properties of immuno-isolated neuronal nicotinic receptors.

Recently we immunoaffinity-purified an ACh receptor from chicken brain using a monoclonal antibody raised against receptors from fish electric organ (Whiting and Lindstrom, 1986). This neuronal receptor could be affinity-labeled with 3H-bromoacetylcholine, and antisera to it specifically blocked ACh-induced depolarization of chicken ciliary ganglion cells. Here we show that this neuronal ACh receptor binds 3H-nicotine with high affinity (KD = 6.61 +/- 0.13 nM). 3H-Nicotine binding was blocked by various nicotinic cholinergic ligands but not by alpha-bungarotoxin or the muscarinic antagonist atropine. Binding was also blocked by affinity labeling the receptor with bromoacetylcholine (after reduction by dithiothreitol). Additionally, we were able to use rat antisera raised against the chicken brain receptor to isolate a component from detergent extracts of rat brain that also bound 3H-nicotine with high affinity (KD = 1.5 nM). The pharmacology of this putative ACh receptor from rat brain was almost identical to the receptor from chicken brain, and its regional distribution was in good agreement with that of 3H-nicotine binding to rodent brain membranes reported by other workers. Thus, by analogy to the receptor we have purified and characterized from chicken brain, this nicotine-binding component from rat brain is probably a functional mammalian neuronal nicotinic ACh receptor.

Animals↗