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1.
Botulinum type F neurotoxin. Large-scale purification and characterization of its binding to rat cerebrocortical synaptosomes. 总被引:3,自引:0,他引:3
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J D Wadsworth M Desai H S Tranter H J King P Hambleton J Melling J O Dolly C C Shone 《The Biochemical journal》1990,268(1):123-128
1. A large-scale purification procedure has been developed for Clostridium botulinum type F neurotoxin. Commencing with 160 litres of bacterial culture, 101 mg of purified type F neurotoxin with a specific toxicity of 2 x 10(7) mouse LD50 (median lethal dose).mg-1 were obtained. 2. Purified type F neurotoxin was labelled to high specific radioactivity (900-1360 Ci/mmol) without loss of biological activity using a chloramine-T procedure. Of the two neurotoxin subunits, the heavy chain was preferentially radiolabelled. 3. Radiolabelled type F neurotoxin displayed specific saturable binding to rat synaptosomes. At least two pools of acceptors were evident: a low content of high-affinity acceptors sites [KD approximately 0.15 nM; Bmax (maximal binding) 20 fmol/mg] and a larger pool of lower-affinity sites (KD greater than 20 nM; Bmax greater than 700 fmol/mg). Both pools of acceptors were sensitive to trypsin and neuraminidase treatment, which suggests that protein and sialic acid residues are components of the synaptosomal acceptors. 4. Experiments investigating competition among botulinum neurotoxin types A, B, E and F for acceptors on rat brain synaptosomes showed that type F neurotoxin binds to acceptor molecules which are completely distinct from those of the other three neurotoxins. 相似文献
2.
The binding of Clostridium botulinum type C neurotoxin to rat brain synaptosomes was determined by the use of 125I-neurotoxin. The binding was independent of the incubation temperature (0 degrees C and 37 degrees C) and was equilibrated in 10 min. The dose dependent of 125I-toxin binding to synaptosomes at 0 degrees C showed that there were two kinds of toxin receptors on the synaptosomal membrane; the association constants and maximum binding values were 1.05 x 10(10 M-1, 5.25 x 10(-13) mol/mg of synaptosomal protein and 5.00 x 10(6) M-1, 5.00 x 10(-12) mol/mg of synaptosomal protein, respectively. When the incubation of toxin with synaptosomes was continued at 37 degrees C after 125I-toxin had been pre-incubated with synaptosomes at 0 degrees C for 10 min, the displacement of labeled toxin by the addition of excess amounts of unlabeled toxin decreased slightly with increasing incubation time, and finally 0.4% of the bound 125I-toxin was not displaced from synaptosomes. The binding of 125I-toxin to synaptosomes was inhibited by anti-heavy chain IgG and a monoclonal antibody which neutralized toxin and recognized heavy chain. These results suggest that the binding sites of toxin to synaptosomes are localized on heavy chain and a small amount of the bound toxin is incorporated into the synaptosomal membrane or synaptosomes. 相似文献
3.
Characterization of cholecystokinin receptors in toad retina 总被引:2,自引:0,他引:2
The binding characteristics, structure, and pharmacologic properties of a cholecystokinin binding protein in toad retinal membranes have been studied. In competition binding studies using 125I-CCK-8, toad retinal membranes exhibited a high affinity binding site having a Ki50 of 1.5 nM using CCK-8 as competitive ligand. The relative potencies of CCK-related peptides in inhibiting radioligand binding were caerulein greater than gastrin II approximately equal to CCK-8 approximately equal to CCK-33 greater than CCK-8-DS approximately equal to gastrin I. L-364,718, a potent inhibitor of peripheral CCK receptors, was ineffective at competition binding at concentrations up to 1 microM; dibutyryl cyclic GMP was modestly effective at competing (KD approximately 10 mM). Covalent binding of 125I-CCK-33 to toad retinal membranes using chemical cross-linkers or UV irradiation resulted in the labeling of a major Mr 62,000 protein and the intermittent labeling of minor components of Mr 105,000 and Mr 40,000 as determined by SDS-PAGE and autoradiography. The binding of 125I-CCK-33 to retinal membranes and the concomitant labeling of the Mr 62,000 component was specifically inhibited by CCK-8 (KD approximately 1.5 nM). Reduction of membranes with DTT abolished specific binding of 125I-CCK. SDS-PAGE analysis of affinity cross-linked membranes under non-reducing conditions revealed that the Mr 62,000 protein migrated with an apparently lower molecular weight. These results suggest that the Mr 62,000 CCK binding protein in the toad retina contains an intramolecular disulfide bond(s). The Mr 62,000 protein was retained on a wheat germ agglutinin-agarose column and eluted with N-acetyl D-glucosamine, suggesting the glycoprotein nature of this protein. Digestion of the Mr 62,000 protein with neuraminidase together with O-glycanase resulted in a discrete product of Mr approximately 60,000. These results indicate that the Mr 62,000 protein is a glycoprotein with O-linked oligosaccharide chains. Taken together, these data indicate that the CCK receptor in toad retina has a distinct structure compared to that described in rat pancreas or brain. It will be important to establish whether this difference is reflected in differences in signal transduction mechanisms. 相似文献
4.
Characterization of Curaremimetic Neurotoxin Binding Sites on Cellular Membrane Fragments Derived from the Rat Pheochromocytoma PC 12 总被引:1,自引:1,他引:0
Ronald J. Lukas 《Journal of neurochemistry》1986,47(6):1768-1773
Studies were conducted on the properties of 125I-labeled alpha-bungarotoxin binding sites on cellular membrane fragments derived from the PC12 rat pheochromocytoma. Two classes of specific toxin binding sites are present at approximately equal densities (50 fmol/mg of membrane protein) and are characterized by apparent dissociation constants of 3 and 60 nM. Nicotine and d-tubocurarine are among the most potent inhibitors of high-affinity toxin binding. The affinity of high-affinity toxin binding sites for nicotinic cholinergic agonists is reversibly or irreversibly decreased, respectively, on treatment with dithiothreitol or dithiothreitol and N-ethylmaleimide. The nicotinic receptor affinity reagent bromoacetylcholine irreversibly blocks high-affinity toxin binding to PC12 cell membranes that have been treated with dithiothreitol. Two polyclonal antisera raised against the nicotinic acetylcholine receptor from Electrophorus electricus inhibit high-affinity toxin binding. These detailed studies confirm that curaremimetic neurotoxin binding sites on the PC12 cell line are comparable to toxin binding sites from neural tissues and to nicotinic acetylcholine receptors from the periphery. Because toxin binding sites are recognized by anti-nicotinic receptor antibodies, the possibility remains that they are functionally analogous to nicotinic receptors. 相似文献
5.
The inhibition of the binding of 125I-labeled Clostridium botulinum type C neurotoxin to synaptosomes by unlabeled toxin indicated that there were two kinds of receptors on the synaptosomal membrane. The dissociation constants (Kd) were calculated as 79 pM and 35 nM from the concentration of unlabeled toxin that induced half-displacement of bound 125I-toxin. These values agree satisfactorily with the values obtained from direct binding experiments (Agui, T, Syuto, B., Oguma, K., Iida, H., & Kubo, S. (1983) J. Biochem. 94, 521-527). The inhibition of the binding of 125I-toxin to synaptosomes and N-acetylneuraminyl(alpha 2-3)galactosyl(beta 1-3)N-acetylgalactosaminyl(beta 1-4) [N-acetylneuraminyl(alpha 2-8) N-acetylneuraminyl(alpha 2-3)]galactosyl(beta 1-4)glucosyl(beta 1-1)ceramide (GT1b) by unlabeled heavy chain indicated that heavy chain facilitates the binding of toxin to synaptosomes and GT1b. The synaptosomal and heavy chain complex Kd values were estimated as 12 nM and 24 microM. Monoclonal antibodies C-9 and CA-12 recognized the binding sites to GT1b and synaptosomes, respectively. Antigenic determinants against the two antibodies are presumably partially overlapping, and the overlapping area seems to be essential to the reaction between toxin and C-9 antibody. 相似文献
6.
Jun Ogasawara Yoichi Kamata Genji Sakaguchi Shunji Kozaki 《FEMS microbiology letters》1991,79(2-3):351-356
To characterize an acceptor for Clostridium botulinum type B neurotoxin, its binding kinetics were examined with mouse brain synaptosomes treated with various enzymes. The amount of 125I-labelled neurotoxin bound to synaptosomes decreased upon treatment with lysyl endopeptidase, neuraminidase, or phospholipase C. The binding of the neurotoxin was partially recovered by incubation of neuraminidase-treated synaptosomes with ganglioside GT1b or GD1a. Gangliosides incorporated into untreated, lysyl endopeptidase-treated, and phospholipase C-treated synaptosomes had no effect on the binding of the neurotoxin. These results may suggest that type B neurotoxin binds to gangliosides in cooperation with a certain protease-sensitive substance on the neural membranes. 相似文献
7.
Phosphatidylcholine Biosynthesis in the Neuroblastoma-Glioma Hybrid Cell Line NG 108-15: Stimulation by Phorbol Esters 总被引:3,自引:1,他引:2
Mordechai Liscovitch rew Freese Jan K. Blusztajn Richard J. Wurtman 《Journal of neurochemistry》1986,47(6):1936-1941
Studies were conducted on curaremimetic neurotoxin binding to the nicotinic acetylcholine receptor present on membrane fractions derived from the human medulloblastoma clonal line, TE671. High-affinity binding sites (KD = 2 nM for 1-h incubation at 20 degrees C) and low-affinity binding sites (KD = 40 nM) for 125I-labeled alpha-bungarotoxin are present in equal quantities (60 fmol/mg membrane protein). The kinetically determined dissociation constant for high-affinity binding of toxin is 0.56 nM (k1 = 6.3 X 10(-3) min-1 nM-1; k-1 = 3.5 X 10(-3) min-1) at 20 degrees C. Nicotine, d-tubocurarine, and acetylcholine are among the most effective inhibitors of high-affinity toxin binding. The quantity of toxin binding sites and their affinity for cholinergic agonists is sensitive to reduction, alkylation, and/or oxidation of membrane sulfhydryl residues. High-affinity toxin binding sites that have been subjected to reaction with the sulfhydryl reagent dithiothreitol are irreversibly blocked by the nicotinic receptor affinity reagent bromoacetylcholine. High-affinity toxin binding is inhibited in the presence of either of two polyclonal antisera or a monoclonal antibody raised against nicotinic acetylcholine receptors from fish electric tissue. Taken together, these results indicate that curaremimetic neurotoxin binding sites on membrane fractions of the TE671 cell line share some properties with nicotinic acetylcholine receptors of peripheral origin and with toxin binding sites on other neuronal tissues. 相似文献
8.
The omega-conotoxin receptor in brain membranes contains components of Mr approximately equal to 310,000, approximately equal to 230,000, and 37,000 as identified by photoaffinity labeling. The toxin specifically bound to two sites with apparent dissociation constants (Kd) of approximately 3 pM and 3.5 nM under the conditions employed. There was about 8 times more of the low affinity site than the high affinity site. Binding was not affected by dihydropyridines or verapamil. However, diltiazem stereospecifically inhibited the binding to the high affinity site. Dissociation of the toxin from the membranes was very slow and only partial. Among the detergents tested, digitonin solubilized the highest toxin-binding activity. The digitonin extract contained only a single class of binding sites with an apparent Kd of about 0.46 nM. Probably only the high affinity binding site was recovered in active form in digitonin extract. The properties of the toxin binding to digitonin extract were in good agreement with those of the binding to the high affinity site in the original membranes. Photoaffinity labeling of the digitonin extract indicated that the solubilized toxin receptor contained the two large components (Mr congruent 310,000 and approximately equal to 230,000) observed in the membranes. 相似文献
9.
Functional unit size of the neurotoxin receptors on the voltage-dependent sodium channel 总被引:1,自引:0,他引:1
K J Angelides T J Nutter L W Elmer E S Kempner 《The Journal of biological chemistry》1985,260(6):3431-3439
Radiation inactivation was used in situ to determine the functional unit sizes of the neurotoxin receptors of the voltage-dependent sodium channel from rat brain. Frozen or lyophilized synaptosomes were irradiated with high energy electrons generated by a linear accelerator and assayed for [3H]saxitoxin, 125I-Leiurus quinquestriatus quinquestriatus (alpha-scorpion toxin), 125I-Centruroides suffusus suffusus (beta-scorpion toxin), and batrachotoxinin-A 20 alpha-[3H]benzoate binding activity. The functional unit size of the neurotoxin receptors determined in situ by target analysis are 220,000 for saxitoxin, 263,000 for alpha-scorpion toxin, and 45,000 for beta-scorpion toxin. Analysis of the inactivation curve for batrachotoxinin-A 20 alpha-benzoate binding to the channel yields two target sizes of Mr approximately 287,000 (50%) and approximately 51,000 (50%). The results are independent of the purity of the membrane preparation. Comparison of the radiation inactivation data with the protein composition of the rat brain sodium channel indicates that there are at least two functional components. 相似文献
10.
Solubilization and physical characterization of acceptors for dendrotoxin and beta-bungarotoxin from synaptic membranes of rat brain 总被引:1,自引:0,他引:1
Dendrotoxin (DTX), an Mr 7000 convulsant polypeptide from the venom of Dendroaspis angusticeps, or its facilitatory homologues act through blockade of certain voltage-sensitive K+ currents in a variety of neurons. High-affinity acceptors for DTX have been demonstrated in synaptic plasma membranes of rat or chick brain, and a fraction of these avidly bind beta-bungarotoxin (beta-BuTX), a presynaptically active protein whose lighter B polypeptide is homologous to this toxin. Extraction of rat synaptic plasma membranes using Triton X-100 in K+-containing buffer yielded binding sites with KD values of approximately 0.5 and 0.7 nM for 125I-labeled DTX and beta-BuTX, respectively. The content of high-affinity sites obtained for beta-BuTX, including the contribution of a lower affinity component, approximates to the Bmax (approximately 1.3 pmol/mg of protein) obtained for the apparent single set of DTX acceptors. On solubilization, the pharmacological specificity of the acceptor for neurotoxic DTX congeners was retained. 125I-beta-BuTX binding (2.1 nM) was blocked efficaciously by DTX (IC50 = 1.6 nM) while the binding of 2.1 nM 125I-DTX was inhibited completely by beta-BuTX (IC50 = 25 nM); the lower potency of the latter could relate to the noncompetitive nature of the mutual competition and to the presence of high- and low-affinity sites for beta-BuTX. On gel filtration, or sedimentation analysis in H2O/sucrose and 2H2O/sucrose gradients, one peak of DTX binding activity was observed, and this was inhibitable by beta-BuTX. From the hydrodynamic properties of the acceptor/detergent/lipid complex (s20,w = 13.2 S; Stokes radius = 8.6 nm), a molecular weight of 405,000-465,000 was estimated.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
11.
Characterization of tetanus toxin binding to rat brain membranes. Evidence for a high-affinity proteinase-sensitive receptor. 总被引:4,自引:0,他引:4
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E J Pierce M D Davison R G Parton W H Habig D R Critchley 《The Biochemical journal》1986,236(3):845-852
Binding of 125I-labelled tetanus toxin to rat brain membranes in 25 mM-Tris/acetate, pH 6.0, was saturable and there was a single class of high-affinity site (KD 0.26-1.14 nM) present in high abundance (Bmax. 0.9-1.89 nmol/mg). The sites were largely resistant to proteolysis and heating but were markedly sensitive to neuraminidase. Trisialogangliosides were effective inhibitors of toxin binding (IC50 10 nM) and trisialogangliosides inserted into membranes lacking a toxin receptor were able to bind toxin with high affinity (KD 2.6 nM). The results are consistent with previous studies and the hypothesis that di- and trisialogangliosides act as the primary receptor for tetanus toxin under these conditions. In contrast, when toxin binding was assayed in Krebs-Ringer buffer, pH 7.4, binding was greatly reduced, was non-saturable and competition binding studies showed evidence for a small number of high-affinity sites (KD 0.42 nM, Bmax. 0.90 pmol/mg) and a larger number of low-affinity sites (KD 146 nM, Bmax. 179 pmol/mg). Treatment of membranes with proteinases, heat, and neuraminidase markedly reduced binding. Trisialogangliosides were poor inhibitors of toxin binding (IC50 11.0 microM), and trisialogangliosides inserted into membranes bound toxin with low affinity. The results suggest that in physiological buffers tetanus toxin binds with high affinity to a protein receptor, and that gangliosides represent only a low-affinity site. 相似文献
12.
Vasoactive intestinal peptide (VIP) receptors have been identified in CNS by their chemical specificity and molecular size. Using synaptosomes isolated from rat cerebral cortex, it was shown that central VIP receptors discriminated among natural and synthetic VIP-related peptides, because half-maximal inhibition of [125I]VIP binding to synaptosomes was obtained for 0.6 nM VIP, 9 nM peptide histidine isoleucineamide (PHI), 50 nM VIP 2-28, 70 nM secretin, 100 nM rat growth hormone-releasing factor (GRF), and 350 nM human GRF. Other peptides of the VIP family, such as glucagon and gastric inhibitory polypeptide, did not interact with cortical VIP receptors. The molecular components of VIP receptors in rat cerebral cortex were identified after [125I]VIP cross-linking to synaptosomes using the cross-linker dithiobis(succinimidyl propionate). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of synaptosomal proteins revealed two major [125I]VIP-protein complexes of Mr 49,000 and 18,000. The labeling of the Mr 49,000 component was specific, because it was abolished by native VIP, whereas the labeling of the Mr 18,000 component was not. Natural VIP agonists reduced the labeling of the Mr 49,000 component with the following order of potency: VIP greater than PHI greater than secretin approximately equal to rat GRF. In contrast, glucagon and octapeptide of cholecystokinin were without effect, a result indicating its peptide specificity. Densitometric scanning of autoradiographs showed that the labeling of the Mr 49,000 component was inhibited by low VIP concentrations between 10(-10) and 10(-6) M (IC50 = 0.8 nM), a result indicating the component's high affinity for VIP.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
13.
《Biochemical and biophysical research communications》1987,143(3):928-933
The binding ability of Cl. botulinum neurotoxin to synaptosomes upon treatment with various enzymes (neuraminidase, trypsin, and β-bungarotoxin containing phospholipase A2 activity) was studied. When synaptosomes were treated with neuraminidase, their ability to bind toxin decreased; trypsin and β-bungarotoxin had slightly week or no effect. The decrease in toxin-binding ability of synaptosomes was paralleled by a release of sialic acid from the synaptosomes by the neuraminidase treatment. The toxin-binding ability of synaptosomes treated with neuraminidase was lower than untreated ones at a high concentration of sodium chloride. The binding of the toxin to synaptosomes occurred at least at the two types of structural sites, one site which contained sialic acid, and other site which was sensitive to high ionic strength. It may be possible that another binding state except these is present at the synapse. 相似文献
14.
《Neurochemistry international》1988,13(1):25-36
Localization in rat CNS of the acceptors for botulinum neurotoxin (types A and B) was examined by lesioning of cholinergic input to the cortex and immuno-affinity purification of cholinergic nerve terminals. Ibotenic acid lesions of the cortical cholinergic tract caused a small reduction in the content of high affinity binding sites for type A neurotoxin and a concomitant decrease in the activities of acetylcholinesterase and choline acetyltransferase. No such change was observed in the level of acceptors for BoNT B or the extent of immuno-labelling of Chol-I, a cholinergic ganglioside. Purification of cholinergic nerve terminals, using anti-(Chol-I) antibodies gave an equivalent enrichment in the acceptors (high and low affinity) for both toxin types and choline acetyltransferase. Neurotoxin type B (but not type A) inhibited binding of anti-(Chol-I) antibodies to this cholinergic ganglioside on nerve terminals and to semi-purified Chol-I. It can be deduced from these collective findings that the high affinity binding sites for BoNT A and possibly B are localized on cholinergic nerve terminals in the CNS and that the Chol-I ganglioside may be associated with the acceptor for type B toxin. 相似文献
15.
Specific inhibition of [3H] saxitoxin binding to skeletal muscle sodium channels by geographutoxin II, a polypeptide channel blocker 总被引:4,自引:0,他引:4
Y Ohizumi H Nakamura J Kobayashi W A Catterall 《The Journal of biological chemistry》1986,261(14):6149-6152
Geographutoxin II (GTX II), a peptide toxin isolated from Conus geographus, inhibited [3H]saxitoxin binding to receptor sites associated with voltage-sensitive Na channels in rat skeletal muscle homogenates and rabbit T-tubular membranes with K0.5 values of 60 nM for homogenates and 35 nM for T-tubular membranes in close agreement with concentrations that block muscle contraction. Scatchard analysis of [3H]saxitoxin binding to T-tubular membranes gave values of KD = 9.3 nM and Bmax = 300 fmol/mg of protein and revealed a primarily competitive mode of inhibition of saxitoxin binding by GTX II. The calculated KD values for GTX II were 24 nM for T-tubules and 35 nM for homogenates, respectively. In rat brain synaptosomes, GTX II caused a similar inhibitory effect on [3H]saxitoxin binding at substantially higher concentrations (K0.5 = 2 microM). In contrast, binding of [3H]batrachotoxin A 20-alpha-benzoate and 125I-labeled scorpion toxin to receptor sites associated with Na channels in synaptosomes was not affected by GTX II at concentrations up to 10 microM. Furthermore, [3H]saxitoxin binding to membranes of rat superior cervical ganglion was only blocked 10% by GTX II at 10 microM. These results indicate that GTX II interacts competitively with saxitoxin in binding at neurotoxin receptor site 1 on the sodium channel in a highly tissue-specific manner. GTX II is the first polypeptide ligand for this receptor site and the first to discriminate between this site on nerve and adult muscle sodium channels. 相似文献
16.
The presynaptically active snake venom neurotoxin beta-bungarotoxin (beta-Butx) is known to affect neurotransmitter release by binding to a subtype of voltage-activated K+ channels. Here we show that mast cell degranulating (MCD) peptide from bee venom inhibits the binding of 125I-labeled beta-Butx to chick and rat brain membranes with apparent Ki values of 180 nM and 1100 nM, respectively. The mechanism of inhibition by MCD peptide is noncompetitive, as is inhibition of 125I-beta-Butx binding by the protease inhibitor homologue from mamba venom, toxin I. Beta-Butx and its binding antagonists thus bind to different sites of the same membrane protein. Removal of Ca2+ by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid inhibits the binding of 125I-beta-Butx by lowering its affinity to brain membranes. 相似文献
17.
The ligand binding subunit of the D2 dopamine receptor (Mr approximately equal to 94,000) can be visualized by autoradiography following photoaffinity labeling with [125I]N-azidophenethylspiperone and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Following removal of sialic acids with the exoglycosidase, neuraminidase, [125I]N-azidophenethylspiperone photoincorporated into a protein of Mr = 54,000 with the appropriate pharmacological profile for D2 receptors. The desialylated D2 receptor bound dopaminergic agonists with high affinity and was capable of coupling to a functional G-protein as indexed by: 1) pertussis-toxin mediated [32P]ADP ribosylation of proteins of Mr = 42,000 and 39,000, and 2) the conversion of the agonist high affinity form of D2 receptors to one displaying low affinity for agonists in the presence of guanine nucleotides. These data suggest that sialic acid residues do not contribute significantly to the ligand binding characteristics of D2 receptors despite the large change produced in the estimated molecular mass of the binding subunit. 相似文献
18.
Bovine Chromaffin Cells Have Insulin-Like Growth Factor-I (IGF-I) Receptors: IGF-I Enhances Catecholamine Secretion 总被引:9,自引:4,他引:5
The binding of 125I-insulin-like growth factor-I (125I-IGF-I) to bovine chromaffin cells was measured. Chromaffin cell cultures contained 111,000 +/- 40,000 IGF-I binding sites/cell. These sites bound IGF-I with a KD of 1.1 +/- 0.3 nM and had a much lower affinity for insulin. Cross-linking studies showed that 125I-IGF-I bound to a protein that had an Mr of approximately 125,000, similar to the Mr of the alpha subunit of the IGF-I receptor in other tissues. Cells cultured with IGF-I (10 nM) for 4 days exhibited an almost twofold increase in high K+-evoked catecholamine secretion. Insulin was much less potent than IGF-I in enhancing catecholamine secretion. These data indicate that binding of IGF-I to its receptors on chromaffin cells can modulate the function of these cells. 相似文献
19.
The Dopamine Transporter Is Absent in Parkinsonian Putamen and Reduced in the Caudate Nucleus 总被引:7,自引:2,他引:5
Hyman B. Niznik Evan F. Fogel Frank F. Fassos Philip Seeman 《Journal of neurochemistry》1991,56(1):192-198
The neuronal dopamine transporter/uptake site can be covalently labeled with the photoaffinity probe 1-(2-[bis-(4-fluorophenyl) methoxy]ethyl)-4-[2-(4-azido-3-[125I]iodophenyl)ethyl]piperazine [( 125I]FAPP) and visualized following sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. Upon photolysis, [125I]FAPP specifically incorporated into a polypeptide of apparent Mr = 62,000 in membranes from both the putamen and the caudate nucleus of control, Alzheimer's, schizophrenia, and Huntington's diseased brain, and following complete deglycosylation, migrated as an Mr approximately 48,000 polypeptide. In parkinsonian postmortem putamen, however, there was no detectable photoincorporation of [125I]FAPP into the ligand binding subunit of the dopamine transporter. [125I]FAPP did specifically label the Mr 62,000 polypeptide of parkinsonian caudate, although with efficiencies of 20-50% of control. The asymmetrical loss of the dopamine transporter in Parkinson's diseased striatum was confirmed in reversible receptor binding experiments using [3H]GBR-12935 (3H-labeled 1-[2-(diphenylmethoxy) ethyl]-4-(3-phenylpropyl)piperazine). In parkinsonian putamen, mazindol competitively inhibited the binding of [3H]GBR-12935 with an estimated affinity (Ki approximately 2,000 nM) 10 times lower than in controls (Ki approximately 30 nM), while the affinity of maxindol for [3H]GBR-12935 binding in the caudate was equal to that seen with controls (Ki approximately 50 nM). The proportion of [3H]GBR-12935 binding sites recognized by mazindol with high affinity in Parkinson's diseased caudate was, however, reduced by 50-80%.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
20.
Two acceptor sub-types for dendrotoxin in chick synaptic membranes distinguishable by beta-bungarotoxin 总被引:5,自引:0,他引:5
Using chick synaptic membranes, proteinaceous acceptors were characterized for dendrotoxin, a polypeptide from Dendroaspis angusticeps with convulsant activity due to its facilitation of transmitter release, resulting from inhibition of A-current K+ channels in brain. Both equilibrium and kinetic measurements of radioiodinated toxin binding showed that two populations of membraneous acceptors were discernible with different affinities (Kd approximately 0.5 nM and 15 nM; Bmax approximately 90 and 400 fmol/mg protein). Only the high-affinity component interacted avidly with beta-bungarotoxin, an inhibitory presynaptic neurotoxin whose lighter chain is homologous to dendrotoxin. Facilitatory homologues of dendrotoxin from Dendroaspis species antagonised its binding to both acceptor sub-types in proportion to their central neurotoxicities, whereas various other toxins (crotoxin, apamin), trypsin inhibitors and lectins proved ineffective. Cross-linking of toxin specifically bound to its membrane acceptors, using bis-imido esters followed by electrophoretic analysis in the presence of sodium dodecyl sulphate, revealed a polypeptide with Mr of 75,000 together with lesser amounts of a 69,000-Mr component. Notably, the covalent labelling of each of these bands was inhibited partially by low concentrations of beta-bungarotoxin, indicating that they are derived from both acceptor species. The demonstrated existence of an acceptor form shared by dendrotoxin and beta-bungarotoxin, together with another sub-type selective for dendrotoxin, is discussed in relation to the known pharmacological interactions of these toxins which exert opposite effects on transmitter release. 相似文献