共查询到20条相似文献,搜索用时 46 毫秒
1.
J P Merlie S Heinemann B Einarson J M Lindstrom 《The Journal of biological chemistry》1979,254(14):6328-6332
The degradation of acetylcholine receptor observed in denervated and innervated normal rat diaphragms in organ culture is stimulated by exogenous antireceptor serum. In this paper we demonstrate that diaphragms from rats with experimental autoimmune myasthenia gravis contain reduced amounts of acetylcholine receptor. Acetylcholine receptor from myasthenic, but not from normal, rats has antibody bound to it and is degraded at an accelerated rate. We conclude that in the chronic phase of experimental autoimmune myasthenia gravis increased acetylcholine receptor degradation can be accounted for by a mechanism involving antigenic modulation, and that such a process can contribute to the clinical symptoms of impaired neuromuscular transmission. 相似文献
2.
Inhibitors of membrane depolarization regulate acetylcholine receptor synthesis by a calcium-dependent, cyclic nucleotide-independent mechanism 总被引:6,自引:0,他引:6
The inhibition of membrane depolarization by tetrodotoxin or the local anesthetic benzocaine elevates the acetylcholine receptor levels in cultured myotubes. The elevated acetylcholine receptor levels are due to increased receptor synthesis rather than to decreased degradation. The effects of tetrodotoxin and benzocaine on acetylcholine receptor levels are not additive, and are not inhibited by exogenously added cyclic GMP analogues or by elevated intracellular levels of cyclic GMP. However, the stimulation of acetylcholine receptor levels by tetrodotoxin or benzocaine is reversed by the addition of the calcium ionophore A23187. In contrast, tetrodotoxin or benzocaine stimulated acetylcholine receptor synthesis beyond the maximal stimulation produced by cholera toxin. These results suggest that the inhibition of membrane depolarization elevates acetylcholine receptor synthesis by a calcium-dependent, cyclic nucleotide-independent mechanism. 相似文献
3.
Prospects for specific immunotherapy in myasthenia gravis 总被引:1,自引:0,他引:1
Myasthenia gravis is an autoimmune disease resulting from a breakdown in T and B cell tolerance to acetylcholine receptor (AChR). Autoantibodies to AChR mediate the disease. Recent advances in experimental immunotherapy of autoimmune disease provide several possibilities for specific intervention in this well-characterized condition. 相似文献
4.
Biochemical characterization of two nicotinic receptors from the optic lobe of the chick 总被引:4,自引:0,他引:4
We have studied putative nicotinic acetylcholine receptors in the optic lobe of the newborn chick, using 125I-labeled alpha-bungarotoxin, a specific blocker of acetylcholine receptors in the neuromuscular junction, and [3H]acetylcholine, a ligand which in the presence of atropine selectively labels binding sites of nicotinic character in rat brain cortex (Schwartz et al., 1982). [3H]Acetylcholine binds reversibly to a single class of high affinity binding sites (KD = 2.2 X 10(-8) M) which occur at a tissue concentration of 5.7 pmol/g. A large fraction (approximately 60%) of these binding sites is solubilized by Triton X-100, sodium cholate, or the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Solubilization increases the affinity for acetylcholine and several nicotinic drugs from 1.5- to 7-fold. The acetylcholine-binding macromolecule resembles the receptor for alpha-bungarotoxin present in the same tissue with respect to subcellular distribution, hydrodynamic properties, lectin binding, and agonist affinity rank order. It differs from the toxin receptor in affinity for nicotinic antagonists, sensitivity to thermal inactivation, and regional distribution. The solubilized [3H]acetylcholine binding activity is separated from the toxin receptor by incubation with agarose-linked acetylcholine, by affinity chromatography on immobilized Naja naja siamensis alpha-toxin, and by precipitation with a monoclonal antibody to chick optic lobe toxin receptor. 相似文献
5.
Effects of inflammatory cells on neuronal M2 muscarinic receptor function in the lung 总被引:6,自引:0,他引:6
In the lungs, acetylcholine released from the parasympathetic nerves stimulates M3 muscarinic receptors on airway smooth muscle inducing contraction and bronchoconstriction. The amount of acetylcholine released from these nerves is limited locally by neuronal M2 muscarinic receptors. These neuronal receptors are dysfunctional in asthma and in animal models of asthma. Decreased M2 muscarinic receptor function results in increased release of acetylcholine and in airway hyperreactivity. Inflammation has long been associated with hyperreactivity and the role of inflammatory cells in loss of neuronal M2 receptor function has been examined. There are several different mechanisms for loss of neuronal M2 receptor function. These include blockade by endogenous antagonists such as eosinophil major basic protein, decreased expression of M2 receptors following infection with viruses or exposure to pro inflammatory cytokines such as gamma interferon. Finally, the affinity of acetylcholine for these receptors can be decreased by exposure to neuraminidase. 相似文献
6.
Average membrane penetration depth of tryptophan residues of the nicotinic acetylcholine receptor by the parallax method. 总被引:5,自引:0,他引:5
The membrane penetration depths of tryptophan residues in the nicotinic acetylcholine receptor from Torpedo californica have been analyzed in reconstituted membranes containing purified receptor and defined lipids. Dioleoylphosphatidylcholine and three spin-labeled phosphatidylcholines with the nitroxide group at three different positions on the fatty acyl chain were used for reconstitution of the receptor. The spin-labeled phospholipids serve as quenchers of tryptophan fluorescence. Differential quenching of the intrinsic fluorescence of the acetylcholine receptor by the spin-labeled phospholipids has been utilized to analyze the average membrane penetration depth of tryptophans by the parallax method [Chattopadhyay, A., & London, E. (1987) Biochemistry 26, 39-45]. Analyses of the quenching data indicate that the tryptophan residues on the average are at a shallow location (10.1 A from the center of the bilayer) in the membrane. In addition, the generally low levels of quenching imply that the majority of tryptophan residues are located in the putative extramembranous region of the receptor. These results are consistent with several proposed models for the tertiary structure of the acetylcholine receptor and are relevant to ongoing analyses of the overall conformation and orientation of the acetylcholine receptor in the membrane. 相似文献
7.
Functional stability of Torpedo acetylcholine receptor. Effects of protease treatment 总被引:4,自引:0,他引:4
The effect of tryptic degradation on structural and functional properties of the membrane-bound acetylcholine receptor from Torpedo californica has been investigated. Under conditions of proteolysis which resulted in extensive degradation of receptor subunits, the membrane preparations retained their full capability of mediating agonist-induced cation flux as measured in rapid kinetic experiments. Low concentrations on trypsin also cleaved receptor dimers to monomers, and this effect was paralleled by degradation of the Mr 65 000 subunits which are known to contain sulfhydryl group(s) involved in receptor dimerization through an interchain disulfide bond(s). This conversion to monomers occurred at lower trypsin concentrations when the enzyme was added to the outside of the vesicles compared with the effects observed when the enzyme was present inside the vesicles. Similarly Mr 43 000 protein consistently found in preparations of the membrane-bound acetylcholine receptor, which can readily be removed without apparent effect on receptor function, displayed greater susceptibility to proteolysis when trypsin was added to the exterior medium rather than inside the vesicles. The results emphasize the full functionality of the monomeric form of the acetylcholine receptor comprised of four polypeptides. 相似文献
8.
Steve Burden 《Developmental biology》1977,61(1):79-85
The turnover of acetylcholine receptors labeled with 125I-labeled α-bungarotoxin was measured in the developing posterior latissimus dorsi muscle of the chick. The degradation rates for acetylcholine receptors at the neuromuscular junction and in extrajunctional regions of the muscle cell were determined. One week after hatching, the rates of junctional and extrajunctional receptor degradation are identical (). Three weeks weeks after hatching, however, the rate of junctional receptor degradation is considerably slower () and different than the rate of extrajunctional receptor degradation (). Thus, receptors which are localized at the neuromuscular junction early in embryonic life only become stable several weeks after hatching. 相似文献
9.
Protease effects on the structure of acetylcholine receptor membranes from Torpedo californica 总被引:14,自引:7,他引:7
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《The Journal of cell biology》1980,85(3):823-838
Protease digestion of acetylcholine receptor-rich membranes derived from Torpedo californica electroplaques by homogenization and isopycnic centrifugation results in degradation of all receptor subunits without any significant effect on the appearance in electron micrographs, the toxin binding ability, or the sedimentation value of the receptor molecule. Such treatment does produce dramatic changes in the morphology of the normally 0.5- to 2-microns-diameter spherical vesicles when observed by either negative-stain or freeze-fracture electron microscopy. Removal of peripheral, apparently nonreceptor polypeptides by alkali stripping (Neubig et al. 1979, Proc. Natl. Acad. Sci. U. S. A. 76:690-694) results in increased sensitivity of the acetylcholine receptor membranes to the protease trypsin as indicated by SDS gel electrophoretic patterns and by the extent of morphologic change observed in vesicle structure. Trypsin digestion of alkali- stripped receptor membranes results in a limit degradation pattern of all four receptor subunits, whereupon all the vesicles undergo the morphological transformation to minivesicles. The protein-induced morphological transformation and the limit digestion pattern of receptor membranes are unaffected by whether the membranes are prepared so as to preserve the receptor as a disulfide bridged dimer, or prepared so as to generate monomeric receptor. 相似文献
10.
11.
Evidence for a voltage-dependent regulatory (inhibitory) site on the nicotinic acetylcholine receptor to which acetylcholine binds was obtained in membrane vesicles prepared from the Torpedo californica electric organ. Two rate coefficients, JA and alpha, which pertain to the receptor-controlled ion flux, were measured. A 1000-fold concentration range of acetylcholine was used in a transmembrane voltage (Vm) range from 0 to -48 mV under a voltage-clamped condition at pH 7.4, 1 degrees C. The following observations were made. (i) At low acetylcholine concentrations, the value of JA, the rate coefficient for ion translocation by the active (nondesensitized) state of the receptor, increased with increasing concentration. (ii) JA decreased at high acetylcholine concentrations. (iii) In contrast, alpha, the rate coefficient for receptor desensitization, did not show such a decrease. (iv) When the transmembrane potential of the vesicle membrane was changed to more negative values, the value of KR (the dissociation constant for binding of acetylcholine to the regulatory site) decreased by a factor of approximately 9 for a 25 mV change in Vm, while KI (the dissociation constant for binding of acetylcholine to the receptor site that controls channel opening) did not show such a change and has a value of 80 microM. When Vm is -48 mV, KR has a value of 8 microM. (v) The effect of a transmembrane voltage on the regulatory site was reversible and occurred within the time resolution (5 ms) of the quench-flow technique used in the measurements.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
12.
In the studies reported here, we investigated whether the degradation of the acetylcholine receptor (AChR) in cultured muscle cells involves similar mechanisms as and is controlled in a manner similar to, the catabolism of the bulk of cell protein. We compared these processes after labeling cell protein with radioactive leucine or phenylalanine for 24 hours, or labeling the acetylcholine receptor with (125I)-bungarotoxin. The apparent average half-life of cell protein was 38 ± 2 hours and that of the receptor-toxin complex was 25 ± 1 hours. Incubation in media lacking serum and embryo extract accelerated the degradation of both average protein and the receptor-toxin complex. Insulin reduced the rate of catabolism of both average protein and the receptor-toxin complex toward levels seen in the presence of serum. However, although these two degradative processes seem to be controlled similarly, they probably involve different mechanisms. The protease inhibitors leupeptin and chymostatin, which slowed overall proteolysis in nongrowing muscles and hepatocytes, reduced the degradation of the ACh receptor by 2–11-fold, but had no, or only slight, effects on the catabolism of average protein, even when overall proteolysis was accelerated by omitting serum and embryo extract. Chloroquine, an inhibitor of lysosomal function, also reduced the degradation of AChR, by about 10-fold, but decreased overall protein breakdown by only 20–30%. Incubation of myotubes at lower temperatures reduced both degradative processes, but affected the breakdown of the receptor to a greater extent. Thus the rate-limiting steps in these processes have different activation energies. Incubation with 2-deoxyglucose, an inhibitor of glycolysis, decreased the breakdown of average protein but not that of the receptor-toxin complex. However, the two degradative processes were sensitive to azide, an inhibitor of oxidative phosphorylation. Although the lysosome is the primary site for AChR degradation and perhaps for degradation of other surface proteins, the breakdown of most proteins in myotubes seems to involve a distinct proteolytic system requiring metabolic energy. 相似文献
13.
Decrease in acetylcholine-receptor content of human myotube cultures mediated by monoclonal antibodies to alpha, beta and gamma subunits 总被引:4,自引:0,他引:4
One of the two main causes of acetylcholine-receptor loss in myasthenia gravis is antigenic modulation, i.e. accelerated internalization and degradation rate by antibody-crosslinking. This phenomenon has been studied only in animal tissues. Therefore, we tested antigenic modulation of the acetylcholine receptor on human embryonic myotubes in cultures. Several monoclonal antibodies to the alpha, beta and gamma subunits of the receptor reduced its concentration, in some cases down to one-third of the control. Some of these antibodies only form complexes of one antibody with two receptor molecules; consequently such small complexes are sufficient to accelerate internalization of the human acetylcholine receptor. This technique might be proved valuable for clinical screening of sera from myasthenic patients. 相似文献
14.
Cortez L Marino-Buslje C de Jiménez Bonino MB Hellman U 《Biochemical and biophysical research communications》2007,355(1):275-279
The muscle-type nicotinic receptor has two distinguishable acetylcholine binding sites at the alpha-gamma and alpha-delta subunit interfaces; alpha-conotoxins can bind them selectively. Moreover, we previously reported that alpha-conotoxin MI can interact with Torpedo californica and Torpedo marmorata receptors showing that conotoxins can also detect receptors from different species of the same genus [L. Cortez, S.G. del Canto, F. Testai, M.B. de Jiménez Bonino, Conotoxin MI inhibits the acetylcholine binding site of the Torpedo marmorata receptor, Biochem. Biophys. Res. Commun. 295 (2002) 791-795]. Herein, to identify T. marmorata receptor regions involved in alpha-conotoxin MI binding, a photoactivatable reagent was used and labeled sites were mapped by enzymatic proteolysis, MALDI-TOF-MS and Edman degradation. alpha-Conotoxin MI binding determinants were found and studies revealed a second binding motif at the alpha/delta interface. A proposal for receptor-toxin interaction is discussed based on experimental results and docking studies. 相似文献
15.
J P Earnest H H Wang M G McNamee 《Biochemical and biophysical research communications》1984,123(2):862-868
Using electron spin resonance spectroscopy and a spin-labeled analog of a tertiary amine local anesthetic, we have identified several populations of the local anesthetic within reconstituted lipid membranes containing purified acetylcholine receptors. These populations represent the local anesthetic interacting with membrane lipid and with the acetylcholine receptor. The data also suggest the existence of at least two classes of binding sites for the local anesthetic on the acetylcholine receptor. 相似文献
16.
W E Thomas J G Townsel 《Comparative biochemistry and physiology. C: Comparative pharmacology》1982,72(1):121-123
1. The interaction of two specific ligands for the vertebrate nicotinic acetylcholine receptor were investigated on the solubilized form of a proposed acetylcholine receptor from the invertebrate Limulus polyphemus. 2. The affinity agent 4-(N-maleimodo)benzyltrimethylammonium iodide exhibited no effect on the binding of alpha-bungarotoxin to the Limulus receptor protein. 3. Torpedo acetylcholine receptor antibody neither inhibited alpha-bungarotoxin binding nor produced any alteration in the sedimentation profile of the Limulus receptor. 4. The lack of interaction of 4-(N-maleimido)benzyltrimethylammonium iodide and Torpedo acetylcholine receptor antibody with the Limulus acetylcholine receptor was interpreted to reflect significant difference between the molecular structures of this invertebrate receptor and the acetylcholine receptor of vertebrate. 相似文献
17.
An accurate and sensitive assay for nicotinic acetylcholine receptor binding sites is described which is based on the specificities of receptor both for an affinity label, 4-(N-maleimido)benzyltrimethylammonium iodide (MBTA), and for α-neurotoxins from Naja venoms. It has been demonstrated that MBTA reacts exclusively with one type of subunit of the acetylcholine receptors isolated from the electric tissue of Electrophorus electricus and Torpedo californica and that this reaction is blocked in the presence of Naja naja siamensis α-neurotoxin and of other ligands of the acetylcholine binding site. Thus, in this assay the difference in the extent of labeling by MBTA in the absence and presence of N. n. siamensis toxin is considered the specific labeling of receptor. Although this assay is more complicated than direct α-neurotoxin binding, it is justified by the wellestablished site specificity of the labeling. The specific activities of several different receptor preparations determined using this assay are one-half of those determined using toxin binding. It is possible to assay accurately as little as 0.25 μg of receptor in the presence of 100-fold as much other protein. 相似文献
18.
Postsynaptic membranes in the electric tissue of Narcine: IV. Isolation and characterization of the nicotinic receptor protein 总被引:7,自引:0,他引:7
The nicotinic receptor protein of the electric tissue of Narcine was purified in several different media by partial isolation of postsynaptic membranes and affinity chromatography. Protease inhibitors were found to be necessary to prevent degradation of the protein, and both EDTA and Tris buffer were used in addition to prevent intramolecular crosslinking of 44,000 and 58,000 dalton subunits by tissue factors. The intact protein was found to have a molecular weight close to 400,000, and appears to be composed of four subunits of 44,000 daltons, two to three of 48,000, one of 58,000 and one of 65,000. All the subunits are glycoproteins and their amino acid compositions show similar hydrophobicity and acidity, suggesting similar positioning in postsynaptic membranes. Crosslinking experiments showed that acetylcholine and alpha-bungarotoxin bind to the smallest subunit, and suggest the juxtaposition of at least two of these subunits, and of all four toxin molecules bound to a receptor molecule. Morphological studies of the protein in membranes and after purification indicated cylindrial molecules with central cores. 相似文献
19.
Regulation of acetylcholine receptor by cyclic AMP 总被引:6,自引:0,他引:6
In primary cultures of chick 11-day embryonic tissue a number of phosphodiesterase inhibitors were found to elevate acetylcholine receptor levels. Of these agents, Ro20-1724 was the most effective, elevating surface receptor content by 2-fold after 48 h of treatment. 8-Br-cAMP and cholera toxin, a natural activator of adenylate cyclase, mimicked the effect of Ro20-1724, while 8-Br-cGMP and dibutyryl cGMP had no effect. Cholera toxin, 8-Br-cAMP, and Ro20-1724 all increased the insertion rate of new receptor into the surface membrane without altering degradation. The enhanced insertion appears related to an actual increase in synthesis since total acetylcholine receptor was elevated by exposure to cholera toxin. In contrast, no change in creatine phosphokinase activity, myosin heavy chain content, or [35S] methionine incorporation into total cellular protein was observed during cholera toxin treatment. These results suggest that cAMP plays a role in the regulation of acetylcholine receptor. 相似文献
20.
Robert Roskoski Jr. Richard Guthrie Jr. Laura M. Roskoski Wojciech Rossowski 《Journal of neurochemistry》1985,45(4):1096-1100
Cholinergic muscarinic receptors undergo proteolytic degradation in vitro under physiological conditions as shown by a loss in [3H]quinuclidinylbenzilate binding activity. The serine protease inhibitor phenylmethylsulfonyl fluoride was very effective in diminishing the receptor loss. Soybean trypsin inhibitor was less effective. Both EDTA and EGTA were also effective in abolishing receptor degradation, suggesting the involvement of metallopeptidases in the process. Calcium-dependent neutral proteases requiring sulfhydryl reducing agents did not seem to be involved in receptor degradation. Dithiothreitol failed to enhance receptor degradation and iodoacetamide, leupeptin, and antipain, inhibitors of this enzyme class, failed to alter receptor loss as measured by radioligand binding. Most of the proteolytic activity occurred in the cytosol and was readily resolved from the receptor in the membrane fraction. We found that [3H]quinuclidinylbenzilate, an antagonist, inhibited the rate of receptor loss. On the other hand, agonists (acetylcholine, methacholine, and muscarine) appeared to enhance the rate of receptor loss. We postulate that these opposite effects are due to differences in receptor conformation in response to ligand binding. Susceptibility to proteolysis may therefore serve as a probe for receptor conformation. 相似文献