scholarly journals Nicotinic postsynaptic membranes from Torpedo: sidedness, permeability to macromolecules, and topography of major polypeptides.

1982 ◽  
Vol 92 (2) ◽  
pp. 333-342 ◽  
Author(s):  
P A St John ◽  
S C Froehner ◽  
D A Goodenough ◽  
J B Cohen
Keyword(s):  
Binding Sites ◽  
Electric Organ ◽  
Postsynaptic Membrane ◽  
Relative Molecular Mass ◽  
Alkaline Extraction ◽  
Before And After ◽  
Torpedo Electric Organ ◽  
Extracellular Side ◽  
Alpha Bungarotoxin

Experiments were conducted to examine the topographic arrangement of the polypeptides of the acetylcholine receptor (AcChR) and the nonreceptor Mr 43,000 protein in postsynaptic membranes isolated from Torpedo electric organ. When examined by electron microscopy, greater than 85% of vesicles were not permeable to ferritin or lactoperoxidase (LPO). Exposure to saponin was identified as a suitable procedure to permeabilize the vesicles to macromolecules with minimal alteration of vesicle size or ultrastructure. The sidedness of vesicles was examined morphologically and biochemically. Comparison of the distribution of intramembrane particles on freeze-fractured vesicles and the distribution found in situ indicated that greater than 85% of the vesicles were extracellular-side out. Vesicles labeled with alpha-bungarotoxin (alpha-Bgtx) were reacted with antibodies against alpha-BgTx or against purified AcChR of Torpedo. Bound antibodies were detected by the use of ferritin-conjugated goat anti-rabbit antibody and were located on the outside of greater than 99% of labeled vesicles. Similar results were obtained for normal vesicles or vesicles exposed to saponin. Quantification of the amount of [3H]-alpha-BgTx bound to vesicles before and after they were made permeable with saponin indicated that less than 5% of alpha-BgTx binding sites were cryptic in normal vesicles. It was concluded that greater than 95% of postsynaptic membranes were oriented extracellular-side out. LPO-catalyzed radioiodinations were performed on normal and saponin-treated vesicles and on vesicles from which the Mr (relative molecular mass) 43,000 protein had been removed by alkaline extraction. In normal vesicles, polypeptides of the AcChR were iodinated while the Mr 43,000 protein was not. In vesicles made permeable with saponin, the pattern of labeling of AcChR polypeptides was unchanged, but the Mr 43,000 protein was heavily iodinated. The relative iodination of AcChR polypeptides was unchanged in membranes equilibrated with agonist or with alpha-BgTx or after alkaline-extraction. It was concluded that the Mr 43,000 protein is present on the intracellular surface of the postsynaptic membrane and that AcChR polypeptides are exposed on the extracellular surface.

scholarly journals Association of the postsynaptic 43K protein with newly formed acetylcholine receptor clusters in cultured muscle cells.

1985 ◽  
Vol 100 (5) ◽  
pp. 1698-1705 ◽  
Author(s):  
H B Peng ◽  
S C Froehner
Keyword(s):  
Acetylcholine Receptor ◽  
Binding Sites ◽  
Electric Organ ◽  
Muscle Cells ◽  
Postsynaptic Membrane ◽  
Latex Beads ◽  
Peripheral Membrane Protein ◽  
Double Label ◽  
Torpedo Electric Organ ◽  
Receptor Clusters

The postsynaptic membrane from Torpedo electric organ contains, in addition to the acetylcholine receptor (AChR), a major peripheral membrane protein of approximately 43,000 mol wt (43K protein). Previous studies have shown that this protein is closely associated with AChR and may be involved in anchoring receptors to the postsynaptic membrane. In this study, binding sites for monoclonal antibodies (mabs) to the 43K protein have been compared to the distribution of AChR in Xenopus laevis muscle cells in culture. In double label immunofluorescence experiments, clusters of AChR that occur spontaneously on these cells were stained with anti-43K mabs. Newly formed receptor clusters induced with positive polypeptide-coated latex beads were also stained with anti-43K mabs as early as 12 h after the application of the beads. Exact correspondence in the distribution of the anti-43K protein binding sites and the AChR was found in both types of clusters. These results suggest that the 43K protein becomes associated with AChR clusters during a period of active postsynaptic membrane differentiation. Thus, this protein may participate in the clustering process.

scholarly journals 300-kD subsynaptic protein copurifies with acetylcholine receptor-rich membranes and is concentrated at neuromuscular synapses

1987 ◽  
Vol 104 (4) ◽  
pp. 939-946 ◽  
Author(s):  
ML Woodruff ◽  
J Theriot ◽  
SJ Burden
Keyword(s):  
Acetylcholine Receptor ◽  
Electric Organ ◽  
Postsynaptic Membrane ◽  
Synaptic Membrane ◽  
Binding Assays ◽  
Western Blots ◽  
Neuromuscular Synapses ◽  
Peripheral Membrane Proteins ◽  
Torpedo Electric Organ ◽  
Skeletal Muscle Antibodies

Acetylcholine receptor-rich membranes from the electric organ of Torpedo californica are enriched in the four different subunits of the acetylcholine receptor and in two peripheral membrane proteins at 43 and 300 kD. We produced monoclonal antibodies against the 300-kD protein and have used these antibodies to determine the location of the protein, both in the electric organ and in skeletal muscle. Antibodies to the 300-kD protein were characterized by Western blots, binding assays to isolated membranes, and immunofluorescence on tissue. In Torpedo electric organ, antibodies to the 300-kD protein stain only the innervated face of the electrocytes. The 300-kD protein is on the intracellular surface of the postsynaptic membrane, since antibodies to the 300-kD protein bind more efficiently to saponin-permeabilized, right side out membranes than to intact membranes. Some antibodies against the Torpedo 300-kD protein cross-react with amphibian and mammalian neuromuscular synapses, and the cross-reacting protein is also highly concentrated on the intracellular surface of the post-synaptic membrane.

scholarly journals A protein homologous to the Torpedo postsynaptic 58K protein is present at the myotendinous junction.

1990 ◽  
Vol 110 (6) ◽  
pp. 2061-2071 ◽  
Author(s):  
Q Chen ◽  
R Sealock ◽  
H B Peng
Keyword(s):  
Muscle Fibers ◽  
Electric Organ ◽  
Protein Band ◽  
Postsynaptic Membrane ◽  
Myotendinous Junction ◽  
Double Labeling ◽  
Torpedo Electric Organ ◽  
Myotomal Muscle

The 58K protein is a peripheral membrane protein enriched in the acetylcholine receptor (AChR)-rich postsynaptic membrane of Torpedo electric organ. Because of its coexistence with AChRs in the postsynaptic membrane in both electrocytes and skeletal muscle, it is thought to be involved in the formation and maintenance of AChR clusters. Using an mAb against the 58K protein of Torpedo electric organ, we have identified a single protein band in SDS-PAGE analysis of Xenopus myotomal muscle with an apparent molecular mass of 48 kD. With this antibody, the distribution of this protein was examined in the myotomal muscle fibers with immunofluorescence techniques. We found that the 48K protein is concentrated at the myotendinous junctions (MTJs) of these muscle fibers. The MTJ is also enriched in talin and vinculin. By double labeling muscle fibers with antibodies against talin and the 48K protein, these two proteins were found to colocalize at the membrane invaginations of the MTJ. In cultured myotomal muscle cells, the 48K protein and talin are also colocalized at sites of membrane-myofibril interaction. The 48K protein is, however, not found at focal adhesion sites in nonmuscle cells, which are enriched in talin. These data suggest that the 48K protein is specifically involved in the interaction of myofibrillar actin filaments with the plasma membrane at the MTJ. In addition to the MTJ localization, 48K protein is also present at AChR clusters both in vivo and in vitro. Thus, this protein is shared by both the MTJ and the neuromuscular junction.

scholarly journals Identification of an intracellular postsynaptic antigen at the frog neuromuscular junction.

1982 ◽  
Vol 94 (3) ◽  
pp. 521-530 ◽  
Author(s):  
S Burden
Keyword(s):  
Monoclonal Antibody ◽  
Intermediate Filaments ◽  
Amorphous Material ◽  
Neuromuscular Synapse ◽  
Electric Organ ◽  
Postsynaptic Membrane ◽  
Frog Neuromuscular Junction ◽  
Intermediate Filament Proteins ◽  
Torpedo Electric Organ ◽  
Whole Muscle

A layer of amorphous, electron-dense material is situated at the cytoplasmic surface of the postsynaptic membrane of vertebrate neuromuscular synapses. The function of this structure is not clear, but its location suggests that it may have an important role in the formation and/or maintenance of the synapse. This paper demonstrates that a monoclonal antibody raised against antigens from Torpedo electric organ binds to an intracellular, postsynaptic protein at the frog neuromuscular synapse. Indirect immunofluorescence on frozen sections of frog muscle was used to demonstrate that the antigen is concentrated at synaptic sites in normal muscle. In denervated muscle, the antigen remains concentrated at synaptic sites, but is also present at extrasynaptic regions of denervated myofibers. The antigen cannot be labeled in intact, whole muscle, but only in whole muscle that has been permeabilized with nonionic detergents. The antibody staining pattern in Triton X-100-permeabilized whole-mounts of the frog neuromuscular synapse is arranged in elongate, arborized areas which are characteristic of the frog neuromuscular synapse. The stained areas are striated and the striations occur with a periodicity that corresponds to the regular folding of the postsynaptic membrane. Immunoferritin labeling of fixed, saponin-permeabilized muscle demonstrates that the antigen is associated with amorphous material that is situated between the postsynaptic membrane and an underlying layer of intermediate filaments. The antigen, solubilized from membrane and an underlying layer of intermediate filaments. The antigen, solubilized from Torpedo electric organ by high ionic strength, was identified by antibody binding to nitrocellulose replicas of SDS gels of Torpedo tissue. In Torpedo tissue, the antibody binds to a single protein band at 51,000 daltons (51 kd). The 51-kd protein shares an antigenic determinant with intermediate filament proteins, since a monoclonal antibody to all intermediate filaments reacts with the same 51-kd protein. The monoclonal antibody also reacts with a 55-kd protein in frog skin which is localized to the perinuclear region of the epithelial cells.

scholarly journals Early events in neuromuscular junction formation in vitro: induction of acetylcholine receptor clusters in the postsynaptic membrane and morphology of newly formed synapses.

1979 ◽  
Vol 83 (1) ◽  
pp. 143-158 ◽  
Author(s):  
E Frank ◽  
G D Fischbach
Keyword(s):  
Synapse Formation ◽  
Synaptic Cleft ◽  
Postsynaptic Membrane ◽  
Active Growth ◽  
Before And After ◽  
Nerve Muscle ◽  
Scanning Em ◽  
Receptor Clusters ◽  
Alpha Bungarotoxin

The development of clusters of acetylcholine (ACh) receptors at newly formed synapses between embryonic chick spinal cord and muscle cells grown in vitro has been studied by iontophoretic mapping with ACh. A semi-automated technique using on-line computer analysis of ACh responses and a photographic system to record the position of each ACh application permit the rapid construction of extensive and detailed maps of ACh sensitivity. Clusters of receptors, evident as peaks of ACh sensitivity, are present on many uninnervated myotubes. The distribution of ACh sensitivity closely parallels the distribution of 125I-alpha-bungarotoxin binding sites on the same muscle cell. In all cases where individual myotubes were adequately mapped before and after synapse formation, ingrowing axons induced new clusters of receptors rather than seeking out preexisting clusters. Synapses can form at active growth cones within 3 h of nerve-muscle contact. New receptor clusters can appear beneath neurites within a few hours. Many of the uninnervated clusters on innervated myotubes disappear with time. In contrast, receptor clusters on uninnervated myotubes remain in the same location for many hours. Synaptic clusters and clusters on uninervated myotubes are stable even though individual receptors are metabolized rapidly. The morphology of several identified sites of transmitter release was examined. At the scanning EM level, synapses appeared as small, rough-surfaced varicosities with filopodia that radiated outwards over the muscle surface. One synapse was studied by transmission EM. Acetylcholinesterase and a basement lamina were present within the synaptic cleft.

MUSCLE RELAXANTS AND BINDING OF [(3) H]-[Greek small letter alpha]-BUNGAROTOXIN IN TORPEDO ELECTRIC ORGAN

1998 ◽  
Vol 89 (Supplement) ◽  
pp. 1014A
Author(s):  
Xing-Guo Sun ◽  
Nguyen Nguyen ◽  
Shaw-Wei Wang ◽  
Laszlo Gyermek
Keyword(s):  
Electric Organ ◽  
Muscle Relaxants ◽  
Small Letter ◽  
Torpedo Electric Organ ◽  
Alpha Bungarotoxin

scholarly journals A novel 87,000-Mr protein associated with acetylcholine receptors in Torpedo electric organ and vertebrate skeletal muscle.

1989 ◽  
Vol 109 (4) ◽  
pp. 1753-1764 ◽  
Author(s):  
C Carr ◽  
G D Fischbach ◽  
J B Cohen
Keyword(s):  
Skeletal Muscle ◽  
Acetylcholine Receptors ◽  
Large Fraction ◽  
Surface Membrane ◽  
Electric Organ ◽  
Postsynaptic Membrane ◽  
Velocity Sedimentation ◽  
Synaptic Membranes ◽  
Torpedo Electric Organ

To identify proteins associated with nicotinic postsynaptic membranes, mAbs have been prepared to proteins extracted by alkaline pH or lithium diiodosalicylate from acetylcholine receptor-rich (AChR) membranes of Torpedo electric organ. Antibodies were obtained that recognized two novel proteins of 87,000 Mr and a 210,000:220,000 doublet as well as previously described proteins of 43,000 Mr, 58,000 (51,000 in our gel system), 270,000, and 37,000 (calelectrin). The 87-kD protein copurified with acetylcholine receptors and with 43- and 51-kD proteins during equilibrium centrifugation on continuous sucrose gradients, whereas a large fraction of the 210/220-kD protein was separated from AChRs. The 87-kD protein remained associated with receptors and 43-kD protein during velocity sedimentation through shallow sucrose gradients, a procedure that separated a significant amount of 51-kD protein from AChRs. The 87- and 270-kD proteins were cleaved by Ca++-activated proteases present in crude preparations and also in highly purified postsynaptic membranes. With the exception of anti-37-kD antibodies, some of the monoclonals raised against Torpedo proteins also recognized determinants in frozen sections of chick and/or rat skeletal muscle fibers and in permeabilized chick myotubes grown in vitro. Anti-87-kD sites were concentrated at chick and rat endplates, but the antibodies also recognized determinants present at lower site density in the extrasynaptic membrane. Anti-210:220-kD labeled chick endplates, but studies of neuron-myotube cocultures showed that this antigen was located on neurites rather than the postsynaptic membrane. As reported in other species, 43-kD determinants were restricted to chick endplates and anti-51-kD and anti-270-kD labeled extrasynaptic as well as synaptic membranes. None of the cross reacting antibodies recognized determinants on intact (unpermeabilized) myotubes, so the antigens must be located on the cytoplasmic aspect of the surface membrane. The role that each intracellular determinant plays in AChR immobilization at developing and mature endplates remains to be investigated.

scholarly journals Dystrophin is a component of the subsynaptic membrane.

1991 ◽  
Vol 115 (4) ◽  
pp. 1069-1076 ◽  
Author(s):  
J E Yeadon ◽  
H Lin ◽  
S M Dyer ◽  
S J Burden
Keyword(s):  
Muscular Dystrophy ◽  
Electric Organ ◽  
Postsynaptic Membrane ◽  
Dystrophic Muscle ◽  
Expression Library ◽  
Neuromuscular Synapses ◽  
Sds Page ◽  
Partial Cdna ◽  
Torpedo Electric Organ

A subsynaptic protein of Mr approximately 300 kD is a major component of Torpedo electric organ postsynaptic membranes and copurifies with the AChR and the 43-kD subsynaptic protein. mAbs against this protein react with neuromuscular synapses in higher vertebrates, but not at synapses in dystrophic muscle. The Torpedo 300-kD protein comigrates in SDS-PAGE with murine dystrophin and reacts with antibodies against murine dystrophin. The sequence of a partial cDNA isolated by screening an expression library with mAbs against the Torpedo 300-kD protein shows striking homology to mammalian dystrophin, and in particular to the b isoform of dystrophin. These results indicate that dystrophin is a component of the postsynaptic membrane at neuromuscular synapses and raise the possibility that loss of dystrophin from synapses in dystrophic muscle may have consequences that contribute to muscular dystrophy.

scholarly journals Arrhenius plots of acetylcholinesterase activity in mammalian erythrocytes and in Torpedo electric organ. Effect of solubilization by proteinases and by a phosphatidylinositol-specific phospholipase C

1985 ◽  
Vol 231 (1) ◽  
pp. 237-240 ◽  
Author(s):  
P L Barton ◽  
A H Futerman ◽  
I Silman
Keyword(s):  
Phospholipase C ◽  
Electric Organ ◽  
Acetylcholinesterase Activity ◽  
Arrhenius Plots ◽  
Before And After ◽  
Break Points ◽  
Membrane Bound ◽  
Torpedo Electric Organ ◽  
Erythrocyte Ache ◽  
Lipid Environment

The temperature-dependence of the catalytic activity of acetylcholinesterase (AChE) from rat erythrocyte-ghost membranes and from Torpedo electric-organ membranes was examined. In the case of rat erythrocyte AChE, a non-linear Arrhenius plot was observed both before and after solubilization by a phosphatidylinositol-specific phospholipase C or by proteinase treatment. Similarly, no significant differences were observed in Arrhenius plots of Torpedo electric-organ AChE before or after solubilization. These results support our suggestion that the catalytic subunit of AChE does not penetrate deeply into the lipid bilayer of the plasma membrane and also suggest that care must be taken in ascribing break points in Arrhenius plots of membrane-bound enzymes to changes in their lipid environment.

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