Cholinergic receptor protein of Electrophorus electricus. II. Physical properties of the cholinergic receptor protein from Electrophorus electricus revealed by a tritiated α-toxin from Naja nigricollis venom

Meunier, Jean Claude; Olsen, Richard W.; Ménèz, Andre; Fromageot, P.; Boquet, P; Changeux, Jean Pierre

doi:10.1021/bi00757a014

Cited by 145 publications

(50 citation statements)

References 17 publications

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“…(45 000 and 54 000) in the range of those already found by various authors with crude extracts [4,5,12] or purified preparations of receptor protein from Electrophorus [2] or Torpedo [13]. The nature of these ~wo bands is not clear.…”

Section: Discussionmentioning

confidence: 73%

“…one would carry the cholinergic receptor site, the other would be more directly involved in ion translocation [14]. In this respect, one might mention that the values already reported for the mol.wt, of the chain labelled by 3H-a-toxin [4,5] or affinity reagents [12] lie between 40 000 and 45 000. In addition, even in the purest preparations, the molecular weight per 3H-a-toxin or [3H] decamethonium-binding site always seems much larger (between 100 000 and 150 000 1-3, I0, 13) than that of the dissociated subunits (45 000 or 54 000).…”

Section: Discussionmentioning

confidence: 99%

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Molecular weight and quaternary structure of the cholinergic receptor protein extracted by detergents from Electrophorus electricus electric tissue

Hucho

1973

FEBS Letters

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The cholinergic receptor protein can now be obtained from Electrophorus electricus electric organ in a highly purified state and in reasonable quantities [1][2][3]. It therefore becomes accessible to thorough biochemical investigation. The hydrodynamic properties of the receptor protein in detergent solution are quite unusual [4--6]: while gel filtration experiments indicate a Stokes radius of 70 A close to that of/~-galactosidase (mol.wt. 550 000), sedimentation in sucrose gradients in the presence of Triton X-100 shows an apparent sedimentation constant of 9.5 S, far lower than the 16 S found for/~-galactosidase. Such a particular behaviour, which has been assigned to a significant binding of detergent to the solubilized protein [7] renders the determination of mol.wt, by hydrodynamic methods particularly difficult. This is why we turned to an entirely different technique: polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) after complete or partial crosslinking of the receptor molecule by suberimidate [8]. In this paper we present estimates of the mol.wt, of the receptor protein given by this method and further show that this protein results from the assembly of several (possibly five) subunits belonging to two different molecular weight classes. Materials and methods Preparation of the purified receptor proteinThe cholinergic receptor protein was purified from the electric organ of Electrophorus electricus as described wlsewhere [1,10]. Its specific activity was 5400 nmo!es o fNa]a nigricollis 3 H-a-toxin-[9] binding site per gram protein, as determined by the Millipore assay for receptor activity and the method of Lowry et al. [ 1 ] for assay of protein concentration. Cross-linking with suberimidateSynthesis of suberimidate, and cross-linking of the receptor protein as well as that of the calibration proteins was performed as described by Davies and Stark [8]. Tris buffer which interferes with the reaction was removed by overnight dialysis against 1000 vol of 0.2 M triethanolamine, pH 8.5, 1% Triton X-100. Subsequently, the protein solution was concentrated to about 0.8 mg/ml with dry Sephadex G100. For complete cross-linking suberimidate was added to a final concentration of 2 mg/ml. For partial cross-linking, the suberimidate concentration was varied between 0.1 and 1.0 mg/ml. The best subunit pattern of the receptor was obtained at 0.2 mg/ml suberimidate. Incubation time was always 3 hr at room temperature.

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Section: Discussionmentioning

confidence: 73%

Section: Discussionmentioning

confidence: 99%

Molecular weight and quaternary structure of the cholinergic receptor protein extracted by detergents from Electrophorus electricus electric tissue

Hucho

1973

FEBS Letters

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“…Affinity-labeling procedures have indicated that there are similarities and differences between the active sites of the receptor and the enzyme (3)(4)(5). Acetylcholinesterase has been purified (6,7), and purified acetylcholine receptors are reported to have no acetylcholinesterase activity (8)(9)(10)(11). The receptor and the enzyme, both of which have sites specific for acetylcholine, may therefore be on different molecules, or there may be different sites on the same molecule that can be experimentally separated (2).…”

mentioning

confidence: 99%

Regulation of Acetylcholine Receptors in Relation to Acetylcholinesterase in Neuroblastoma Cells

Simantov

Sachs

1973

Proc. Natl. Acad. Sci. U.S.A.

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Purified α-toxin from Naja nigricollis snake venom labeled by [ 3 H]acetylation binds specifically to the acetylcholine receptors of mouse neuroblastoma cells. Toxin binding was inhibited by inhibitors for nicotinic and muscarinic acetylcholine receptors. Clones of neuroblastoma cells were selected for low acetylcholinesterase (EC 3.1.1.7) activity with antibodies against this enzyme. Selection for an 80-fold decrease in acetylcholinesterase activity was not associated with any decrease in the number of acetylcholine receptors (3.4 × 10 7 per cell). Removal or inactivation of 80% of the acetylcholine receptors by proteolytic enzymes or by compounds that block sulfhydryl groups did not change the activity of acetylcholinesterase on the cell surface. In addition to these results on the separation between acetylcholine receptors and acetylcholinesterase, a common regulation was found in that both the number of acetylcholine receptors and the activity of acetylcholinesterase were increased 5- to 10-fold when the cells stopped to multiply or were induced to differentiate by dibutyryl-cyclic AMP. It is suggested that there are different genes for the acetylcholine receptor and acetylcholinesterase, and that both are regulated during growth and differentiation by a common regulatory gene.

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“…Thus, the labeling did not correspond to a low affinity reversible association between cr-Bgt and receptor polypeptides [24,25] but to the covalent attachment of the o-toxin.…”

Section: Resultsmentioning

confidence: 98%

Crosslinking of α‐bungarotoxin to the acetylcholine receptor from Torpedo marmorata by ultraviolet light irradiation

Oswald

Changeux

1982

FEBS Letters

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Cholinergic receptor protein of Electrophorus electricus. II. Physical properties of the cholinergic receptor protein from Electrophorus electricus revealed by a tritiated α-toxin from Naja nigricollis venom

Cited by 145 publications

References 17 publications

Molecular weight and quaternary structure of the cholinergic receptor protein extracted by detergents from Electrophorus electricus electric tissue

Molecular weight and quaternary structure of the cholinergic receptor protein extracted by detergents from Electrophorus electricus electric tissue

Regulation of Acetylcholine Receptors in Relation to Acetylcholinesterase in Neuroblastoma Cells

Crosslinking of α‐bungarotoxin to the acetylcholine receptor from Torpedo marmorata by ultraviolet light irradiation

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