Mohyee E. Eldefrawi

Purification and molecular properties of the acetylcholine receptor from Torpedo electroplax

The acetylcholine receptor of Torpedo electroplax is purified by affinity adsorption using cobra toxin (Naja naja siamensis) covalently attached to Sepharose 4B. Desorption by 10 mm benzoquinonium produces a protein that binds α-[125I]bungarotoxin but not [3H]acetylcholine or other reversible cholinergic ligands. On the other hand, desorption by 1 m carbamylcholine produces an acetylcholine receptor protein that binds [3H]acetylcholine, [3H]decamethonium, [3H]nicotine, [14C]dimethyl-d-tubocurarine, and α-[125I]bungarotoxin. The batch method of affinity adsorption employed gives recoveries of acetylcholine receptor (as measured by acetylcholine binding) averaging 69.2 ± 14.6%. The purity of the isolated acetylcholine receptor protein is estimated to be at best 87% as judged by disc gel electrophoresis and electrofocusing. The purified acetylcholine receptor binds 7.8 nmoles acetylcholine/mg protein based on estimation of protein concentration by a spectrophotometric method. Of these, 2.7 nmoles exhibit high affinity (KD = 0.02 μM) and 5.1 nmoles a lower affinity (KD = 1.97 μM. If the protein concentration used is that obtained by amino acid analysis, the total specific activity would be 10.4 nmoles acetylcholine bound per milligram protein. The subunit carrying one acetylcholine binding site is estimated to range between 83,000 and 112,000 daltons. In contrast to the membrane-bound or Lubrol-solubilized acetylcholine receptor, the purified acetylcholine receptor shows no autoinhibition with acetylcholine concentrations up to 10 μm. Binding of acetylcholine was totally inhibited by α-bungarotoxin or cobra toxin and was partially blocked by four nicotinic drugs, but not by two muscarinic ones. The amino acids of the acetylcholine receptor are analyzed and compared to those of acetylcholinesterase.

Interaction between calcium and ligand-binding sites of the purified acetylcholine receptor studied by use of a fluorescent lanthanide.

Abstract The acetylcholine receptor isolated from Torpedo ocellata binds about 10 moles of a fluorescent lanthanide, terbium, per mole α-bungarotoxin-binding site, a process which is accompanied by a fluorescence enhancement (λexcitation 295 nm, λemission 546 nm) which allows detection of receptor-Tb 3+ complexes at μM concentrations. In presence of calcium two types of terbium-binding site are revealed, both with terbium dissociation constants of 18 ± 0.5 μM. About 60% of the sites bind calcium with an apparent dissociation constant of 1.1 ± 0.1 mM. Sites which interact with calcium also interact with activators of neural transmission, carbamylcholine and decamethonium, but not with the inhibitors, d-tubocurarine and α-bungarotoxin. Whether the displacement of calcium by chemical mediators is directly responsible for activator-induced changes in ion permeability of neural membranes is an important question raised by our experiments. The results show that fluorescent lanthanides can be an important tool in such studies.

A simple, rapid, and quantitative radiometric assay of acetylcholinesterase.

Abstract A thin-layer chromatographic method has been developed for the radiometric assay of minute quantities of acetylcholinesterase. It is simple, rapid, and allows the simultaneous assay and total recoveries of acetylcholine and acetate.

Properties of Lubrol-solubilized acetylcholine receptor from Torpedo electroplax

Abstract The efficiencies of two nonionic and four anionic detergents in solubilizing acetylcholine receptors (AChR) (as detected by ACh-binding) from electric tissue of Torpedo were compared. Lubrol WX solubilized the highest concentration of active AChR/mg protein and sodium dodecyl sulfate (SDS) abolished ACh-binding; however, removal of the latter detergent resulted in a small recovery of ACh binding. The Lubrol-solubilized AChR was similar to the particulate AChR in binding ACh at two sites reversibly, with high affinities, and in showing autoinhibition at ACh concentrations higher than 1 μ m . Also, binding of ACh was abolished by boiling and was reduced by pretreatment with trypsin, pronase, and phospholipases. However, “kinetics” of ACh binding to solubilized AChR were changed: there were increases in the affinity of the high-affinity site and in the ratio of the concentrations of the high- to low-affinity site, and the Hill coefficient was reduced to 0.66. Its nicotinic nature was demonstrated by blockade of ACh binding by 13 nicotinic drugs including α-bungarotoxin and cobrotoxin. Anticholinesterases were also blockers, but 21 noncholinergic drugs had no effect. Ultrafiltration and gel chromatography were used to separate the ACh-binding macromolecules. Sepharose 6B gave partial separation of AChR from AChE. The molecular weight of the smallest functional AChR was between 50,000 and 100,000 daltons, and had a high tendency to aggregate. The majority of Lubrol-solubilized AChR had molecular weights above 300,000 daltons.

Acetylcholine Binding to Torpedo Electroplax: Relationship to Acetylcholine Receptors

Binding of [3H]acetylcholine to a particulate fraction of Torpedo electroplax was measured by equilibrium dialysis. Two high-affinity sites present on phospholipoproteins bound acetylcholine reversibly, and binding was blocked by nicotinic drugs. Characteristics of this binding suggest that these phospholipoproteins may be acetylcholine receptors.

Action of organophosphates on binding of cholinergic ligands

Abstract Four organophosphates: DFP, Guthoxon, Tetram and 2-( O,S -dimethylthiophosphorylimino-)-3-ethyl-5-methyl-1,3-oxazolidine, were found (at 10 −4 M ) to block the binding of 3 H-nicotine and 3 H-decamethonium (at 10 −7 M ) to macromolecules suggested to be acetylcholine receptors (ACR) in preparations of the housefly brain and electroplaxes of Torpedo and Electrophorus . Differences in blocking abilities were attributed to variations in ACR of the different tissues, structural differences amongst the organophosphates and ligand affinities. Organophosphates showed much lower affinities for ACR than for acetylcholinesterase. Binding of 3 H-muscarone and 3 H-acetylcholine to Torpedo electroplax was measured in the presence of increasing amounts of Tetram. The organophosphate blocked the binding of both ligands to ACR at concentrations higher than 5 × 10 −4 M . At low Tetram concentrations ( −5 M ) acetylcholine binding was reduced because of its hydrolysis by some acetylcholinesterase. Whereas the binding of organophosphates to acetylcholinesterase was irreversible, their binding to ACR was completely reversible.

Binding of calcium and zinc to the acetylcholine receptor purified from Torpedo Californica

Abstract Binding of [65Zn++] and [45Ca++] to the acetylcholine (ACh)-receptor, purified from the Torpedo electric organ, was studied by equilibrium dialysis. Whereas [65Zn++] bound to 56 nmoles of sites per mg protein with a dissociation constant of 2.5 × 10−6M, no binding of [45Ca++] at concentrations up to 10−3M could be detected with this method. However, the binding of [acetyl-3H]choline to the receptor was blocked equally by very high Zn++ or Ca++ concentrations, and the Ki for this low affinity binding was 7 × 10−3M. The high affinity binding of [65Zn++] to the receptor was blocked best by Cd++ then Co++ and Mn++, but least by Mg++ and Ca++. When the purified ACh-receptor itself was analyzed for the presence of cations by atomic absorption, it was discovered that 4.7% of its weight was due to bound Ca++ that could not be removed even by extensive dialysis. When Ca++-free solutions (containing 1 mM EDTA) were used during purification, 0.6% of the molecular weight of the receptor was still due to bound Ca++. This was equivalent to 15 moles of Ca++ for each mole of ACh bound at saturation. It is suggested that the source of this Ca++ is endogenous, and that it is tightly bound to the ACh-receptor molecule.

Studies on the structure of the acetylcholine receptor from Topredo marmorata

Abstract The purified acetylcholine receptor of Torpedo marmorata has been characterized by sedimentation velocity measurements on dilute solutions using an ultracentrifuge and scanner. Several preparations were studied and all exhibited sedimentation coefficients in the vicinity of 24S. In a number of experiments the receptor could be resolved into two sedimenting boundaries of 18S and 26S, corresponding to minimum molecular weights of about 5 × 10 5 and 10 6 , respectively. Additions of sodium dodecyl sulfate or Triton X-100 resulted in marked decreases in sedimentation coefficient, while treatment with Lubrol-WX had only a slight effect on the S values. Small changes in S 20,w were produced by guanidine hydrochloride alone, although addition of dithiothreitol with 6 M guanidine hydrochloride resulted in an 8.8S component. Electrophoresis in sodium dodecyl sulfate gave one principal band with a molecular weight of 46,000.

Differential effects of mercurial compounds on excitable tissues

Sarcoplasmic reticulum (SR), Ca2+ plus Mg2+-ATPase, and Ca2+-ionophore were obtained from white rabbit skeletal muscles. Methylmercury inhibited the Ca2+ plus Mg2+-ATPase and Ca2+-transport but had no effect on the Ca2+-ionophore. Mercuric chloride inhibited all three functions (i.e., ATPase, transport and ionophoric activity). The mechanism of HgCl2 inhibition of the Ca2+-ionophore was by competition with Ca2+ for Ca2+-ionophoric site whereas its inhibition of the enzyme and Ca2+-transport was due to the blockage of essential sulfhydryl (--SH) groups. Ca2+ plus Mg2+-ATPase and Ca2+-transport were more sensitive to methylmercury than to HgCl2. Acetylcholine receptor (AChR) was obtained for the electric organ of T. californica. Methylmercury inhibited the ACh binding to AChR WITH Ki = 5.7 - 10(-6) M. This effect was not due to mercuric ion alone since mercuric chloride up to 10(-4) M did not affect ACh binding to AChR. It is concluded that: the Ca2+ plus Mg2+-ATPase and Ca2+-transport contain --SH groups essential for their activity, and that the two functions are tightly coupled; the Ca2+-ionophore contains no --SH groups essential for its activity; CH3HgCl inhibition of Ca2+ plus Mg2+-ATPase and Ca2+-transport is partly due to its reactivity with --SH groups in hydrophobic environment; the Ca2+-transport is inhibited by HgCl2 through two processes, one which is the blockage of --SH groups and another which is the inhibition of the Ca2+-ionophoric site; and the inhibition of ACh binding to AChR is due to the blockage of --SH groups in hydrophobic environment, which is inaccessible to Hg2+. Our data present for the first time a molecular basis for the myopathy associated with mercurial compounds toxicity.

Identification of a calcium-binding subunit of the acetylcholine receptor

Abstract Four subunits of the acetylcholine receptor molecule, obtained from the electric organ of Torpedo ocellata , have been isolated using polyacrylamide gel electrophoresis, and assayed by titration with a fluorescent lanthanide, terbium, and by affinity-labeling with p -( N -maleimido)benzyl [trimethyl-3H] ammonium iodide. The site with which the activator-analogue affinity label reacts, as well as the terbium-binding sites, are mainly associated with the smallest of the subunits of an apparent molecular weight of 40,000. Calcium competes with terbium for these binding sites. The affinity for terbium is the same in the intact molecule as in the subunit (KTb ⋍ 19 ± 1 μM), but the affinity for calcium decreases by a factor of 4 (KCa ⋍ 4 mM) in the subunit. Hydrolysis of the receptor, catalyzed by trypsin and chymotrypsin, to peptides with an apparent molecular weight of 8000 or less, does not affect the terbium-binding sites. These experiments indicate that the binding sites for neural activators and for calcium are associated with the same subunit, and that the terbium- and calcium-binding sites reflect structural properties of the polypeptide chain rather than the three-dimensional structure of the protein.