Three-dimensional structure of a complex of E2020 with acetylcholinesterase from Torpedo californica

The 3D structure of a complex of the anti-Alzheimer drug, E2020, also known as Aricep^®, with Torpedo californica acetylcholinesterase is reported. The X-ray structure, at 2.5 Å resolution, shows that the elongated E2020 molecule spans the entire length of the active-site gorge of the enzyme. It thus interacts with both the ‘anionic’ subsite, at the bottom of the gorge, and with the peripheral anionic site, near its entrance, via aromatic stacking interactions with conserved aromatic residues. It does not interact directly with either the catalytic triad or with the ‘oxyanion hole’. Although E2020 is a chiral molecule, and both the S and R enantiomers have similar affinity for the enzyme, only the R enantiomer is bound within the active-site gorge when the racemate is soaked into the crystal. The selectivity of E2020 for acetylcholinesterase, relative to butyrylcholinesterase, can be ascribed primarily to its interactions with Trp279 and Phe330, which are absent in the latter.     

Structure of acetylcholinesterase complexed with E2020 (Aricept): implications for the design of new anti-Alzheimer drugs.

BACKGROUND: Several cholinesterase inhibitors are either being utilized for symptomatic treatment of Alzheimer's disease or are in advanced clinical trials. E2020, marketed as Aricept, is a member of a large family of N-benzylpiperidine-based acetylcholinesterase (AChE) inhibitors developed, synthesized and evaluated by the Eisai Company in Japan. These inhibitors were designed on the basis of QSAR studies, prior to elucidation of the three-dimensional structure of Torpedo californica AChE (TcAChE). It significantly enhances performance in animal models of cholinergic hypofunction and has a high affinity for AChE, binding to both electric eel and mouse AChE in the nanomolar range. RESULTS: Our experimental structure of the E2020-TcAChE complex pinpoints specific interactions responsible for the high affinity and selectivity demonstrated previously. It shows that E2020 has a unique orientation along the active-site gorge, extending from the anionic subsite of the active site, at the bottom, to the peripheral anionic site, at the top, via aromatic stacking interactions with conserved aromatic acid residues. E2020 does not, however, interact directly with either the catalytic triad or the 'oxyanion hole', but only indirectly via solvent molecules. CONCLUSIONS: Our study shows, a posteriori, that the design of E2020 took advantage of several important features of the active-site gorge of AChE to produce a drug with both high affinity for AChE and a high degree of selectivity for AChE versus butyrylcholinesterase (BChE). It also delineates voids within the gorge that are not occupied by E2020 and could provide sites for potential modification of E2020 to produce drugs with improved pharmacological profiles.     

Kinetics of Torpedo californica acetylcholinesterase inhibition by bisnorcymserine and crystal structure of the complex with its leaving group

Natural and synthetic carbamates act as pseudo-irreversible inhibitors of AChE (acetylcholinesterase) as well as BChE (butyrylcholinesterase), two enzymes involved in neuronal function as well as in the development and progression of AD (Alzheimer's disease). The AChE mode of action is characterized by a rapid carbamoylation of the active-site Ser(200) with release of a leaving group followed by a slow regeneration of enzyme action due to subsequent decarbamoylation. The experimental AD therapeutic bisnorcymserine, a synthetic carbamate, shows an interesting activity and selectivity for BChE, and its clinical development is currently being pursued. We undertook detailed kinetic studies on the activity of the carbamate bisnorcymserine with Tc (Torpedo californica) AChE and, on the basis of the results, crystallized the complex between TcAChE and bisnorcymserine. The X-ray crystal structure showed only the leaving group, bisnoreseroline, trapped at the bottom of the aromatic enzyme gorge. Specifically, bisnoreseroline interacts in a non-covalent way with Ser(200) and His(440), disrupting the existing interactions within the catalytic triad, and it stacks with Trp(84) at the bottom of the gorge, giving rise to an unprecedented hydrogen-bonding contact. These interactions point to a dominant reversible inhibition mechanism attributable to the leaving group, bisnoreseroline, as revealed by kinetic analysis.     

Conformation similarities of the globular and tailed forms of acetylcholinesterase from Torpedo californica

The conformation of the globular dimer (G2), the tailed asymmetric dodecamer (A12, also containing some tailed octamer A8) and the globular tetramer (G4, prepared by removing the collagen-like tail from A12) of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) was studied by circular dichroism (CD) in the ultraviolet region. The G2 and G4 forms had similar conformation with about 40% alpha-helix, 35% beta-sheets and 4% beta-turns; the tailed form had a lower helicity (about 34%) and beta-form (about 25%) content probably because of the presence of the tail whose CD spectrum resembles that of an unordered form, but it had about the same amount of beta-turns as the other two forms. All three forms also had similar CD spectra in the near-ultraviolet region due to their non-peptide chromophores. The pH, thermal and urea denaturation of the three acetylcholinesterase forms was also similar to each other. The pH-dependency of both the enzymatic activity and CD intensity of the three forms showed bell-shaped curves with a plateau at pH 7-8. The activity was completely lost at pH below 5 or above 10, but the corresponding CD spectra retained 70-80% of the original magnitudes. Thermal denaturation of the three forms at pH 7.5 showed a conformational transition and loss of activity between 30 and 40 degrees C, but the CD intensity of the helical band at 222 nm was reduced by only 20-30%. Urea denaturation of the three forms began at 1 M urea; it was protein concentration- and time-dependent. Again, the activity disappeared faster than the decreasing CD intensity. Thus, the overall conformation of the three acetylcholinesterase forms appears to be relatively stable, but their active site is easily perturbed by changing the environment. The loss of activity correlated well with the disappearance of the CD band of tryptophan(s) in the near-ultraviolet region, suggesting that the Trp residue(s) might be at or near the active center of the enzyme.     

Molecular forms of acetylcholinesterase from Torpedo californica: their relationship to synaptic membranes.

The 16S and 8S forms of acetylcholinesterase (AchE), which are composed of an elongated tail structure in addition to the more globular catalytic subunits, were extracted and purified from membranes from Torpedo californica electric organs. Their subunit compositions and quaternary structures were compared with 11S lytic enzyme which is derived from collagenase or trypsin treatment of the membranes and devoid of the tail unit. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence of reducing agent, appreciable populations of monomeric through tetrameric species are observed for the 11S form. Under the same conditions, the 16S form yields only monomer and dimer in addition to a higher molecular weight species. If complete reduction is effected, only the 80,000 molecular weight monomer is dominant for both the 11S and 16S forms. Cross-linking of the 11S form by dimethyl suberimidate followed by reduction yields monomer through tetramer in descending frequency, while the 16S form again shows a high molecular weight species. A comparison of the composition of the 11S and 16S forms reveals that the latter has an increased glycine content, and 1.1 and 0.3 mol % hydroxyproline and hydroxylysine, respectively. Collagenases that have been purified to homogencity and are devoid of amidase and caseinolytic activity, but active against native collagen, will convert 16S acetylcholinesterase to the 11S form. Thus, composition and substrate behavior of the 16S enzyme are indicative of the tail unit containing a collagen-like sequence. A membrane fraction enriched in acetylcholinesterase and components of basement membrane can be separated from the major portion of the membrane protein. The 16S but not the 11S form reassociates selectively with this membrane fraction. These findings reveal distinct similarities between the tail unit of acetylcholinesterase and basement membrane components and suggest a primary association of AchE with the basement membrane.     

The antigenic structure of the acetylcholine receptor from Torpedo californica

The immunological structure of the acetylcholine receptor (AChR) from the electric organ of Torpedo californica was studied using a large number of monoclonal antibodies which were initially selected for their abilities to bind to intact AChRs. The monoclonal antibodies were tested for their ability to bind to denatured AChR subunits labeled with 125I. Antibodies derived from rats immunized with individual denatured subunits or a mixture of subunits of Torpedo AChR reacted well in the assay. A much smaller proportion of antibodies derived from rats immunized with native Torpedo AChR or native AChR from Electrophorus electricus electric organ, bovine muscle, or human muscle reacted with denatured subunits of Torpedo AChR. Many monoclonal antibodies reacted with more than one subunit, but they always reacted best with the subunit used for immunization. Those monoclonal antibodies that bound to intact subunits were mapped more precisely by their ability to bind characteristic fragments of each subunit generated by proteolysis with Staphylococcal V8 protease. These fragments were analyzed by SDS polyacrylamide gel electrophoresis, and monoclonal antibodies that precipitated the same fragment pattern were placed in groups. By this method, we define a minimum of 28 determinants on Torpedo AChR.     

A neutral molecule in a cation-binding site: specific binding of a PEG-SH to acetylcholinesterase from Torpedo californica

The crystal structure of acetylcholinesterase from Torpedo californica complexed with the uncharged inhibitor, PEG-SH-350 (containing mainly heptameric polyethylene glycol with a terminal thiol group) is determined at 2.3 A resolution. This is an untypical acetylcholinesterase inhibitor, since it lacks the cationic moiety typical of the substrate (acetylcholine). In the crystal structure, the elongated ligand extends along the whole of the deep and narrow active-site gorge, with the terminal thiol group bound near the bottom, close to the catalytic site. Unexpectedly, the cation-binding site (formed by the faces of aromatic side-chains) is occupied by CH(2) groups of the inhibitor, which are engaged in C-H...pi interactions that structurally mimic the cation-pi interactions made by the choline moiety of acetylcholine. In addition, the PEG-SH molecule makes numerous other weak but specific interactions of the C-H...O and C-H...pi types.     

Raman spectroscopic study on the conformation of 11 S form acetylcholinesterase from Torpedo californica

Vibrational Raman spectroscopy has been used to study the conformation of the 11 S form of acetylcholinesterase from Torpedo californica. Secondary structure analysis by the method of Williams [(1983) J. Mol. Biol. 166, 581-603] shows 49% alpha-helical structure, 23% beta-sheets, 11% turns and 15% undefined structure. Secondary structure estimates obtained for this enzyme by Raman spectroscopy and circular dichroism have been analyzed.     

Purification and crystallization of a dimeric form of acetylcholinesterase from Torpedo californica subsequent to solubilization with phosphatidylinositol-specific phospholipase C

A dimeric form of acetylcholinesterase from Torpedo californica was purified to homogeneity by affinity chromatography subsequent to solubilization with a phosphatidylinositol-specific phospholipase C of bacterial origin. Bipyramidal crystals of the enzyme were obtained from solutions in polyethylene glycol 200. The crystals diffract to 2.0 A (1 A = 0.1 nm) resolution. They were found to be orthorhombic, space group P2221, with a = 163.4(+/- 0.2) A, b = 112.1(+/- 0.2) A, c = 81.3(+/- 0.1) A.     

Protease effects on the structure of acetylcholine receptor membranes from Torpedo californica

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.     

Comparison of asymmetric forms of acetylcholinesterase from the electric organ of Narke japonica and Torpedo californica

The asymmetric forms of acetylcholinesterase were purified from the electric organs of the electric rays Narke japonica and Torpedo californica, and their properties were compared. Asymmetric acetylcholinesterase was purified by immunoaffinity chromatography with a monoclonal antibody (Nj-601) to acetylcholinesterase. The MgCl2 extracts of these electric organs were applied to a column of Nj-601-Sepharose, and the bound acetylcholinesterase was eluted by lowering the pH of the eluent to 2.8. The purified asymmetric acetylcholinesterases gave peaks of 17 S (A12) and 13 S (A8) on sucrose density gradients. The enzyme from N. japonica contained more A8 than A12, while that of T. californica contained more A12. After treatment with collagenase, the enzymes gave three peaks on sedimentation; 20 S, 16 S and 11 S for N. japonica, and 19 S, 15 S and 11 S for T. californica, indicating the presence of collagen-like tails. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the asymmetric acetylcholinesterase from N. japonica gave bands of Mr 140 000, 100 000, 70 000 and 60 000, while that from T. californica gave bands of Mr 140 000, 100 000, 70 000 and 55 000. The bands of Mr 70 000 and 140 000 were monomers and non-reducible dimers, respectively, of the catalytic subunits. The bands of Mr 60 000 and 55 000 were the tail subunits, since collagenase treatment of the purified enzymes markedly decreased the amounts of these components. The Mr 100 000 subunit constituted less than 3% of the total asymmetric acetylcholinesterase from N. japonica but 18% of that from T. californica. The tail subunits constituted 6-8% of the two preparations. The catalytic subunits and the Mr 100 000 subunits bound concanavalin A, indicating that they are glycoproteins. The amino acid compositions of the enzymes from N. japonica and T. californica were very similar. Both contained hydroxyproline and hydroxylysine, characteristic of the collagen-like tails. The enzyme required divalent metal ions for activity, but only Mn2+, Mg2+ and Ca2+ were effective. Mn2+ was effective at the lowest concentrations, while Mg2+ gave the highest activity.     

3D structure of Torpedo californica acetylcholinesterase complexed with huprine X at 2.1 A resolution: kinetic and molecular dynamic correlates

Huprine X is a novel acetylcholinesterase (AChE) inhibitor, with one of the highest affinities reported for a reversible inhibitor. It is a synthetic hybrid that contains the 4-aminoquinoline substructure of one anti-Alzheimer drug, tacrine, and a carbobicyclic moiety resembling that of another AChE inhibitor, (-)-huperzine A. Cocrystallization of huprine X with Torpedo californica AChE yielded crystals whose 3D structure was determined to 2.1 A resolution. The inhibitor binds to the anionic site and also hinders access to the esteratic site. Its aromatic portion occupies the same binding site as tacrine, stacking between the aromatic rings of Trp84 and Phe330, whereas the carbobicyclic unit occupies the same binding pocket as (-)-huperzine A. Its chlorine substituent was found to lie in a hydrophobic pocket interacting with rings of the aromatic residues Trp432 and Phe330 and with the methyl groups of Met436 and Ile439. Steady-state inhibition data show that huprine X binds to human AChE and Torpedo AChE 28- and 54-fold, respectively, more tightly than tacrine. This difference stems from the fact that the aminoquinoline moiety of huprine X makes interactions similar to those made by tacrine, but additional bonds to the enzyme are made by the huperzine-like substructure and the chlorine atom. Furthermore, both tacrine and huprine X bind more tightly to Torpedo than to human AChE, suggesting that their quinoline substructures interact better with Phe330 than with Tyr337, the corresponding residue in the human AChE structure. Both (-)-huperzine A and huprine X display slow binding properties, but only binding of the former causes a peptide flip of Gly117.     

Anionic subsites of the acetylcholinesterase from Torpedo californica: affinity labelling with the cationic reagent N,N-dimethyl-2-phenyl-aziridinium.

Several peptides of acetylcholinesterase of Torpedo californica labelled with the alkylating reagent [3H]N,N-dimethyl-2-phenyl-aziridinium (DPA) were localized within the primary structure. One peptide had the sequence KPQELIDVE (positions 270-278); the incorporation of DPA into this peptide could be specifically suppressed by propidium, which suggests that it is part of the peripheral anionic site. The incorporation of DPA into two other peptides was insensitive to propidium but could be prevented by edrophonium; the sequence of one of the peptides assumed to be part of the anionic site in the catalytic centre was found to be DLFR (positions 217-220). Decamethonium efficiently blocked alkylation by DPA in all three investigated peptides.     

X-ray structures of Torpedo californica acetylcholinesterase complexed with (+)-huperzine A and (-)-huperzine B: structural evidence for an active site rearrangement

Kinetic and structural data are presented on the interaction with Torpedo californica acetylcholinesterase (TcAChE) of (+)-huperzine A, a synthetic enantiomer of the anti-Alzheimer drug, (-)-huperzine A, and of its natural homologue (-)-huperzine B. (+)-Huperzine A and (-)-huperzine B bind to the enzyme with dissociation constants of 4.30 and 0.33 microM, respectively, compared to 0.18 microM for (-)-huperzine A. The X-ray structures of the complexes of (+)-huperzine A and (-)-huperzine B with TcAChE were determined to 2.1 and 2.35 A resolution, respectively, and compared to the previously determined structure of the (-)-huperzine A complex. All three interact with the "anionic" subsite of the active site, primarily through pi-pi stacking and through van der Waals or C-H.pi interactions with Trp84 and Phe330. Since their alpha-pyridone moieties are responsible for their key interactions with the active site via hydrogen bonding, and possibly via C-H.pi interactions, all three maintain similar positions and orientations with respect to it. The carbonyl oxygens of all three appear to repel the carbonyl oxygen of Gly117, thus causing the peptide bond between Gly117 and Gly118 to undergo a peptide flip. As a consequence, the position of the main chain nitrogen of Gly118 in the "oxyanion" hole in the native enzyme becomes occupied by the carbonyl of Gly117. Furthermore, the flipped conformation is stabilized by hydrogen bonding of Gly117O to Gly119N and Ala201N, the other two functional elements of the three-pronged "oxyanion hole" characteristic of cholinesterases. All three inhibitors thus would be expected to abolish hydrolysis of all ester substrates, whether charged or neutral.     

Immunospecific identification and three-dimensional structure of a membrane-bound acetylcholine receptor from Torpedo californica.

Antibodies, raised against affinity column-purified acetylcholine receptor from Torpedo californica, were used as a basis for immunospecific identification of the receptor in membrane fragments. Rabbit and goat anti-receptor antibodies were coupled directly or indirectly via goat anti-rabbit antibody to colloidal gold spheres or to ferritin. The labeled membranes were visualized by negative stain electron microscopy, and show that the receptor corresponds to the 85 Å diameter rosette seen in membranes derived from electroplaques.Electron micrographs of immunospecifically labeled receptor, in the plane perpendicular to the membrane surface, confirm and extend our previous conclusions based on X-ray diffraction analysis, that the molecule extends above the extracellular membrane surface by approximately 55 Å, and little on the cytoplasmic side. Calculated molecular volumes based on X-ray diffraction and electron microscopy indicate that the membrane receptor has a molecular weight in the range of 250,000 to 310,000, a range consistent with current estimates of detergent-solubilized monomer molecular weight.     

Kinetic and structural studies on the interactions of Torpedo californica acetylcholinesterase with two donepezil-like rigid analogues

Acetylcholinesterase inhibitors were introduced for the symptomatic treatment of Alzheimer’s disease (AD). Among the currently approved inhibitors, donepezil (DNP) is one of the most preferred choices in AD therapy. The X-ray crystal structures of Torpedo californica AChE in complex with two novel rigid DNP-like analogs, compounds 1 and 2, have been determined. Kinetic studies indicated that compounds 1 and 2 show a mixed-type inhibition against TcAChE, with K_i values of 11.12?±?2.88 and 29.86?±?1.12?nM, respectively. The DNP rigidification results in a likely entropy-enthalpy compensation with solvation effects contributing primarily to AChE binding affinity. Molecular docking evidenced the molecular basis for the binding of compounds 1 and 2 to the active site of β-secretase-1. Overall, these simplified DNP derivatives may represent new structural templates for the design of lead compounds for a more effective therapeutic strategy against AD by foreseeing a dual AChE and BACE-1 inhibitory activity.     

The 2.1 A structure of Torpedo californica creatine kinase complexed with the ADP-Mg(2+)-NO(3)(-)-creatine transition-state analogue complex

Creatine kinase (CK) catalyzes the reversible conversion of creatine and ATP to phosphocreatine and ADP, thereby helping maintain energy homeostasis in the cell. Here we report the first X-ray structure of CK bound to a transition-state analogue complex (CK-TSAC). Cocrystallization of the enzyme from Torpedo californica (TcCK) with ADP-Mg(2+), nitrate, and creatine yielded a homodimer, one monomer of which was liganded to a TSAC complex while the second monomer was bound to ADP-Mg(2+) alone. The structures of both monomers were determined to 2.1 A resolution. The creatine is located with the guanidino nitrogen cis to the methyl group positioned to perform in-line attack at the gamma-phosphate of ATP-Mg(2+), while the ADP-Mg(2+) is in a conformation similar to that found in the TSAC-bound structure of the homologue arginine kinase (AK). Three ligands to Mg(2+) are contributed by ADP and nitrate and three by ordered water molecules. The most striking difference between the substrate-bound and TSAC-bound structures is the movement of two loops, comprising residues 60-70 and residues 323-332. In the TSAC-bound structure, both loops move into the active site, resulting in the positioning of two hydrophobic residues (one from each loop), Ile69 and Val325, near the methyl group of creatine. This apparently provides a specificity pocket for optimal creatine binding as this interaction is missing in the AK structure. In addition, the active site of the transition-state analogue complex is completely occluded from solvent, unlike the ADP-Mg(2+)-bound monomer and the unliganded structures reported previously.     

Structural and functional characterization of the interaction of the photosensitizing probe methylene blue with Torpedo californica acetylcholinesterase

The photosensitizer, methylene blue (MB), generates singlet oxygen that irreversibly inhibits Torpedo californica acetylcholinesterase (TcAChE). In the dark, it inhibits reversibly. Binding is accompanied by a bathochromic absorption shift, used to demonstrate displacement by other acetylcholinesterase inhibitors interacting with the catalytic "anionic" subsite (CAS), the peripheral "anionic" subsite (PAS), or bridging them. MB is a noncompetitive inhibitor of TcAChE, competing with reversible inhibitors directed at both "anionic" subsites, but a single site is involved in inhibition. MB also quenches TcAChE's intrinsic fluorescence. It binds to TcAChE covalently inhibited by a small organophosphate (OP), but not an OP containing a bulky pyrene. Differential scanning calorimetry shows an ~8° increase in the denaturation temperature of the MB/TcAChE complex relative to native TcAChE, and a less than twofold increase in cooperativity of the transition. The crystal structure reveals a single MB stacked against Trp279 in the PAS, oriented down the gorge toward the CAS; it is plausible that irreversible inhibition is associated with photooxidation of this residue and others within the active-site gorge. The kinetic and spectroscopic data showing that inhibitors binding at the CAS can impede binding of MB are reconciled by docking studies showing that the conformation adopted by Phe330, midway down the gorge, in the MB/TcAChE crystal structure, precludes simultaneous binding of a second MB at the CAS. Conversely, binding of ligands at the CAS dislodges MB from its preferred locus at the PAS. The data presented demonstrate that TcAChE is a valuable model for understanding the molecular basis of local photooxidative damage.     

Reaction of quinacrine mustard with the acetylcholine receptor from Torpedo californica

Amines with local anesthetic activity are typically also noncompetitive inhibitors of the agonist-induced increase in cation permeability mediated by the nicotinic acetylcholine receptor. Quinacrine is such an agent, and we have synthesized tritiated quinacrine mustard, a derivative capable of reacting with nucleophiles. Quinacrine mustard was reacted with receptor-rich membrane from torpedo electric tissue, excess reagent was removed by partition into liposomes, and the modified receptor was extracted and reconstituted with exogenous phospholipid. After reaction of the native membrane with 10 microM quinacrine mustard for 5 min, binding of cobratoxin to the acetylcholine binding sites is inhibited 15%; in contrast, receptor-mediated 86Rb uptake in the reconstituted vesicles is inhibited 70%. When the reaction with quinacrine mustard is carried out in the presence of 10 microM carbamylcholine or 10 microM d-tubocurarine, there is no block of the acetylcholine binding sites; nevertheless, the inhibition of Rb uptake is greater than that resulting from reaction in the absence of acetylcholine binding site ligands. Conversely, when the reaction is carried out in the presence of either 100 microM quinacrine or 100 microM proadifen (also a potent noncompetitive inhibitor), either with or without carbamylcholine or d-tubocurarine, the inhibition of 86Rb uptake is about 70% smaller. Under the same conditions that we used in the functional studies, quinacrine mustard reacts with the four types of chains that constitute the receptor complex, alpha 2 beta gamma delta. The presence of the acetylcholine binding site ligands, however, results in increased reaction with the alpha and beta chains, while the presence of the noncompetitive inhibitors, with or without the acetylcholine binding site ligands, results in decreased reaction with the alpha and beta chains. We conclude that the alpha and beta chains contribute to one or more functionally significant binding sites for noncompetitively inhibiting amines.     

Acetylcholine receptor: characterization of the voltage-dependent regulatory (inhibitory) site for acetylcholine in membrane vesicles from Torpedo californica electroplax

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)