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.     

Electrogenic behavior of synaptic vesicles from Torpedo californica.

Electrical potential changes in pure synaptic vesicles from Torpedo californica were monitored with the fluorescent dye 3,3'-dipropylthiadicarbocyanine iodide. Vesicles resuspended in variable external sodium ion in the presence of gramicidin established sodium ion membrane diffusion potentials. Vesicles resuspended in choline or acetylcholine chloride became hyperpolarized upon addition of gramicidin. Hyperpolarization was subsequently partially reversed spontaneously by choline or acetylcholine influx, which was confirmed by gel filtration, to yield a new, less negative, stable membrane potential. Thus, acetylcholine and choline are taken up electrogenically by synaptic vesicles.     

Affinity partitioning of membranes. Cholinergic receptor-containing membranes from Torpedo californica.

Affinity partitioning has been employed in the purification of membranes rich in cholinergic receptor from Torpedo californica electric organs. The procedure involves a modification of poly(ethylene oxide)-dextran aqueous phase partitioning systems where a ligand selective for the receptor is conjugated to the poly(ethylene oxide). Specific partitioning of the receptor-containing membranes into the poly(ethylene oxide)-rich phase occurs when bis-alpha,omega-trimethylamino poly(ethylene oxide) or bis-rho-tri-methylammonium phenylamino poly(ethylene oxide) was added to the phase system in low mole ratio. bis-alpha,omega-Methylamino poly(ethylene oxide), which should impart equivalent interfacial electromotive potential to the system but bind poorly to the receptor sites, was much less effective in producing phase distribution changes. The ligand-polymer-dependent phase distribution shifts were blocked by bisquaternary methonium ligands at concentrations consistent with their relative affinities for the cholinergic receptor. Titration or receptor sites with cobra alpha-toxin decreased the phase distribution changes in a linear fashion up to the point of stoichiometry. These observations are consistent with the phase distribution changes being consequent to ligand-polymer association with the pharmacologically important site on the receptor. The affinity partitioning procedure, when employed following an initial purification of the membranes by differential and density gradient centrifugation, yields membrane preparations with a high degree of morphological uniformity and a specific activity between 2.9 and 4.6 nmol of bound cobra alpha-toxin/mg of protein.     

Two partially unfolded states of Torpedo californica acetylcholinesterase.

Chemical modification with sulfhydryl reagents of the single, nonconserved cysteine residue Cys231 in each subunit of a disulfide-linked dimer of Torpedo californica acetylcholinesterase produces a partially unfolded inactive state. Another partially unfolded state can be obtained by exposure of the enzyme to 1-2 M guanidine hydrochloride. Both these states display several important features of a molten globule, but differ in their spectroscopic (CD, intrinsic fluorescence) and hydrodynamic (Stokes radii) characteristics. With reversal of chemical modification of the former state or removal of denaturant from the latter, both states retain their physiochemical characteristics. Thus, acetylcholinesterase can exist in two molten globule states, both of which are long-lived under physiologic conditions without aggregating, and without either intraconverting or reverting to the native state. Both states undergo spontaneous intramolecular thioldisulfide exchange, implying that they are flexible. As revealed by differential scanning calorimetry, the state produced by chemical modification lacks any heat capacity peak, presumably due to aggregation during scanning, whereas the state produced by guanidine hydrochloride unfolds as a single cooperative unit, thermal transition being completely reversible. Sucrose gradient centrifugation reveals that reduction of the interchain disulfide of the native acetylcholinesterase dimer converts it to monomers, whereas, after such reduction, the two subunits remain completely associated in the partially unfolded state generated by guanidine hydrochloride, and partially associated in that produced by chemical modification. It is suggested that a novel hydrophobic core, generated across the subunit interfaces, is responsible for this noncovalent association. Transition from the unfolded state generated by chemical modification to that produced by guanidine hydrochloride is observed only in the presence of the denaturant, yielding, on extrapolation to zero guanidine hydrochloride, a high free energy barrier (ca. 23.8 kcal/mol) separating these two flexible, partially unfolded states.     

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.     

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.     

A differential scanning calorimetry study of acetylcholine receptor-rich membranes from Torpedo californica

Various acetylcholine receptor-rich membrane preparations from Torpedo californica electroplax tissue were examined using the techniques of differential scanning calorimetry coupled with gel electrophoretic analysis of heat-denaturing material and functional assays following passage through discrete transitions. In unfractionated membranes, four irreversible calorimetric transitions were observed, one of which (Td = 59 degrees C) could be assigned to a complete loss of acetylcholine receptor function. A second lower temperature transition apparently corresponds to loss of certain peripheral membrane proteins including the Mr = 43,000 polypeptide and the acetylcholinesterase activity. Membrane preparations highly enriched in acetylcholine receptor polypeptides contained a major transition at 59 degrees C which could be shown to be sensitive to the presence of added ligands of the acetylcholine receptor, supporting its assignment to structural alterations of the receptor protein or its arrangement in the membrane.     

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.     

Preparation of right-side-out, acetylcholine receptor enriched intact vesicles from Torpedo californica electroplaque membranes.

Intact vesicles enriched in acetylcholine receptor from Torpedo californica electroplaque membranes can be separated from collapsed or leaky vesicles and membrane sheets on sucrose density gradients. alpha-Bungarotoxin binding in intact vesicles reveals that approximately 95% of the acetylcholine receptor containing vesicles are formed outside-out (with the synaptic membrane face exposed on the vesicle exterior). The binding data also indicated that only 5% or less of the sites for alpha-bungarotoxin binding to synaptic membranes are located on the interior, cytoplasmic face. Intact vesicles are stable to gentle pelleting and resuspension but are easily osmotically shocked. The vesicles are impermeable to sucrose and Ficoll, but glycerol readily transverses to membrane barrier. Intact vesicles provide a sealed, oriented membrane preparation for studies of vectorial acetylcholine receptor mediated processes.     

Torpedo marmorata acetylcholinesterase; a comparison with the Electrophorus electricus enzyme. Molecular forms, subunits, electron microscopy, immunological relationship.

Electron microscopy, sequential degradation by hydrolytic enzymes and the physical-chemical properties of the molecular forms of Torpedo acetylcholinesterase indicate that these molecules are structurally related to each other in the same way as the molecular forms of Electrophorus acetylcholinesterase: all are derived from a complex structure in which three tetrameric groups of subunits are associated with a rod-like 'tail'. In aged preparations the catalytic subunits are split into fragments in a manner similar to those of Electrophorus acetylcholinesterase. Immunological cross-reaction between both enzymes demonstrates the occurrence of common antigenic sites. The enzymes from the two sources, however, are different in their molecular weights and susceptibility to hydrolytic enzymes. Also, Torpedo acetylcholinesterase does not precipitate with either isologous or heterologous antibodies.     

Interaction of partially unfolded forms of Torpedo acetylcholinesterase with liposomes.

A water-soluble dimeric form of acetylcholinesterase from electric organ tissue of Torpedo californica was obtained by solubilization with phosphatidylinositol-specific phospholipase C of the glycophosphatidylinositol-anchored species, followed by purification by affinity chromatography. The water-soluble species, in its catalytically active native conformation, did not interact with unilamellar vesicles of dimyristoylphosphatidylcholine. We previously showed that either chemical modification or exposure to low concentrations of guanidine hydrochloride converted the native enzyme to compact, partially unfolded species with the physicochemical characteristics of the molten globule state. In the present study, it was shown that such molten globule species, whether produced by mild denaturation or by chemical modification, interacted efficiently with small unilamellar vesicles. Binding was not accompanied by significant vesicle fusion, but transient leakage occurred at the time of binding. The bound acetylcholinesterase reduced the transition temperature of the vesicles slightly, and NMR data suggested that it interacted primarily with the head-group region of the bilayer. The effects of tryptic digestion of the bound acetycholinesterase were monitored by gel electrophoresis under denaturing conditions. It was found that a single polypeptide, of mass approximately 5 kDa, remained associated with the vesicles. Sequencing revealed that this is a tryptic peptide corresponding to the sequence Glu 268-Lys 315. This polypeptide contains the longest hydrophobic sequence in the protein, Leu 274-Met 308, as identified on the basis of hydropathy plots. Inspection of the three-dimensional structure of acetylcholinesterase reveals that this hydrophobic sequence is largely devoid of tertiary structure and is localized primarily on the surface of the protein. It is suggested that this hydrophobic sequence is aligned parallel to the surface of the vesicle membrane, with nonpolar residues undergoing shallow penetration into the bilayer.     

Labeling of functionally sensitive sulfhydryl-containing domains of acetylcholine receptor from Torpedo californica membranes

N-(1-Pyrene)maleimide, a fluorescent, lipophilic, alkylating agent, was used as a probe for the nicotinic acetylcholine receptor (AChR). Preincubation with N-(1-pyrene)maleimide under nonreducing conditions inhibits agonist-induced cation permeability of AChR-enriched membranes. This inhibition is dependent on the concentration of N-(1-pyrene)maleimide used. This correlation was also exhibited by resonance energy transfer of tryptophan fluorescence to N-(1-pyrene)maleimide and by the labeling stoichiometries. However, agonist-induced desensitization, as based on the time-dependent inhibition of alpha-bungarotoxin binding upon preincubation with the agonist carbamylcholine, was unaffected by N-(1-pyrene)maleimide. Alkylation of the AChR by N-(1-pyrene)maleimide is pH-dependent with an apparent pKa of 7.5 and is unaffected by preincubation with carbamylcholine, alpha-bungarotoxin, tubocurarine, or decamethonium. Preincubation with a 25-fold molar excess of N-ethylmaleimide partially protects against N-(1-pyrene)maleimide, yet simultaneous incubation with an equimolar concentration does not protect. In contrast, simultaneous incubation with equimolar concentrations of phenylmaleimide or naphthylmaleimide inhibited N-(1-pyrene)maleimide alkylation by 52 and 67%, respectively. Each AChR subunit is labeled by N-(1-pyrene)maleimide. Prior alkylation with N-ethylmaleimide does not alter the labeling profile but lowers the amount of labeling of all subunits. Reductive methylation of membranes under conditions which dimethylate all or most protein amino groups does not inhibit alkylation by N-(1-pyrene)maleimide. The above results, as well as amino acid analysis of N-(1-pyrene)maleimide-alkylated receptor, indicate that a homologous class of cysteines, which reside in each subunit within the AChR domain embedded in the membrane, are involved in the reaction with N-(1-pyrene)maleimide.     

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.     

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.     

Molecular forms of acetylcholinesterase: their de novo synthesis in mouse neuroblastoma cells

Rat mouse AChE molecular forms are indistinguishable with respect to their sedimentation coefficients and their evolutive proportions during brain maturation. Among rat or mouse erythrocytes, rat C6 glial cells, and mouse 2A and NS 20 neuroblastoma cells, only neuroblastoma cells showed both the ES and HS molecular forms with a 1:1 proportion for NS 20 cells. All these cells lack a third molecular form (16S), which is present in rat and mouse superior cervical ganglia. After irreversible inhibition of pre-existing NS 20 neuroblastoma AchE, the ES form is first synthesized (de novo synthesis). The HS form begins to appear after a lag time of several hours and represents, 24 h after inhibition, only 15% of the total recovered activity, which is near the initial level. The initial relative proportions return by 2 to 3 days after inhibition. The recovery of the HS form is, for the most part, blocked by actinomycin D, which does not block the recovery of activity itself, which remains as an ES form. It seems that integration of the ES form into the HS form more probably depends on the synthesis of a new messenger RNA, which is required for the synthesis of either new AChE polypeptide chain, polymerization initiating protein or activating enzyme.     

Biochemical properties of acteylcholine receptor subunits from Torpedo californica.

Four polypeptide chains composing acetylcholine receptors from the electric organ of Torpedo californica were purified by preparative electrophoresis in sodium dodecyl sulfate. Their apparent mole ratio alpha/beta/gamma/delta is 2:1:1:1. These chains are not readily distinguished by amino acid or carbohydrate composition but are distinguished by apparent molecular weight and polypeptide maps. By peptide maps, no extensive homology is evident between these chains or between any of these chains and higher molecular weight chains found in receptor-enriched membrane fragments.     

Affinity-labeling of purified acetylcholine receptor from Torpedo californica

The receptor for acetylcholine purified from electric tissue of $\text{Torpedo californica}$ has been assayed both by affinity-alkylation and by neurotoxin binding. The specific activity by the latter method is about twice that by the former. Four major components of apparent molecular weights of 39,000, 48,000, 58,000 and 64,000 are separated by dodecyl sulfate-acrylamide gel electrophoresis. Reduction and affinity-alkylation of the receptor with a tritiated quaternary ammonium maleimide derivative results in the exclusive labeling of the 39,000 dalton subunit. This subunit, it is concluded, contains all or part of the acetylcholine binding site.     

Activation and inactivation kinetics of Torpedo californica acetylcholine receptor in reconstituted membranes

By use of a quench-flow technique to measure tracer ion flux rates in a physiologically significant time domain, the kinetics of activation and inactivation of purified reconstituted acetylcholine receptor (AChR) were investigated. After solubilization in sodium cholate, purification by affinity chromatography, and reconstitution into soybean lipids, the AChR from Torpedo californica displayed a characteristically fast rate of ion influx measured with 86Rb+. At 4 degrees C 1 mM carbamoylcholine (Carb) stimulated a fast (t1/2 = 7 ms) first-order filling of vesicle internal volume that presented a 10(4)-fold stimulation of ion flux rate by Carb. The concentration dependence of activation was sigmoidal with a half-maximal value at 3 X 10(-4) M Carb. In the presence of Carb, the purified AChR also underwent a two-step inactivation (desensitization) process. Inactivation was measured by preincubating AChR with Carb for various times (milliseconds to minutes) and then measuring the 86Rb+ influx rate. The two inactivation processes were each characterized by a distinct maximum rate (5.3 and 0.10 s-1) and by a different dependence on Carb concentration. The slow phase of inactivation gave a half-maximal rate at 2.5 X 10(-4) M Carb, and the fast inactivation was half-maximal at 1.3 X 10(-3) M Carb. The concentration dependence curves for both inactivation processes were approximately hyperbolic. The results are discussed in terms of models that describe the relationship between ligand binding and the processes of channel activation and desensitization.     

Creatine kinase isoenzymes in Torpedo californica: absence of the major brain isoenzyme from nicotinic acetylcholine receptor membranes

Creatine kinase, actin, and nu 1 are three proteins of Mr 43 000 associated with membranes from electric organ highly enriched in nicotinic acetylcholine receptor. High levels of creatine kinase are required to maintain adequate ATP levels, while actin may play a role in maintaining the synaptic cytoskeleton. Previous investigations have prompted the conclusion that postsynaptic specializations at the receptor-enriched membrane domains in electroplax contain the brain form of creatine kinase rather than the form of creatine kinase predominantly found in muscle. We have examined this conclusion by purifying Torpedo brain creatine kinase to virtual homogeneity in order to examine its immunochemical, molecular, and electrophoretic properties. On the basis of immunological cross-reactivity and isozyme analysis, the receptor-associated creatine kinase is identified to be of the muscle type. When the molecular characteristics of Torpedo brain and muscle creatine kinase are compared, the brain enzyme is positioned at a more basic pH during chromatofocusing and on two-dimensional gel electrophoresis (pI = 7.5-7.9). Furthermore, electrophoretic mobilities of the brain and muscle forms of creatine kinase differ in sodium dodecyl sulfate electrophoresis: the brain isozyme of creatine kinase has lower apparent molecular weight (Mr 41 000) when compared with the muscle enzyme (Mr 43 000). On the basis of the results of our current investigations, the hypothesis that the brain isozyme of creatine kinase is a component of the postsynaptic specializations of the Torpedo californica electroplax must be abandoned. Recent sequence data have established close homology between Torpedo and mammalian muscle creatine kinases. On the basis of electrophoretic criteria, our results indicate that a lower degree of homology exists between the brain isozymes.