Monoclonal Antibodies Allow Precipitation of Esterasic but Not Peptidasic Activities Associated with Butyrylcholinesterase

Commercially available and affinity-purified butyrylcholinesterases isolated from human serum were examined for their esterasic activity and their ability to hydrolyze various neuropeptides, including neurotensin, substance P, and leucine-enkephalin. The three pools that displayed the lowest esterasic activities were shown to hydrolyze neurotensin with the same HPLC degradative pattern. By contrast, noticeable qualitative and quantitative discrepancies were observed when hydrolyses of substance P and leucine-enkephalin by these three butyrylcholinesterase pools were studied. The pool that exhibited the highest esterasic activity appeared to be homogeneously constituted by 90- and 180-kDa protein bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and was totally unable to hydrolyze these three neuropeptides. This suggested that the three other butyrylcholinesterase preparations could be contaminated by exogenous peptidases. This was confirmed by means of three distinct monoclonal antibodies directed toward human serum butyrylcholinesterase. The three IgG-purified fractions precipitated the esterasic activity, whereas they failed to precipitate the neuropeptide-hydrolyzing activities whatever the substrate examined. Altogether, these results demonstrate that peptidases associated with butyrylcholinesterase are contaminating enzymes that cannot be considered as intrinsic activities of this enzyme.     

Studies on a reconstituted acetylcholine receptor system: effect of agonists

An impure preparation of acetylcholinesterase from electroplax of the electric eel can be incorporated into a bimolecular lipid membrane. The acetylcholinesterase-modified bimolecular lipid membrane shows a concentration-dependent increase in membrane conductance elicited by several agonists (acetylcholine, carbamylcholine, phenyltrimethylammonium ion, tetraethylammonium ion, decamethonium ion, and nicotine) added to the compartment opposite that to which acetylcholinesterase was originally added. Affinity and efficacy of the various agonists in generating the conductance increase were measured from dose-response curves; these are in good quantitative agreement with corresponding values observed for depolarization of intact eel electroplax. The ion conduction pathways induced by agonists in the modified bimolecular lipid membrane show a slight cation selectivity, Na ? K > Cl (3:3:1), similar to that observed for the depolarized electroplax membrane. Evidence is presented that suggests that some components other than acetylcholinesterase induce the acetylcholine receptor response in the bimolecular lipid membrane.     

Acetylcholine, ATP, and Proteoglycan Are Common to Synaptic Vesicles Isolated from the Electric Organs of Electric Eel and Electric Catfish as Well as from Rat Diaphragm

Cholinergic synaptic vesicles were isolated from the electric organs of the electric eel (Electrophorus electricus) and the electric catfish (Malapterurus electricus) as well as from the diaphragm of the rat by density gradient centrifugation followed by column chromatography on Sephacryl-1000. This was verified by both biochemical and electron microscopic criteria. Differences in size between synaptic vesicles from the various tissue sources were reflected by their elution pattern from the Sephacryl column. Specific activities of acetylcholine (ACh; in nmol/mg of protein) of chromatography-purified vesicle fractions were 36 (electric eel), 2 (electric catfish), and 1 (rat diaphragm). Synaptic vesicles from all three sources contained ATP in addition to ACh (molar ratios of ACh/ATP, 9-12) as well as binding activity for an antibody raised against Torpedo cholinergic synaptic vesicle proteoglycan. Synaptic vesicles from rat diaphragm contained binding activity for the monoclonal antibody asv 48 raised against a rat brain 65-kilodalton synaptic vesicle protein. Antibody asv 48 binding was absent from electric eel and electric catfish synaptic vesicles. These antibody binding results, which were obtained by a dot blot assay on isolated vesicles, directly correspond to the immunocytochemical results demonstrating fluorescein isothiocyanate staining in the respective nerve terminals. Our results imply that ACh, ATP, and proteoglycan are common molecular constituents of motor nerve terminal-derived synaptic vesicles from Torpedo to rat. In addition to ACh, both ATP and proteoglycan may play a specific role in the process of cholinergic signal transmission.     

Synthesis of tetrakis(multifluoro-4-pyridyl)porphin derivatives as acetylcholinesterase inhibitors

New tetrakis(multifluoro-4-pyridyl)porphin derivatives (2-4) and water soluble porphyrin (5) were synthesized to investigate their interactions with acetylcholinesterase from electric eel. These compounds have been found to be the potent reversible inhibitors of the enzyme with Ki values of microM range. In addition, porphyrin (5) showed broad spectrum of anticancer activities.     

Reversible inhibition of acetylcholinesterases of different origins by quaternary phosphonium compounds

Studies have been made on the reversible inhibition of acetylcholinesterase activity from the erythrocytes of man, horse and camel, the electric organ of the skate Torpedo marmorata and eel Electrophorus electricus, the venom of the snakes Naja naja and Vipera lebetina, the brain of the pigeon Columba livia by tetraphenyl-, triphenylalkyl- and tributyrylalkyl-phosphonium salts. The investigated phosphonium inhibitors exhibit an evident specificity in their action: they were more effective in inhibiting the acetylcholinesterase from human erythrocytes than that from the erythrocytes of horse and camel. These salts were more effective with respect to the acetylcholinesterase activity of the electric organ of the skate than that of the electric organ of the eel. Acetylcholinesterases from the venom of the snakes exhibited practically identical sensitivity to all the phosphonium compounds investigated. The present work is the first attempt to use quaternary phosphonium salts (the so-called "hydrophobic ions") in comparative enzymological investigation.     

Acetylcholinesterase inhibition by pitofenone: a spasmolytic compound.

Pitofenone, a spasmolytic compound, inhibited the acetylcholinesterase activity from bovine erythrocytes and from electric eel. It is a potent inhibitor of this enzyme from the two sources, with Ki values of 36 and 45 microM, respectively. Of the five compounds structurally related to pitofenone, only those containing a piperidine moiety show acetylcholinesterase inhibition. All these inhibitions are reversible, linear, and noncompetitive in nature. A qualitative correlation between the anticholinesterase and the corresponding antimuscarinic activity for some of these compounds was apparent. Good separation of these two effects would be a desirable feature for newer muscarinic antagonists.     

Separation of protease activity from acetylcholinesterase of the electric EEL

We examined the protease activity reported to be associated with acetylcholinesterase (AChE) by extensive purification of the electric eel enzyme. Upon edrophonium-Sepharose chromatography of a commercial preparation, a majority of the protease activity was recovered in the effluent with no AChE activity, while a marginal activity was detected in the AChE fraction eluted with edrophonium chloride. Further chromatography of the edrophonium eluate on hydroxyapatite gave partially overlapping peaks of protease and AChE activities. Finally, the protease activity was mostly removed from the AChE fraction by passing through an ovoinhibitor-agarose column. The protease activity in the edrophonium eluate was inhibited by various serine protease inhibitors, but not by AChE inhibitors. These results suggest that the AChE and protease activities are physically separable, and thus that the protease activity, so far reported as intrinsic to AChE, is probably due to contaminants.     

Monoclonal Antibodies to Rat Brain Acetylcholinesterase: Comparative Affinity for Soluble and Membrane-Associated Enzyme and for Enzyme from Different Vertebrate Species

Seven unique monoclonal antibodies were generated to rat brain acetylcholinesterase. Upon density gradient ultracentrifugation, immunoglobulin complexes with the monomeric enzyme appeared as single peaks of acetylcholinesterase activity with a sedimentation coefficient approximately 3S greater than that of the free enzyme. This behavior is consistent with the assumption of one binding site per enzyme molecule. Apparent dissociation constants of these antibodies for rat brain acetylcholinesterase calculated on the basis of this assumption ranged from about 10 nM to more than 1,000 nM. Some of the antibodies were less able to bind the membrane-associated enzyme that required detergent for solubilization than the naturally soluble acetylcholinesterase of detergent-free brain extracts. Species cross-reactivity was investigated with crude brain extracts from mammals (human, mouse, rabbit, guinea pig, cow, and cat) and from other vertebrates (chicken, frog, and electric eel). Three antibodies bound rat acetylcholinesterase exclusively; one had nearly the same affinity for all mammalian acetylcholinesterases investigated; the remaining three showed irregular binding patterns. None of the antibodies recognized frog and electric eel enzyme. Pooled antibody was found to be suitable for specific immunofluorescence staining of large neurons in the ventral horn of the rat spinal cord and smaller cells in the caudate nucleus. Other potential applications of these antibodies are discussed.     

Chemical modification of acetylcholinesterase from eel and basal ganglia: effect on the acetylcholinesterase and aryl acylamidase activities

The effect of chemical modification on the acetylcholinesterase and the aryl acylamidase activities of purified acetylcholinesterase from electric eel and basal ganglia was investigated in the presence and absence of acetylcholine, the substrate of acetylcholinesterase, and 1,5-bis[4-(allyldimethylammonium)phenyl]pentan-3-one dibromide (BW284C51), a reversible competitive inhibitor of acetylcholinesterase. Trinitrobenzenesulfonic acid, pyridoxal phosphate, acetic anhydride, diethyl pyrocarbonate, and 2-hydroxy-5-nitrobenzyl bromide under specified conditions inactivated both acetylcholinesterase and aryl acylamidase in the absence of acetylcholine and BW284C51. Chemical modifications in the presence of acetylcholine and BW284C51 by all the above except diethyl pyrocarbonate selectively prevented the loss of acetylcholinesterase but not aryl acylamidase activity; modification by diethyl pyrocarbonate in the presence of acetylcholine and BW284C51 prevented the loss of both acetylcholinesterase and aryl acylamidase activities. Treatment with N-acetylimidazole resulted in the inactivation of acetylcholinesterase and the activation of aryl acylamidase. These changes in both the activities could be prevented by acetylcholine and BW284C51. Modification by phenylglyoxal, 2,4-pentanedione, or N-ethylmaleimide did not affect the enzyme activities. Indophenylacetate hydrolase activity followed a pattern similar to that of acetylcholinesterase in all the above modification studies. The results suggested essential lysine, tyrosine, tryptophan, and histidine residues for the active center of acetylcholinesterase and essential lysine, histidine, and tryptophan residues for the active center of aryl acylamidase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulatory effects of polyamines on membrane-bound acetylcholinesterase

The effects of putrescene, spermidine and spermine on membrane-bound acetylcholinesterase from human erythrocyte ;ghosts' and the solubilized enzyme of the electric organ of the electric eel were studied by kinetic methods. Measurements were made by using a photometric method which made it possible to record the enzyme reaction in the steady-state phase. Substrate-concentration-dependent activation and inhibition of acetylcholinesterase by polyamines is similar to that by Na(+), K(+), Ca(2+), Mg(2+) and certain quaternary and bisquaternary amines. The kinetics suggest an allosteric reaction mechanism. On the basis of the kinetic results a role for the polyamines as modulators of synaptic acetylcholinesterase is proposed.     

Enthalpy of acetylcholine hydrolysis by acetylcholinesterase

Direct microcalorimetric measurements were made of the reaction between acetylcholine chloride and acetylcholinesterase (EC 3.1.1.7) that was extracted from electric eel (Electrophorus electricus) and purified by affinity chromatography. Tris-HCl, sodium phosphate and potassium phosphate were used as buffers and sources of ions for the reaction. At pH 7.2 and in 0.1-0.2 M phosphate buffer, the delta H for acetylcholine hydrolysis was found to be -0.107 kcal/mol (under buffered conditions) and -0.931 kcal/mol under unbuffered conditions (water). At pH 8.0 in 0.1 M Tris-HCl buffer, values greater than -2.5 kcal/mol were obtained, with the highest value of -9.2 kcal/mol being seen with bovine erythrocyte acetylcholinesterase. Tris-HCl buffer at 4 X 10(-2) M enhanced the reaction velocity by 51.2% over that of 4 X 10(-3) M buffer. Enzyme purity, pH and ionic milieu of reaction mixture, and substrate concentration affected the measured delta H value.     

The inactivation of acetylcholinesterase by trimethyloxonium ion, an active-site-directed methylating agent

Trimethyloxonium ion inactivates acetylcholinesterase from the electric eel and acetylcholinesterase on the surface of human red blood cells. Tetramethylammonium ion, which is a competitive inhibitor of acetylcholinesterase, protects against this inactivation. Trimethyloxonium ion does $\text{not}$ inactivate the system that transports choline into the red blood cell. We conclude that trimethyloxonium ion is an affinity-labeling reagent for acetylcholinesterase and that red blood cell acetylcholinesterase is probably not a component of the choline transport system.     

Immunological cross-reactivity between electric-eel acetylcholinesterase and rat-tail-tendon collagen.

Immunological cross-reactivity between acetylcholinesterase from the electric organ of the electric eel and rat tail tendon collagen was examined both on the cellular and humoral levels. 1. Guinea pigs immunized with rat tail tendon collagen displayed a strong delayed-type skin reaction when tested with the elongated acetylcholinesterase preparation (i.e. 14-S + 18-S molecular forms). However, when the glubular 11-S enzyme was tested, almost no cross-reactivity was obtained. Similarly, guinea pigs immunized with 14-S + 18-S preparation exhibited skin sensitization to rat tail tendon collagen. 2. Using a radioimmunoassay, it was observed that 125I-labeled 14-S + 18-S acetylcholinesterase binds efficiently to rabbit antiserum elicited against rat tail tendon collagen, whereas 125I-labeled 11-S enzyme does not bind at all to this antiserum. Similar results were obtained by passive hemagglutination assay. The experiments suggest that 14-S + 18-S acetylcholinesterase, but not 11-S enzyme, which is devoid of the tail structure, has antigenic determinants in common with collagen from rat tail tendon.     

Immunochemical studies of mammalian brain acetylcholinesterase.

Abstract— Specific antibodies were raised in rabbits to acetylcholinesterase (AChE) from bovine caudate nucleus and the‘native’(14S + 18S) and globular (11S) forms of AChE from eel electric tissue. All AChE preparations were purified by affinity chromatography to a specific activity of 100–400 mmol acetylthiocholine hydrolyzed/mg protein/h. Antigenic specificities of the different enzyme forms were studied by immunodiffusion, Immunoelectrophoresis and micro-complement fixation. Minor differences in antigenic determinants were observed between the different molecular forms of electric tissue AChE. In crossover experiments using both eel AChE and bovine caudate AChE antisera there was complete absence of cross reactivity between the mammalian brain AChE and the different molecular forms of the electric tissue enzyme. Brain AChE activity was inhibited up to 50% in the presence of its antiserum.     

Cholinesterases Display Genuine Arylacylamidase Activity but Are Totally Devoid of Intrinsic Peptidase Activities

Abstract: The purpose of this article was to evaluate the intrinsic character of arylacylamidase and peptidase activities that are often detected along with cholinesterase activities. Various pools of commercial or affinity-purified acetylcholinesterases (AChEs) were examined. Affinity-purified AChE displays esterase- and amidase-specific activities that are similarly enriched when compared with commercial AChE. By contrast, commercial AChE exhibits much higher tryptic-like and carboxypeptidase-specific activities than the affinity-purified enzyme. The parallel enrichment in esterase and arylacylamidase suggests that these two activities are copurified, whereas peptidases do not seem to behave similarly. We show that trypsinolysis or spontaneous degradation of affinity-purified AChE leads to the conversion of the 75-kDa monomer protein into two fragments of 50 and 25 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. However, these modifications are without effect on the esterase, arylacylamidase, and peptidase activities. This clearly shows that AChE does not behave as a zymogen of peptidases that would have been activated on autolysis of AChE. Immunoprecipitation of AChEs with a purified monoclonal antibody directed toward electric eel AChE totally separated the esterase and arylacylamidase activities (pellet) from peptidase activities (supernatant). The immunoprecipitated AChEs could be dissociated from the interaction with IgGs. These resolubilized AChE preparations have kept the same percentage of initial esterase and arylacylamidase activities but were totally devoid of peptidase activities. These data clearly indicate that commercial and affinity-purified AChEs from Electrophorus electricus bear an intrinsic arylacylamidase activity but that the peptidase activity detected in these preparations is not an integral property of the AChE molecule and most probably represents a contaminating activity. It appears therefore unlikely that AChE may participate to the processing of the β-amyloid protein precursor (β-APP) leading to the secretion of protease nexin II and therefore acts as an APP secretase, as was recently suggested. By a similar approach, we established that human butyrylcholinesterase recovered after immunoprecipitation retained its esterase activity but was no longer able to act as a peptidase.     

Effects of substance P on acetylcholinesterase activity

The effects of substance P on acetylcholinesterase activity have been examined. The neuropeptide produced a significant increase in the activity of the enzyme in rat cerebral cortex. Pretreatment of rats with either actinomycin-D or cycloheximide did not fully abolish the substance P-mediated stimulation of cerebral acetylcholinesterase. Substance P increased the enzyme activity in rat brain slices; moreover, substance P increased the activity of electric eel acetylcholinesterase in in vitro experiments. These observations indicate that substance P produces an induction of acetylcholinesterase in cerebral cortex of rats and in addition indicate that a direct action on the enzyme takes place.     

Visualization of collagenase-sensitive acetylcholinesterase in isolated cardiomyocytes and in heart tissue

Summary Previous studies have indicated that the asymmetric form of acetylcholinesterase (collagen-tailed) is localized in the basal lamina of the neuromuscular junction of skeletal muscle. The present study shows localization of the asymmetric acetylcholinesterase in the heart of the rat. Antiserum to 14+18 S acetylcholinesterase of the electric eel was raised in rabbits. The purified antibody did not react with collagen type I or laminin. Collagenase reduced the immunoreactivity of the enzyme with the purified antibody. Isolated cardiomyocytes and frozen sections of the heart were stained for acetylcholinesterase with the antibody. Diffuse immunofluorescence appeared over the surface of the cardiomyocytes. In the frozen sections, the immunofluorescence was most intense at the cell boundaries. These data suggest that collagenase-sensitive acetylcholinesterase in the heart is present in the myocytes and occurs in the vicinity of the basal lamina.Abbreviations AChE acetylcholinesterase - BSA bovine serum albumin - PBS phosphate-buffered saline - DME Dulbecco's Modified Eagle Medium     

Structural composition and anticoagulant activity of dermatan sulfate from the skin of the electric eel, Electrophorus electricus (L.)

We determined the disaccharide composition of dermatan sulfate (DS) purified from the skin of the electric eel Electrophorus electricus. DS obtained from the electric eel was composed of non-sulfated, mono-sulfated disaccharides bearing esterified sulfate groups at positions C-4 or C-6 of N-acetyl galactosamine (GalNAc), and disulfated disaccharides bearing esterified sulfate groups at positions C-2 of the uronic acid and at position C-4 or C-6 of GalNAc. The anticoagulant, antithrombotic and bleeding effects of electric eel skin DS were compared to those of porcine DS and also to those described previously for DS purified from skin of eel, Anguilla japonica. DS from electric eel is a potent anticoagulant due to a high heparin co-factor II (HC II) activity. The electric eel DS has a higher potency to prevent thrombus formation on an experimental model and a lower bleeding effect in rats than the porcine DS. Interestingly, it was recently demonstrated that DS obtained from skin of the eel Anguilla japonica, which possesses a disaccharide composition very similar to that of electric eel skin DS described here, did not show anticoagulant activity. Thus, the anticoagulant activity of electric eel skin DS is not merely a consequence of its charge density. We speculate that the differences among the anticoagulant activities of these three DS may be related to different arrangements of the disulfated disaccharide domain for binding to HC II within their polysaccharide chains and that it may be more efficiently arranged along the carbohydrate chain in electric eel skin DS than in the two other types of DS.     

Production in Large Quantities of Actinorhodin and Undecyl-prodigiosin Induced by afsB in Streptomyces lividans

Paraquat inhibited the acetylcholinesterase activity of human erythrocytes and electric organs of Electrophorus electricus. The inhibition of acetylcholinesterase activity was reversible, as shown from the following two experimental results: [I] The degree of inhibition was not affected by changing the preincubation time of the enzyme and paraquat before the addition of the substrate. [II] The enzyme, preincubated with paraquat and subsequently freed from inhibitor by gel filtration on Sephadex G-25, showed the same activity as the untreated enzyme. Paraquat gave effective protection against the inhibition by an irreversible anionic site inhibitor, dibenamine, but not by irreversible esteratic site inhibitors, dichlorvos and methanesulfonyl chloride. These results indicate that paraquat functions as a reversible inhibitor for the anionic site. The inhibitory powers and Hill coefficients of paraquat and diquat were compared with the other quaternary ammonium compounds. Although secondary to edrophonium, paraquat strongly inhibited acetylcholinesterases of human erythrocytes and electric eel, and showed higher inhibition selectivity for both acetylcholinesterases than for human plasma butyrylcholinesterase. The Hill coefficients concerning the interaction of paraquat with acetylcholinesterases of human erythrocytes and electric eel were given as 0.83 and 0.73, respectively. This indicates negative cooperativity between these enzymes and paraquat, which is similar to the case with d-tubocurarine. On the other hand, diquat showed weak inhibitory power and low inhibition selectivity, and its Hill coefficients were almost 1.0, indicating a competitive inhibition mode.     

Identification of isoenzymes in cholinesterase preparations using kinetic data of organophosphate inhibition

Commercial preparations of acetylcholinesterase (EC 3.1.1.7) and of cholinesterase (EC 3.1.1.8) were characterized by organophosphate inhibition. Cholinesterase activities were inhibited by varying organophosphate concentration and time of inhibition. Bimolecular rate constants were determined by plotting log activity vs inhibitor concentration or inhibition time. Inhibition of acetylcholinesterase from bovine erythrocytes by diethyl p-nitrophenyl phosphate (Paraoxon), diisopropylphosphorofluoridate (DFP), and N,N′-diisopropylphosphorodiamidic fluoride (Mipafox) in semilogarithmic plots showed a linear decay of activity. Inhibition of acetylcholinesterase from electric eel (Electrophorus electricus) and of cholinesterases from horse serum and from human serum did not show linear characteristics, indicating the presence of more than one single enzyme in these preparations. The corresponding inhibition curves were resolved by subtraction of exponential functions. In each case two different activity components were identified and characterized in respect to partial activity, substrate specificity, and reactivity with organophosphorous compounds. The suitability of the method for application on crude homogenates is discussed.