Monoclonal Antibodies to Rabbit Brain Acetylcholinesterase: Selective Enzyme Inhibition, Differential Affinity for Enzyme Forms, and Cross-Reactivity with Other Mammalian Cholinesterases

Eleven unique monoclonal IgG antibodies were raised against rabbit brain acetylcholinesterase (AChE, EC 3.1.1.7), purified to electrophoretic homogeneity by a two-step procedure involving immunoaffinity chromatography. The apparent dissociation constants of these antibodies for rabbit AChE ranged from about 10 nM to more than 100 nM (assuming one binding site per catalytic subunit). Species cross-reactivity was investigated with crude brain extracts from rabbit, rat, mouse cat, guinea pig, and human. One antibody bound rabbit AChE exclusively; most bound AChE from three or four species; two bound enzyme from all species tested. Identical, moderate affinity for rat and mouse brain AChE was displayed by two antibodies; two others were able to distinguish between these similar antigens. Nine of the antibodies had lowered affinity for AChE in the presence of 1 M NaCl, but two were salt resistant. Analysis of mutual interferences in AChE binding suggested that certain of the antibodies were competing for nearby epitopes on the AChE surface. One antibody was a potent AChE inhibitor (IC50 = 10(-8) M), blocking up to 90% of the enzyme activity. Most of the antibodies were less able to bind the readily soluble AChE of detergent-free brain extracts than the AChE which required detergent for solubilization. The extreme case, an antibody that was unable to recognize nearly half of the "soluble" AChE, was suspected of lacking affinity for the hydrophilic enzyme form.     

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.     

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.     

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.     

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.     

Generation of subunit-specific antibody probes for Torpedo acetylcholinesterase: cross-species reactivity and use in cell-free translations

The assembly of the collagen tailed A12 form of acetylcholinesterase (AChE) is regulated by muscle contraction. To begin to study this regulation, we derived antibody probes for the three subunits (100 kd, catalytic, and collagen tail) of AChE purified from Torpedo californica electric tissue. These included a polyclonal antiserum that recognizes all 3 subunits and 19 monoclonal antibodies; 16 of the monoclonals recognized the catalytic subunit, 2 recognized the tail subunit, and 1 recognized the 100 kd subunit on Western blots. We used immunohistochemical procedures to show that several of the anticatalytic and one of the antitail monoclonals cross-reacted with frog muscle AChE and Western blotting to show that several of the anticatalytic monoclonals cross-react with rat brain AChE. These antibodies were then used to immunoprecipitate AChE precursors from a cell-free translation system. There were generally three primary translation products, corresponding to the three enzyme subunits. Therefore, each subunit is probably derived from a separate mRNA. Occasionally there were two translation products corresponding to the catalytic subunit alone. The catalytic subunit was glycosylated following addition of canine microsomal membranes to the translation mix. The mRNA coding for this subunit appeared to be present in the poly(A)- RNA pool.     

Peptidasic Activities Associated with Acetylcholinesterase Are Due to Contaminating Enzymes

The esterasic and peptidasic activities of two different sources of acetylcholinesterase purified from electric eel were examined. Hydrolyses of leucine-enkephalin and neurotensin indicated that both sources exhibited exopeptidasic and tryptic-like activities. However, the enzyme preparation which appeared 10-fold enriched with regard to the esterasic activity was found to display a 50- and 185-fold lower tryptic-like and exopeptidasic function, respectively. This lack of parallelism in the enrichment of the various activities seemed to indicate that they were not co-purified. Immunoprecipitation experiments performed with monoclonal antibodies directed towards the catalytic subunit of globular or asymmetric forms of electric eel acetylcholinesterase allowed the physical dissociation of esterasic and peptidasic functions and therefore confirmed that the ability of acetylcholinesterase to hydrolyze various neuropeptides was likely due to contaminating peptidases.     

Atypical effect of some spin trapping agents: reversible inhibition of acetylcholinesterase

N-tert-butyl-alpha-phenylnitrone (PBN), a widely used nitrone-based free radical trap was recently shown to prevent acetylcholinesterase (AChE) inhibitors induced muscle fasciculations and brain seizures while being ineffective against glutamergic or cholinergic receptor agonist induced seizures. In the present study we compared the effects on AChE activity of four free radical spin traps PBN, alpha-(4-pyridil-1)-N-tert-butyl nitrone (POBN), N-tert-butyl-alpha-(2-sulfophenyl)-nitrone (S-PBN) and 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO). The kinetics of AChE inhibition were studied in vitro using a spectrophotometric kinetic assay with AChE from rat brain, diaphragm, electric eel and mouse brain. Spin trapping compounds S-PBN and DEPMPO, in concentrations up to 3 mM did not inhibit hydrolysis of ACh, while PBN and POBN inhibited hydrolysis of ACh in a reversible and concentration-dependent manner. Double reciprocal plots of the reaction velocity against varying ACh concentrations at each inhibitor concentration were linear and generally indicated mixed type inhibition. PBN was the most potent inhibitor of mouse AChE with Ki and Ki' of 0.58 and 2.99 mM, respectively, and the weakest inhibitor of electric eel AChE. In contrast, POBN showed the highest affinity for electric eel enzyme, with Ki and Ki' values of 1.065 and 3.15 mM, respectively. These findings suggest that the effect of PBN and POBN on AChE activity does not depend on trapping of damaging reactive oxygen and that in addition to their antioxidant action other pharmacological effects of these compounds should be considered when neuroprotective actions of PBN or POBN are investigated.     

Isolation, localization, and cloning of a kainic acid binding protein from frog brain

Excitatory amino acids (EAA) are major neurotransmitters in the vertebrate central nervous system. EAA receptors have been divided into three major subtypes on the basis of electrophysiological and ligand binding studies: N-methyl-D-aspartate, kainate, and quisqualate receptors. To understand their molecular properties, we undertook a project aimed at isolation and cloning of these receptor subtypes. We purified a kainate binding protein (KBP) from frog brain, in which kainate binding sites are about fortyfold more abundant than in rat brain, using domoic acid affinity chromatography, and made monoclonal and polyclonal antibodies to the purified protein. These antibodies immunoprecipitate the frog KBP but not KBPs from other species. Immunocytochemical analyses show that KBP has a synaptic and extrasynaptic localization in frog optic tectum, with most labeling being extrasynaptic. The cDNA encoding frog brain KBP was isolated by screening a frog brain cDNA library with oligonucleotide probes that were based on the amino acid sequence of the purified protein. The deduced amino acid sequence of the KBP has a hydrophobic profile similar to those of other ligand-gated ion channel subunits, such as the nicotinic acetylcholine receptor, the GABAA receptor, and the glycine receptor. Frog brain KBP is very similar (36% amino acid identity to the carboxyl half) to rat brain kainate receptor, suggesting that these two proteins evolved from a common ancestor. The function of KBP in frog brain remains a major question. Preliminary results showed that Xenopus laevis oocytes injected with KBP RNA did not produce a detectable electrophysiological response when perfused with kainate. These results suggest that additional subunits may be required to form a functional receptor or that KBP is not functionally related to a neurotransmitter receptor.     

A Library of Monoclonal Antibodies to Torpedo Cholinergic Synaptosomes

Abstract: A library of monoclonal antibodies was generated to the cholinergic synaptosome. The immunogen was a preparation of highly purified synaptosomes from Torpedo electric organ. One hundred forty-one hybridoma cell lines were generated from the fusion of a single mouse. Tests reveal these cells produce antibodies with a vast range of neuronal specificities. The initial screen for specificity of antibody production was solid phase radioimmune binding to the original, highly purified synaptosome preparation. Subsequent tissue specificity tests have indicated that most antibodies are synaptosome-specific amongst the fish tissues tested: brain, liver, and purified synaptic vesicles. Less than 11% cross-react with liver. Many antibodies cross-react with frog and rat CNS. Localization within the frog and rat nervous tissue has revealed a vast array of antibody staining patterns. Some antibodies stain in a synaptic fashion. A few stain a restricted set of mammalian CNS neurons. Others define a broader set of CNS neurons. These Torpedo antibodies promise to be valuable probes with which to describe the molecular cell biology of the nervous system, of neurons in general, and of cholinergic neurons in particular.     

An inhibitory monoclonal antibody to human acetylcholinesterases

The monoclonal antibody AE-2 raised against acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) from human erythrocytes is shown to inhibit the enzyme activity. The reaction of the antibody with a structural epitope is investigated further. The epitope resides on monomeric, dimeric and tetrameric species of the enzyme. The rate of phosphorylation of the enzyme by diisopropylfluorophosphate was not affected by the antibody. On the other hand, inhibitors directed towards the anionic site(s) competed with antibody binding, suggesting that one of these is the epitope. The titration with antibody is biphasic and yields about 80% inhibition even in the presence of a large excess of antibody. Inhibition is fully reversible upon dilution, in a time-dependent manner. AE-2 also inhibited human adult and fetal brain acetylcholinesterase (to the same extent). However bovine brain acetylcholinesterase was inhibited to a lesser extent and rat brain acetylcholinesterase did not interact with the antibody. Butyrylcholinesterase (EC 3.1.1.8) also showed no reactivity towards the antibody.     

An Immunological Study of Rat Acetylcholinesterase: Comparison with Acetylcholinesterases from Other Vertebrates

We have examined the immunoreactivity of acetylcholinesterase from different vertebrate species with a rabbit antiserum raised against the purified rat brain hydrophobic enzyme (G4 form). We found no significant interaction with enzymes from Electrophorus, Torpedo, chicken, and rabbit. The antiserum reacted with acetylcholinesterases from the brains of the other mammalian species studied, with titers decreasing in the following order: rat = mouse greater than human greater than bovine. The serum was inhibitory with murine and human acetylcholinesterases, but not with the bovine enzyme. The inhibition was partially depressed in the presence of salt (e.g., 1 M NaCl). In those species whose acetylcholinesterase was recognized by the antiserum, both soluble and detergent-soluble fractions behaved in essentially the same manner, interacting with the same antibodies. The apparent immunoprecipitation titer was decreased in the presence of salt, and it did not make any difference whether NaCl was included in the solubilization procedure or added to the extracts. Both G1 and G4 forms of acetylcholinesterase in the soluble and detergent-soluble fractions were recognized by the antiserum, and in the case of the human enzyme, by monoclonal antibodies produced against human erythrocyte acetylcholinesterase. However, the monomer G1 showed a clear tendency to form smaller complexes and precipitate less readily than the tetramer G4. Although we cannot exclude the existence of significant differences between the various molecular forms of acetylcholinesterase, our results are consistent with the hypothesis that they all derive from the same gene or set of genes by posttranslational modifications.     

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.     

Crucial role of cytoskeleton reorganization in the negative inotropic effect of chromogranin A-derived peptides in eel and frog hearts

Vasostatins (VSs), i.e. the main biologically active peptides generated by the proteolytic processing of chromogranin A (CGA) N-terminus, exert negative inotropism in vertebrate hearts. Here, using isolated working eel (Anguilla anguilla) and frog (Rana esculenta) heart preparations, we have studied the role of the cytoskeleton in the VSs-mediated inotropic response. In both eel and frog hearts, VSs-mediated-negative inotropy was abolished by treatment with inhibitors of cytoskeleton reorganization, such as cytochalasin-D (eel: 10 nM; frog: 1 nM), an inhibitor of actin polymerisation, wortmannin (0.01 nM), an inhibitor of PI3-kinase (PI3-K)/protein kinase B (Akt) signal-transduction cascade, butanedione 2-monoxime (BDM) (eel: 100 nM; frog: 10 nM), an antagonist of myosin ATPase, and N-(6-aminohexil)-5-chloro-1-naphthalenesulfonamide (W7) (eel: 100 nM; frog: 1 nM), a calcium-calmodulin antagonist. These results demonstrate that changes in cytoskeletal dynamics play a crucial role in the negative inotropic influence of VSs on eel and frog hearts.     

Active-site determinations on forms of mammalian brain and eel acetylcholinesterase.

Three forms of brain acetylcholinesterase were purified from bovine caudate-nucleus tissue and determined by calibrated gel filtration to have mol.wts. of approx. 120 000 (C), 230 000 (B) and 330 000 (A). [3H]Di-isopropyl phosphorofluoridate (isopropyl moiety labelled) was purified from commercial preparations and its concentration estimated by an enzyme-titration procedure. Brain acetylcholinesterase preparations and enzyme from eel electric tissue were allowed to react with [3H]di-isopropyl phosphorofluridate in phosphate buffer until enzyme activity was inhibited by 98%. Excess of [3H]di-isopropyl phosphorofluoridate that had not reacted was separated from the labelled enzyme protein by gel filtration, or by vacuum filtration or by extensive dialysis. The specificity of active-site labelling was confirmed by use of the enzyme reactivator, pyridine 2-aldoxime. The forms of brain acetylcholinesterase were calculted to contain approximately two (C) four (B) and six (A) active sites per molecule respectively. Acetylcholinesterase (mol.wt. 250 000) from electric-eel tissue was estimated to contain two active sites per molecule. Gradient-gel electrophoresis was used to confirm the estimation of molecular weights of brain acetylcholinesterase forms made by gel filtration. Under the conditions of electrophoresis acetylcholinesterase form A was stable, but form B was converted into a species of approx. 120 000 mol. wt. Similarly, form C of the brain enzyme was converted into a 60 000-mol.wt. form during electrophoresis. These results are in general accord with the suggestion that the multiple forms of brain acetylcholinesterase may be related to the aggregation of a single low-molecular-weight species.     

Biochemical properties of sodium channels in a wide range of excitable tissues studied with site-directed antibodies

Antibodies against a peptide (SP19) corresponding to a highly conserved, predicted intracellular region of the sodium channel alpha subunit bind rat brain sodium channels with a similar affinity as the peptide antigen, indicating that the corresponding segment of the alpha subunit is fully accessible in the intact channel structure. These antibodies recognize sodium channel alpha subunits from rat or eel brain, rat skeletal muscle, rat heart, eel electroplax, and locust nervous system. alpha subunits from all these tissues except rat skeletal muscle are substrates for phosphorylation by cAMP-dependent protein kinase. Disulfide linkage of alpha and beta 2 subunits was observed for both the RI and RII subtypes of rat brain sodium channels and for sodium channels from eel brain but not for sodium channels from rat heart, eel electroplax, or locust nerve cord. Treatment with neuraminidase reduced the apparent molecular weight of sodium channel alpha subunits from rat and eel brain and eel electroplax by 22,000-58,000, those from heart by 8000, and those from locust nerve cord by less than 4000. Our results provide the first identification of sodium channel alpha subunits from rat heart and locust brain and nerve cord and show that sodium channel alpha subunits are expressed with different subunit associations and posttranslational modifications in different excitable tissues.     

Analysis of 3H-kainic acid binding to brain membranes in rats and frogs

It has been shown that H3-kainic acid (3H-KA) specifically binds with membrane preparations from various parts of rat brain or whole frog brain. The saturation isotherms of 3H-KA binding revealed the presence of two sites with a high and low affinity. An exception was for rat cerebellum where Scatchard analysis showed but one low affinity site. The density of 3H-KA binding sites in frog brain was 5 to 10 times higher than in rat brain. Among the drugs studied, KA itself, L-glutamate and folic acid were the most potent inhibitors of specific binding. Methyltetrahydrofolate, quinolinic acid, kynurenine, GABA, taurine, L-aspartate were ineffective in this respect. The kinetic analysis of the binding data in the presence or absence of L-glutamate and folic acid showed, however, that these drugs inhibited 3H-KA binding in a noncompetitive manner. In the light of these findings L-glutamate or folate cannot be considered as endogenous ligands for hypothetic "kainate receptors".     

Ecdysterone induces acetylcholinesterase in mammalian brain

The effects of ecdysterone on brain acetylcholinesterase (AChE) in immature and adult rats of both sexes have been studied in in vitro conditions. Ecdysterone produced an increase of AChE in rat brain slices. The most remarkable effect was found in immature male rats. In vitro assay using a purified AChE from electric eel showed no effect. Pretreatment with cycloheximide or actinomycin D abolished the ecdysterone action on brain AChE. These results support the idea that induction of AChE may be involved in the heterophilic action of ecdysterone.     

Immunochemical differences among molecular forms of acetylcholinesterase in brain and blood

Molecular forms of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) differ in their solubility properties as well as in the number of their catalytic subunits. We used monoclonal antibodies to investigate the structure of acetylcholinesterase forms in brain, erythrocytes and serum of rats, rabbits and other mammals. Two antibodies were found to bind tetrameric acetylcholinesterase in preference to the monomeric enzyme. These antibodies also displayed lower affinity for certain forms of 'soluble' brain acetylcholinesterase than for the 'membrane-associated' counterparts. Furthermore, one of them was virtually lacking in affinity for the membrane-associated enzyme of erythrocytes. The basis for the antibody specificity was not fully determined. However, the immunochemical results were supported by measurements of enzyme thermolability, which showed that the catalytic activity of 'soluble' acetylcholinesterase was comparatively heat-resistant. These observations point toward structural differences among the solubility classes of acetylcholinesterase.     

Partial purification and reconstitution of the (Ca2+ + Mg2+)-ATPase of erythrocyte membranes.

Benzenemethane Sulfonylfluoride (329-98-6) is an irreversible inactivator of many esterases including mammalian acetylcholinesterases. However, previous reports indicated that acetylcholinesterase from the electric eel, Electrophorus electricus (EC 3.1.1.7) failed to react with benzenemethane sulfonylfluoride at measurable rates. We report here that eel acetylcholinesterase reacts with this inactivator at a low rate. Hydrolysis of the sulfonylating agent is so much faster than enzyme inactivation that, under most conditions, there will be only slight inactivation. Like the reaction of other active site acylating agents with this enzyme, inactivation can be accelerated in the presence of certain organic cations. We introduce a rate equation for enzyme sulfonylation which incorporates both the hydrolysis of the inactivator and the complication that fluoride resulting from hydrolysis of the inactivator is a potent competitive inhibitor of this enzyme. This rate equation accurately describes the time course of enzyme inactivation.