Novel allosteric sites on human erythrocyte acetylcholinesterase identified by two monoclonal antibodies.

Monoclonal antibodies against human erythrocyte acetylcholinesterase (acetylcholine acetylhydrolase EC 3.1.1.7) have been examined for inhibition of enzyme activity. Of sixteen antibodies analyzed, only one (C1B7) inhibited enzyme activity, indicating selection of an unusual susceptible site. The inhibitory activity of C1B7 was characterized and compared to another inhibitory antibody, AE-2, previously described by Fambrough et al. (Proc. Natl. Acad. Sci. USA 79, 1078, 1982). Maximal demonstrated inhibition was 84% for C1B7 and 72% for AE-2 and antibody inhibition of enzyme activity was equivalent for the reduced and alkylated acetylcholinesterase monomer and the intact dimer. The Ki (stoichiometry of the enzyme-antibody reaction estimated from enzyme kinetics) was 1.0 for C1B7 and 4.8 molecules of antibody per monomer of acetylcholinesterase for AE-2. The antibodies did not compete with one another for binding to acetylcholinesterase, indicating that they have different target epitopes on the enzyme. Antibody binding to the enzyme was not specifically affected by any of the anticholinesterase agents tested: (a) the irreversible esteratic site-directed inhibitor diisopropylfluorophosphate; (b) the reversible active site-directed inhibitors edrophonium, neostigmine, BW284c51, and carbachol; and (c) allosteric site-directed compounds propidium and gallamine. Kinetic analysis of their effects provide evidence that both antibodies decrease the catalytic rate of enzyme activity and have little or no effect on substrate binding.     

The monoclonal antibody 2G8 is carbohydrate-specific and distinguishes between different forms of vertebrate cholinesterases

The monoclonal antibody (mAb) 2G8 (subclass IgG2a) raised against acetylcholinesterase (AChE, EC 3.1.1.7) from electric organ of Torpedo nacline timilei crossreacted with AChE from Torpedo marmorata, electric eel (Electrophorus electricus), flounder (Platichthys flesus) body muscle, rat brain, bovine brain, and human brain, this suggests that the epitope to which mAb 2G8 bound had been highly conserved during evolution. No crossreaction was found with AChE from human and bovine erythrocytes, nor with butyrylcholinesterase (BtChE, EC 3.1.1.8) from human serum. Binding of mAb 2G8 to the globular G2 form of AChE from T. marmorata strongly decreased enzyme activity, while no significant inhibition was found with either collagen-tailed, asymmetric forms, or with the enzymes from flounder body muscle or mammalian sources. The possibility that mAb 2G8 bound to anionic sites of AChE could be excluded since neither edrophonium chloride nor decamethonium bromide influenced the binding of 2G8 to the enzymes. Enzyme-linked immunosorbent assay and Western blot showed that heat-denatured, diisopropylfluorophosphate-treated, CNBr- and trypsin-digested AChE from T. marmorata still reacted with mAb 2G8; this indicates that the epitope to which 2G8 bound, at least partially, belonged to a continuous determinant. Treatment of cholinesterases with N-glycosidase F abolished crossreaction with 2G8, showing that an essential part of the epitope consisted of N-linked carbohydrates.     

Human acetylcholinesterase. Immunochemical studies with monoclonal antibodies

Monoclonal antibodies were used to investigate the immunochemistry of human erythrocyte acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7). A series of experiments on the sedimentation velocity and Stokes radius of acetylcholinesterase and its immune complexes indicated that each antibody recognized a single high-affinity binding site (epitope) on the monomeric enzyme. Further analysis suggested that the antibody-binding sites were replicated on multimeric enzyme forms but were subject to steric hindrance between nearby IgG molecules or adjacent enzyme subunits. The cellular localization of the epitopes was studied by measuring the binding of monoclonal antibodies to the cholinesterase of intact erythrocytes. The results implied that most of the epitopes are exposed to the external media. However, one antibody failed to bind to intact cells, despite a relatively high affinity for detergent-solubilized antigen, possibly because its epitope is buried in the lipid bilayer.     

Acetylcholinesterase from fetal bovine serum. Purification and characterization of soluble G4 enzyme

Acetylcholinesterase (EC 3.1.1.7) from fetal bovine serum (FBS) was purified to electrophoretic homogeneity. The procedure involved procainamide affinity chromatography with native FBS, followed by chromatography on Sepharose 6B and DEAE-Sephadex. The acetylcholinesterase was purified approximately 44,000-fold, and 13 mg was obtained corresponding to an overall yield of about 45%. The purified acetylcholinesterase was stable at 4 degrees C for at least 8 weeks but was labile to freezing; however, in 50% glycerol the enzyme was stable at -20 degrees C for at least 12 weeks. FBS acetylcholinesterase exhibited typical substrate inhibition, had a Km of 120 microM, and a turnover number of 5300 s-1 with the substrate acetylthiocholine. The enzyme was highly sensitive to the specific acetylcholinesterase inhibitor 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one. FBS acetylcholinesterase was characterized as a G4 form of acetylcholinesterase and was distinguished from bovine erythrocyte acetylcholinesterase on the basis of lectin gel binding, [3H] Triton X-100 binding, amino acid composition, number of catalytic subunits/molecule, and hydrodynamic properties. FBS acetylcholinesterase had a Stokes radius of 76 A as judged by gel filtration, and from this a molecular weight of 340,000 daltons was calculated. The enzyme had a subunit weight of approximately 83,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; paraoxon titration indicated a relative active site mass of 75,000 daltons. The amino acid composition of FBS acetylcholinesterase was similar to the human erythrocyte acetylcholinesterase (Rosenberry, T. L., and Scoggin, D. M. (1984) J. Biol. Chem. 259, 5643-5652). A monoclonal antibody directed against human erythrocyte acetylcholinesterase, AE-2, (Fambrough, D. M., Engel, A. G., and Rosenberry, T. L. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 1078-1082) cross-reacted with FBS acetylcholinesterase.     

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.     

The monoclonal antibody AE-2 modulates fetal bovine serum acetylcholinesterase substrate hydrolysis

The monoclonal antibody (mAb) AE-2 decreases the rate of hydrolysis of acetylthiocholine (ATC) by fetal bovine serum acetylcholinesterase (acetylcholine acetylhydrolase EC 3.1.1.7) (FBS-AChE) (Doctor, B.P. et al. (1989) Proc. 32nd Oholo Conf., Eilat, Israel, in press), but increases the rate of hydrolysis (Vmax) of the nonpolar substrate, indophenyl acetate (IPA) approx. 15-fold. The affinity (Km) of FBS-AChE for IPA changes minimally in comparison with the increase in the rate of hydrolysis. The complex is dissociated, and the modulation of substrate hydrolysis is reversed by the active-center ligand, 1-methyl-2-hydroxyiminomethylpyridinium chloride (2-PAM).     

Two-Site Immunoassay for Acetylcholinesterase in Brain, Nerve, and Muscle

Two-site methods were developed for immunoassay of acetylcholinesterase (AChE; EC 3.1.1.7) in crude extracts of rat and human tissues. A radiometric assay for human AChE utilized a specific monoclonal AChE antibody adsorbed to polystyrene microtiter wells at alkaline pH. AChE bound strongly to this antibody after 24 h at 4 degrees C. Bound enzyme was detected with an 125I-labeled antibody against a different AChE epitope. The assay signal was quasi-linearly related to AChE concentration in purified and crude samples, with a detection threshold near 100 pg. Tetrameric and dimeric AChE behaved equivalently in the assay. Two-site methods with a different pair of species-selective antibodies worked equally well for immunoassay of rat AChE. Assays of the rat enzyme showed that immunoreactivity was lost as rapidly as enzyme activity during heating to 54 degrees C. On the other hand, immunoreactivity was preserved despite loss of enzyme activity after exposure to anticholinesterases or trypsin. A biotinylated second antibody detected by alkaline-phosphatase-conjugated avidin was used to develop an AChE enzyme-linked immunosorbent assay (ELISA) with a sensitivity similar to that of the radiometric assay. Either the ELISA or the radiometric immunoassay may be useful whenever proteolysis or other mechanisms are suspected of dissociating enzyme activity and immunoreactivity. In denervated muscle and ligated peripheral nerve, application of the two-site method showed closely parallel variations in immunoreactivity and enzyme activity.     

A monoclonal antibody against a native conformation of the porcine renal Na+/K(+)-ATPase alpha-subunit protein

A monoclonal antibody (mAb50c) against the native porcine renal Na+/K(+)-transporting adenosinetriphosphatase (EC 3.6.1.37, ATP phosphohydrolase) (Na+/K(+)-ATPase) was characterized. The antibody could be classified as a conformation-dependent antibody, since it did not bind to Na+/K(+)-ATPase denatured by detergent and its binding was affected by the normal conformational changes of the enzyme induced by ligands. The binding was the greatest in the presence of Na+, ATP or Mg2+ (E1 form), slightly less in the presence of K+ (E2K form) and the least when the enzyme was phosphorylated, especially in the actively hydrolyzing form in the presence of Na+, Mg2+ and ATP. The antibody inhibited both the Na+,K(+)-ATPase activity and the K(+)-dependent p-nitrophenylphosphatase activity by 25%, but it had no effect on Na(+)-dependent ATPase activity. The antibody partially inhibited the fluorescence changes of the enzyme labeled with 5'-isothiocyanatofluorescein after the addition of orthophosphate and Mg2+, and after the addition of ouabain. Proteolytic studies suggest that a part of the epitope is located on the cytoplasmic surface of the N-terminal half of the alpha-subunit.     

Characterization of monoclonal antibodies against human tissue plasminogen activator (tPA): quantitation of free tPA in human cell cultures by an ELISA.

Seven murine monoclonal antibodies produced against tissue plasminogen activator (tPA) were evaluated by means of enzyme-linked immunosorbent assays (ELISAs), and their effects on the enzymatic activities of tPA towards a synthetic substrate (S-2288) and plasminogen were investigated. One of the antibodies, TPA1-70, strongly inhibited the enzymatic activity of tPA in a fibrin agarose plate assay, while it did not affect the enzymatic activity towards the synthetic substrate or plasminogen. The antibody is directed to an epitope on the B-chain of tPA, which is necessary for the formation of a ternary complex of tPA, fibrin and plasminogen, but probably not to the active site. Another antibody, TPA2-14, partially inhibited the enzymatic activities of tPA towards the synthetic substrate and plasminogen, but it was not able to bind to the inactive tPA complexed with plasminogen activator inhibitor-1 (PAI-1). The antibody is directed to an epitope on the second kringle region, which is probably one of the PAI-1 binding sites. This property of the antibody enabled us to develop an ELISA for selective quantitation of free tPA in culture media conditioned with several human cell lines. The results indicate that tPA in these media exists either partially or almost entirely in a complex with PAI-1.     

An Amphiphile-Dependent Form of Human Brain Caudate Nucleus Acetylcholinesterase: Purification and Properties

Abstract: Different forms of acetylcholinesterase (AChE), EC 3.1.1.7, were demonstrated in human brain caudate nucleus. One form was solubilized at high ionic strength, the other with Triton X-100. The detergent-extractable form was purified to homogeneity by affinity chromatography. This form of AChE is amphiphile-dependent; i.e., it was active only in the presence of amphiphiles (detergents or lipids). Further, the enzyme was shown to bind detergents and to interact hydrophobically with Phenyl-Sepharose. In the presence of detergents the enzyme is a tetramer (subunit molecular weight, 78,000) which aggregates on the removal of detergents. Human brain AChE showed a reaction of identity with human erythrocyte AChE in crossed-line immunoelectrophoresis. The high-salt-soluble brain enzyme did not cross-react with the erythrocyte enzyme. The two classes of AChE seem not to be related, as they show no common antigenic determinant.     

Substance P Hydrolysis by Human Serum Cholinesterase

Highly purified human serum cholinesterase (EC 3.1.1.8, also known as pseudocholinesterase and butyrylcholinesterase) had peptidase activity toward substance P. Digestion of substance P was monitored by high performance liquid chromatography, which separated three product peptides. The cleavages occurred sequentially. The first peptide to appear as Arg1-Pro2. The Km for this hydrolysis was 0.3 mM; maximum activity was 7.9 nmol min-1 mg-1 of protein, which corresponded to a turnover number of 0.6 min-1. A second cleavage yielded Lys3-Pro4. A third cleavage occurred at the C-terminal, where the amide was removed from Met11 to yield a peptide containing residues 5-11. Both the peptidase and esterase activities of the enzyme were completely inhibited by the anticholinesterase agent, diisopropylfluorophosphate. Substance P inhibited the hydrolysis of benzoylcholine (a good ester substrate) with a KI of 0.17 mM, indicating that substance P interacted with cholinesterase rather than with a trace contaminant. Peptidase and amidase activities for serum cholinesterase are novel activities for this enzyme. It was demonstrated previously that the related enzyme acetylcholinesterase (EC 3.1.1.7) catalyzed the hydrolysis of substance P, but at entirely different cleavage sites from those reported in the present work. Since butyrylcholinesterase is present in brain and muscle, as well as in serum, it may be involved in the physiological regulation of substance P.     

Development, validation and application of an enzyme immunoassay (EIA) of atriopeptin

A rapid, convenient, and sensitive enzyme immunoassay (EIA) for atriopeptin (AP) has been developed. The tracer-ligand for the assay is the 24-amino acid peptide, AP24, which has been covalently coupled to the tetrameric form of acetylcholinesterase (AChE) (EC 3.1.1.7). Tracer, unknown, and primary antibody are incubated in a 96-well microtiter plate precoated with secondary antibody. After washing, a colorimetric reaction is used to measure acetylcholinesterase activity. A direct linear correlation was obtained when comparing the conventional radioimmunoassay and the EIA by using the same primary antibody to assay: plasma samples (rat or human), HPLC column fractions, or atrial extracts. Besides being technically much less demanding and not requiring the use of the radioisotopes, the EIA is more sensitive than the radioimmunoassay and thereby lends itself to a "flash" same-day assay of samples.     

Energy-Metabolising Enzymes in Brain Regions of Adult and Aging Rats

Abstract: The regional enzyme activities of glucose metabolism in the rat brain were investigated. Hexokinase (EC 2.7.1.1) and pyruvate dehydrogenase (EC 1.2.4.1), key enzymes for glucose metabolism, showed no changes in activity in all the regions studied of the aging brain as compared with the adult brain. However, the activity of d -3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) is low throughout the adult brain and, in contrast with hexokinase and pyruvate dehydrogenase, its activity decreases significantly during aging. Other enzymes that showed significant decreases during aging are aldolase (EC 4.1.2.13), lactate dehydrogenase (EC 1.1.1.27), citrate synthase (EC 4.1.3.7), and NAD⁺-linked isocitrate dehydrogenase (EC 1.1.1.41). The catabolic enzyme in cholinergic metabolism, acetylcholinesterase (EC 3.1.1.7), selected as an example of a non-energy-metabolising enzyme, also showed significant decreases in all regions of the brain in aging, although its highest activity remained in the striatum. These results are discussed with respect to the energy metabolism in various brain regions and their status with aging.     

Distribution and tissue specificity of glutamate decarboxylase (EC 4.1.1.15).

Abstract— The activity of L–glutamate decarboxylase (EC 4.1.1.15) (GAD) in various mouse tissues was determined by five different methods, namely, the radiometric CO₂ method, column separation, electro–phoretic separation, the filtration method, and amino acid analysis. Results from the latter four methods agreed well, showing that brain had the highest activity, 4.27 nmol/min/mg protein (100%), followed by heart (7.4%), kidney (6.3%) and liver (1.5%). Measurement of brain GAD using the radiometric CO₂ assay method agreed with the other techniques. However, in heart, kidney, and liver, the GAD activities measured by the CO₂ method were about 3–4 times higher than those obtained by the GABA method, suggesting that the CO₂ method does not give a valid measurement of GAD activity in a crude non–neural tissue preparation. GAD activity also was detected in adrenal gland but not in pituitary, stomach, testis, muscle, uterus, lung, salivary gland, or spleen. GAD from brain, spinal cord, heart, kidney and liver were further compared by double immunodiffusion, enzyme inhibition by antibody, and microcomplement fixation using antibody against GAD purified from mouse brain. GAD from brain and spinal cord appear to be identical as judged from the following results: the immunoprecipitin bands fused together without a spur; the enzyme activity was inhibited by anti–GAD to the same extent; and the microcomplement fixation curves were similar in both the shape of the curve and the extent of fixation. No crossreactivity was observed between GAD from heart, kidney or liver and antibody against brain GAD in all the immunochemical tests described above, suggesting that GAD in non–neural tissues is different from that in brain and spinal cord.     

Characterization of antibody to human phosphatidylcholine: cholesterol acyltransferase.

Purified preparations of phosphatidylcholine (lecithin): cholesterol acyltransferase (EC 2.3.1.43), were injected into goats to produce antisera reacting with this enzyme. The antisera and the gamma-globulin derived thereform were examined by the technics of immunodiffusion, immunoelectrophoresis and immunoinhibition of the enzyme. The antisera gave no precipitation lines with human high density lipoproteins (HDL) and human low density lipoproteins (LDL). A weak antibody titer towards human serum albumin was noted only after prolonged immunization. The enzymatically active band isolated from acrylamide gels gave a single arc in immunodiffusion and immunoelectrophoresis. The gamma-globulin derived from the antisera inhibited human phosphatidylcholine:cholesterol acyltransferase activity.     

A monoclonal antibody against COOH-terminal peptide of human liver manganese superoxide dismutase

Three stable hybridoma cell lines producing monoclonal antibodies specific for human liver manganese superoxide dismutase were established, and one monoclonal antibody, PG 11, was chosen for immunochemical studies. Immunoblotting demonstrated that the monoclonal antibody binds exclusively to the manganese superoxide dismutase. Immunohistochemical studies indicated that the enzyme is localized in the matrix of human liver mitochondria. To localize antibody-binding epitope, synthetic peptides of the NH2-terminal (residues 1-16) and COOH-terminal (residues 182-189, 190-196, and 182-196) parts of the enzyme were synthesized, and then their effects on the binding were studied using an enzyme-linked immunosorbent assay method. All of the above COOH-terminal peptides inhibited the binding whereas the NH2-terminal ones did not, indicating that PG 11 recognizes several peptides of COOH termini of manganese superoxide dismutase. This is the first report of monoclonal antibodies against human manganese superoxide dismutase with a distinct epitope and of the immunocytochemical demonstration of manganese superoxide dismutase.     

Properties of catechol O-methyltransferases from brain and liver of rat and human.

Kinetic and electrophoretic properties of catechol O-methyltransferases (EC 2.1.1.6) from brain and liver were studied. The enzyme of either rat or human tissues exhibited a single molecular form when subjected to electrophoresis at pH7.9. At pH9 a second, apparently oxidized, form was detected. Isoelectric-focusing experiments also indicated only one enzyme form, which was identical from extracts of brain and liver of each species (pI = 5.2 for rat, 5.5 for human). Similarities between brain and liver catechol O-methyltransferase of a given species were also demonstrated by kinetic parameters, meta/para ratios of products, and inhibitor potencies. Human catechol O-methyltransferase exhibited lower Km values than did the rat enzyme for S-adenosyl-L-methionine, dopamine and dihydroxybenzoic acid. Adrenochrome inhibited both rat and human enzyme. It was concluded (1) that only a single enzyme form could be demonstrated in the physiological pH region; (2) that catechol O-methyltransferase of brain could not be distinguished from the liver enzyme of the same species; and (3) that species differences exist between the enzymes of rat and human tissues.     

A monoclonal antibody to bovine brain inositol monophosphatase. Immunoaffinity purification of the brain and kidney enzymes and evidence for their structural identity.

A monoclonal IgG2b(K) antibody, G-2A4, has been generated against bovine brain myo-inositol monophosphatase (EC 3.1.3.25). The identity of the antigen recognized by the antibody was established by using e.l.i.s.a. and Western blotting procedures, and by immunoprecipitation of enzyme activity from crude brain supernatant. In addition, the hydrolysis of Ins1P by crude brain extract was inhibited by up to 83% by the pure antibody. Under identical conditions, the hydrolysis of Ins(1,4)P2 was unaffected. An immunoadsorbent column containing monoclonal antibody G-2A4 covalently attached to CNBr-activated Sepharose 4B has been used for rapid purification of the brain enzyme. Elution conditions have been optimized to allow isolation of the enzyme in high yield (54%) with full retention of column-binding capacity. The enzyme was electrophoretically homogeneous, Mr 30,000 and of higher specific activity than that purified conventionally. Chromatography of the pure enzyme on high resolution ion-exchange columns revealed some charge heterogeneity, possibly indicative of some type of post-translational modification. The immunoadsorbent column has also been used to purify the bovine kidney cortex enzyme to homogeneity. Partial proteolytic fragmentation patterns of the brain and kidney enzymes using endoprotease glu-C were identical, suggesting that they are almost certainly products of the same gene.     

Time-dependence of the effect of X-irradiation on the formation of glycerol phosphate dehydrogenase and other dehydrogenases in the developing rat brain

X-irradiation (100-1500 r) administered to the heads of rats 8-30 days of age inhibited the development of glycerol phosphate dehydrogenase (l-glycerol 3-phosphate-NAD oxidoreductase, EC 1.1.1.8) in the brain stem and cerebral hemispheres. At 40 days of age and older no effect was observed. This inhibition was a delayed phenomenon, dose-dependent and with no recovery. It is proposed that the inhibition of enzyme formation is related to radiation damage caused to DNA. Actinomycin D inhibited the development of glycerol phosphate dehydrogenase in a manner similar to ionizing radiation. Four other dehydrogenases also showed age-dependent radiosensitivities. ;Malic enzyme' (EC 1.1.1.40), lactate dehydrogenase (EC 1.1.1.27) and malate dehydrogenase (EC 1.1.1.37) ceased to be radiosensitive at about 8 days of age and isocitrate dehydrogenase (NADP) (EC 1.1.1.42) at 16 days. The correlation between developmental increase in enzyme activity and radiosensitivity held closely for glycerol phosphate dehydrogenase and isocitrate dehydrogenase and to a smaller extent for the others.     

Isolation of a novel neutralizing antibody fragment against human vascular endothelial growth factor from a phage-displayed human antibody repertoire using an epitope disturbing strategy

Following the clinical success of Bevacizumab, a humanized monoclonal antibody that blocks the interaction between vascular endothelial growth factor (VEGF) and its receptors, the search for new neutralizing antibodies targeting this molecule has continued until now. We used a human VEGF variant containing three mutations in the region recognized by Bevacizumab to direct antibody selection towards recognition of other epitopes. A total of seven phage-displayed antibody fragments with diverse binding properties in terms of inter-species cross-reactivity and sensitivity to chemical modifications of the antigen were obtained from a human phage display library. All of them were able to recognize not only the selector mutated antigen, but also native VEGF. One of these phage-displayed antibody fragments, denominated 2H1, was shown to compete with the VEGF receptor 2 for VEGF binding. Purified soluble 2H1 inhibited in a dose dependent manner the ligand-receptor interaction and abolished VEGF-dependent proliferation of human umbilical vein endothelial cells. Our epitope disturbing strategy based on a triple mutant target antigen was successful to focus selection on epitopes different from a known one. Similar approaches could be used to direct phage isolation towards the desired specificity in other antigenic systems.