Inhibitor Report for: Tetramethylammonium

People with genetic variants of cholinesterase respond abnormally to succinylcholine, experiencing substantial prolongation of muscle paralysis with apnea rather than the usual 2-6 min. The structure of usual cholinesterase has been determined including the complete amino acid and nucleotide sequence. This has allowed identification of altered amino acids and nucleotides. The variant most frequently found in patients who respond abnormally to succinylcholine is atypical cholinesterase, which occurs in homozygous form in 1 out of 3500 Caucasians. Atypical cholinesterase has a single substitution at nucleotide 209 which changes aspartic acid 70 to glycine. This suggests that Asp 70 is part of the anionic site, and that the absence of this negatively charged amino acid explains the reduced affinity of atypical cholinesterase for positively charged substrates and inhibitors. The clinical consequence of reduced affinity for succinylcholine is that none of the succinylcholine is hydrolyzed in blood and a large overdose reaches the nerve-muscle junction where it causes prolonged muscle paralysis. Silent cholinesterase has a frame shift mutation at glycine 117 which prematurely terminates protein synthesis and yields no active enzyme. The K variant, named in honor of W. Kalow, has threonine in place of alanine 539. The K variant is associated with 33% lower activity. All variants arise from a single locus as there is only one gene for human cholinesterase (EC 3.1.1.8). Comparison of amino acid sequences of esterases and proteases shows that cholinesterase belongs to a new family of serine esterases which is different from the serine proteases.

A steroid, the dibutyrate analogue of pancuronium bromide (9.8 X 10(-8)M), has been used as differential inhibitor in the study of the plasma cholinesterase variants. Pancuronium dibutyrate numbers have been measured for 190 individuals, and the mean values for six of the known genotypes, E1uE1u, E1uE1f, E1uE1a, E1fE1a, E1aE1a, and E1fE1f, have been calculated. Evidence is presented that a combination of the pancuronium dibutyrate number and the fluoride number give better resolution of the six genotypes than the combination of the pancuronium dibutyrate and the dibucaine number. This new differential inhibitor has real potential for revealing the probable existence of new genotypes.

The dynamics of ligand movement through the constricted region of the acetylcholinesterase gorge is important in understanding how the ligand gains access to and is released from the active site of the enzyme. Molecular dynamics simulations of the simple ligand, tetramethylammonium, crossing this bottleneck region are conducted using umbrella potential sampling and activated flux techniques. The low potential of mean force obtained is consistent with the fast reaction rate of acetylcholinesterase observed experimentally. From the results of the activated dynamics simulations, local conformational fluctuations of the gorge residues and larger scale collective motions of the protein are found to correlate highly with the ligand crossing.

We describe three catalytic cholinesterase-like catalytic antibodies (Ab1), as well as anti-idiotypic (Ab2) and idiotypic (Ab3) antibodies, to one of the Ab1s. The Ab1s were raised against the human erythrocyte acetylcholinesterase (AChE), and are unusual in that they both recognise and resemble acetylcholinesterase in their catalytic activity. No contamination of the antibody preparations with either acetylcholinesterase or butyrylcholinesterase (BChE) was found. None of the Ab2s showed catalytic activity, whereas four Ab3s did (an incidence of 1.26% of all Ab3s). Although there is considerable resemblance between Ab1s and Ab3s, there are significant differences between the two groups. All the antibodies were inhibited by phenylmethylsulphonyl fluoride (PMSF), indicating the presence of a serine residue in their active sites, and were inhibited by the cholinesterase active site inhibitors iso-OMPA and pyridostigmine, suggesting the similarity of the sites to those of cholinesterases. The Ab3s resemble the Ab1s in their ability to hydrolyse both acetyl and butyrylthiocholine (BTCh). However, the Ab3s appear to be better catalysts, having significantly reduced K(m) values (for acetyl, but not for butyrylthiocholine) and increased turnover numbers (K(cat)), rate enhancements (K(cat)/K(uncat)) and K(cat)/K(m) ratios, for both substrates, although these values by no means approach those of the natural enzymes. The Ab1s appear to have structures resembling the anionic sites of cholinesterases, as shown by their reaction with the anionic site inhibitors (edrophonium and tetramethylammonium). No such reactions were observed in the Ab3s. None of the antibodies show evidence of the sites resembling the peripheral anionic site (PAS) of acetylcholinesterase. All the antibodies recognise, to varying degrees, the peripheral anionic site of acetylcholinesterase. This was shown by their ability to inhibit acetylcholinesterase, to compete with peripheral site inhibitors, and to block acetylcholinesterase-mediated cell adhesion, a property of this site. The results indicate idiotypic mimicry of a catalytic antibody's active site, and suggest that the development of the catalytic activity in the anti-acetylcholinesterase antibodies may be related to the structural features of the peripheral anionic site of acetylcholinesterase.

We have previously described three catalytic antibodies (Ab1s) raised against human erythrocyte acetylcholinesterase (AChE). These antibodies both recognise and resemble AChE in their reaction with substrates and appear with a relatively high frequency. We do not know, however, why catalytic activity should have developed in response to a ground state antigen. This question has implication for autoimmune disorders, which are frequently characterised by the presence of catalytic antibodies, many of which have cytotoxic effects. In this study, we raised anti-idiotypic (Ab2) and anti-anti-idiotypic (Ab3) antibodies to a catalytic Ab1 and examined their properties. None of the Ab2s showed catalytic activity, whereas four of the Ab3s did, an incidence of 1.26%. No contamination of antibody preparations with either AChE or butyrylcholinesterase (BChE) was found. Immunisation of mice with AChE, as well as AChE complexed with various inhibitors, resulted in a significant increase in catalytic immunoglobulins in the serum, compared with non-immunised mice and mice immunised with the Ab1. There appears to be considerable resemblance between Ab1s and Ab3s, but there are also significant differences between the two groups. All the antibodies were inhibited by phenylmethylsulphonyl fluoride (PMSF), indicating the presence of a serine residue in their active sites and were inhibited by the cholinesterase active site inhibitors tetraisopropyl pyrophosphoramide (iso-OMPA) and pyridostigmine. The Ab3s resembled the Ab1s in their ability to hydrolyse both acetylthiocholine (ATCh) and butyrylthiocholine (BTCh). However, the Ab3s appear to be better catalysts, having significantly reduced K(M) values (for ATCh but not BTCh) and increased turnover numbers (K(cat)), rate enhancements (K(cat)/K(uncat)) and K(cat)/K(M) ratios. The Ab3s also had reduced affinities for cholinesterase anionic site inhibitors (edrophonium, tetramethylammonium and BW284c51) and no affinity at all for the AChE peripheral anionic site (PAS) inhibitor fasciculin. All the antibodies recognise, to some degree, the PAS of AChE, shown by their ability to inhibit AChE, to compete with peripheral site inhibitors and to block AChE-mediated cell adhesion, a property of the site. These results indicate idiotypic mimicry of the catalytic antibody's active site, suggesting that the catalytic activity is due to affinity maturation of immunoglobulin genes in response to a specific antigen, namely, the PAS of AChE. Further studies are required to determine the structural features of this ground state antigen responsible for the development of catalytic activity.

A liquid chromatography (LC) detector based on a fast response and sensitive bienzyme amperometric biosensor for acetylcholine (ACh) and choline (Ch) is described. The detector fabrication consisted of glutaraldehyde co-crosslinking of acetylcholinesterase and choline oxidase with bovine serum albumin on the Pt working electrode of a conventional thin-layer electrochemical flow cell. The influence of some experimental parameters (e.g., enzyme loading, thickness of the bienzyme layer, flow rate) on the detector characteristics has been studied in order to optimize the analyte response while minimizing band-broadening and distortion. A mobile phase consisting of a phosphate buffer (I, 0.1 M; pH, 6.5) containing 5 mM sodium hexane sulfonate and 10 mM tetramethylammonium phosphate was found to give very satisfactory resolution and peak shape in ion-pair, reversed-phase LC. Linear responses were observed over at least four decades and absolute detection limits (at a signal-to-noise ratio of 3) were 12 and 27 fmol injected for Ch and ACh, respectively. After one month of intensive use in the LC system, the detector retained about 70% of its initial sensitivity. The potential of the described approach is demonstrated by the simultaneous determination of Ch and ACh in rat brain tissue homogenates.

The peripheral anionic site (PAS) of human butyrylcholinesterase is involved in the mechanism of substrate activation by positively charged substrates and ligands. Two substrate binding loci, D70 in the PAS and W82 in the active site, are connected by the Omega loop. To determine whether the Omega loop plays a role in the signal transduction between the PAS and the active site, residues involved in stabilization of the loop, N83, K339 and W430, were mutated. Mutations N83A and N83Q caused loss of substrate activation, suggesting that N83 which interacts with the D70 backbone may be an element of the transducing system. The K339M and W430A mutant enzymes retained substrate activation. Residues W82, E197, and A328 in the active site gorge have been reported to be involved in substrate activation. At butyrylthiocholine concentrations greater then 2 mM, W82A showed apparent substrate activation. Mutations E197Q and E197G strongly reduced substrate activation, while mutation E197D caused a moderate effect, suggesting that the carboxylate of residue E197 is involved in substrate activation. Mutations A328F and A328Y showed no substrate activation, whereas A328G retained substrate activation. Substrate activation can result from an allosteric effect due to binding of the second substrate molecule on the PAS. Mutation W430A was of special interest because this residue hydrogen bonds to W82 and Y332. W430A had strongly reduced affinity for tetramethylammonium. The bimolecular rate constant for reaction with diisopropyl fluorophosphate was reduced 10000-fold, indicating severe alteration in the binding area in W430A. The kcat values for butyrylthiocholine, o-nitrophenyl butyrate, and succinyldithiocholine were lower. This suggested that the mutation had caused misfolding of the active site gorge without altering the Omega loop conformation/dynamics. W430 as well as W231 and W82 appear to form the wall of the active site gorge. Mutation of any of these tryptophans disrupts the architecture of the active site.

We have previously described a catalytic monoclonal antibody, raised against acetylcholinesterase (AChE) and capable of hydrolysing acetylthiocholine. Here, we describe two more such antibodies. All three antibodies were raised against the same antigen, human erythrocyte AChE, a commercial product purified using the cholinesterase anionic site inhibitor, tetramethylammonium. IgG was purified on Protein A-Sepharose, and lack of contamination with AChE or butyrylcholinesterase (BChE) was demonstrated on sucrose density gradients and immunoassay of the fractions. The antibodies recognised AchE and were capable of hydrolysing acetylthiocholine and the larger butyrylthiocholine substrate, and were inactivated by phenylmethylsulphonyl fluoride (PMSF), indicating a serine residue in the active site. K(m), K(cat), K(cat)/K(uncat) and K(cat)/K(m) values were obtained for both substrates. The active sites of the antibodies were probed with anti-cholinesterases known to react with the active and anionic sites of acetyl- and BChE, and the peripheral anionic site of AChE. The antibodies were inactivated to varying degrees by the BChE inhibitors iso-OMPA, ethopropazine and tetracaine, indicating a less sterically constrained site than AChE and the lack of an acyl-binding pocket. They were also partially inhibited by the AChE-specific inhibitors, BW284c51 and propidium. No peripheral anionic site, as seen in AChE, was observed, shown by the almost complete lack of reaction with fasciculin. All three antibodies appear to have structures resembling the anionic sites of the cholinesterases, seen by their inhibition by quaternary and tricyclic compounds. Further work is required to determine whether the catalytic activity shown by these antibodies is germline-encoded, or is the result of complexation of the antigen with an inhibitor at a peripheral site.

The clearance of seven different ligands from the deeply buried active-site of Torpedo californica acetylcholinesterase is investigated by combining multiple copy sampling molecular dynamics simulations, with the analysis of protein-ligand interactions, protein motion and the electrostatic potential sampled by the ligand copies along their journey outwards. The considered ligands are the cations ammonium, methylammonium, and tetramethylammonium, the hydrophobic methane and neopentane, and the anionic product acetate and its neutral form, acetic acid. We find that the pathways explored by the different ligands vary with ligand size and chemical properties. Very small ligands, such as ammonium and methane, exit through several routes. One involves the main exit through the mouth of the enzyme gorge, another is through the so-called back door near Trp84, and a third uses a side door at a direction of approximately 45 degrees to the main exit. The larger polar ligands, methylammonium and acetic acid, leave through the main exit, but the bulkiest, tetramethylammonium and neopentane, as well as the smaller acetate ion, remain trapped in the enzyme gorge during the time of the simulations. The pattern of protein-ligand contacts during the diffusion process is highly non-random and differs for different ligands. A majority is made with aromatic side-chains, but classical H-bonds are also formed. In the case of acetate, but not acetic acid, the anionic and neutral form, respectively, of one of the reaction products, specific electrostatic interactions with protein groups, seem to slow ligand motion and interfere with protein flexibility; protonation of the acetate ion is therefore suggested to facilitate clearance. The Poisson-Boltzmann formalism is used to compute the electrostatic potential of the thermally fluctuating acetylcholinesterase protein at positions actually visited by the diffusing ligand copies. Ligands of different charge and size are shown to sample somewhat different electrostatic potentials during their migration, because they explore different microscopic routes. The potential along the clearance route of a cation such as methylammonium displays two clear minima at the active and peripheral anionic site. We find moreover that the electrostatic energy barrier that the cation needs to overcome when moving between these two sites is small in both directions, being of the order of the ligand kinetic energy. The peripheral site thus appears to play a role in trapping inbound cationic ligands as well as in cation clearance, and hence in product release.

Human butyrylcholinesterase displays substrate activation with positively charged butyrylthiocholine (BTC) as the substrate. Peripheral anionic site (PAS) residues D70 and Y332 appear to be involved in the initial binding of charged substrates and in activation control. To determine the contribution of PAS residues to binding and hydrolysis of quaternary substrates and activation control, the single mutants D70G/Y and Y332F/A/D and the double mutants Y332A/D70G and Y332D/D70Y were studied. Steady-state hydrolysis of the charged substrates, BTC and succinyldithiocholine, and the neutral ester o-nitrophenyl butyrate was measured. In addition, inhibition of wild-type and mutant enzymes by tetramethylammonium was investigated, at low concentrations of BTC. Single and double mutants of D70 and Y332 showed little or no substrate activation, suggesting that both residues were important for activation control. The effects of double mutations on D70 and Y332 were complex. Double-mutant cycle analysis provided evidence for interaction between these residues. The category of interaction (either synergistic, additive, partially additive or antagonistic) was found to depend on the nature of the substrate and on measured binding or kinetic parameters. This complexity reflects both the cross-talk between residues involved in the sequential formation of productive Michaelian complexes and the effect of peripheral site residues on catalysis. It is concluded that double mutations on the PAS induce a conformational change in the active site gorge of butyrylcholinesterase that can alter both substrate binding and enzyme acylation.

Cholinesterases display a non-Michaelian behaviour with respect to substrate concentration. With the insect enzyme, there is an activation at low substrate concentrations and an inhibition at high concentrations. Previous studies allow us to propose a kinetic model involving a secondary non-productive binding site for the substrate. Unexpectedly, this secondary site has a very high affinity for the substrate when the enzyme is free. On the contrary, when the catalytic site of the enzyme is occupied a strong decrease of this affinity was observed. Moreover, a substrate molecule bound to the peripheral site results in a global decrease of the acylation and/or the deacylation step. Kinetic studies with three reversible inhibitors, tetramethylammonium, edrophonium and choline supported the kinetic model and enable its further refinement.

A 6-coumarin diazonium salt was synthesized and tested on Torpedo acetylcholinesterase as a site-directed irreversible probe for quaternary ammonium binding. The rate of the inactivation was examined as a function of time, inhibitor concentration, and pH, which allowed the determination of the dissociation and the rate constants of this efficient affinity labeling process. Protection experiments using tetramethylammonium, edrophonium, and propidium demonstrated that the labeling reaction occurred exclusively at the peripheral quaternary ammonium binding site of the enzyme. This result was confirmed by the modification of propidium binding at the peripheral site after inactivation reaction, as directly determined by fluorescence. Mutations of the likely labeled amino acid residues, Tyr70 and Tyr121, by histidine and phenylalanine indicated a predominant involvement of Tyr70 over Tyr121 in the coupling reaction.

We describe a method of detecting human DNA mutations with nonradioactive, biotinylated allele-specific oligonucleotide probes. This method can detect seven different mutations in the butyrylcholinesterase, cystic fibrosis, and N-acetyltransferase genes under identical assay conditions. This indicates that it may be used to detect mutations responsible for a wide variety of genetic diseases and pharmacogenetic conditions. The method involves first amplifying selected DNA fragments by the polymerase chain reaction and dot blotting the amplified DNA in duplicate onto small nitrocellulose squares. Each dot blot is then hybridized in individual wells containing a tetramethylammonium chloride solution with short biotinylated probes specific for either the normal or mutant allele. Successfully hybridized probes are detected by a simple colorimetric reaction using an avidin-alkaline phosphatase conjugate, which yields a very strong, clear signal. DNA from homozygous normal or mutant individuals hybridizes only to the normal- or mutant-specific probes respectively, while DNA from heterozygous individuals hybridizes equally well with both probes. These results can be easily interpreted to assign a genotype to the sample DNA. This method is amenable to automation, and may be useful in clinical laboratories for diagnosis of a wide variety of DNA mutations responsible for unusual reactions to drugs and environmental chemicals.

We describe an affinity chromatography method in which dimethylaminoethylbenzoic acid-Sepharose 4B is used, making it possible to separate in one step the molecular forms of globular acetylcholinesterase (AChE, EC 3.1.1.7) or butyrylcholinesterase (ChE, EC 3.1.1.8). A crude extract containing these enzymes was deposited onto the chromatography gel, washed, and eluted by a linear gradient of tetramethylammonium chloride (0-0.3 M). With rat brain AChE, two well-separated peaks were eluted in the presence of 1% Triton X-100; the first peak corresponded to 4 S forms and the second to 11 S forms. This separation was very efficient for salt-soluble activity and less efficient for the detergent-soluble AChE. In this case, the 4 S peak represented only 6.5% of total detergent-soluble activity and was cross-contaminated by the 11 S form. Rat serum ChE was efficiently separated into two peaks of 7 S and 11 S. This method could potentially be adapted to separate other multimeric proteins with varying numbers of affinity sites.

To assess the relative importance of binding to enzyme-substrate complex (E.S) and to acetylenzyme (EA), noncompetitive inhibition has been studied in hydrolysis by acetylcholinesterase (AcChE) of cationic and uncharged substrates - acetylcholine (AcCh), 3,3-dimethylbutyl acetate, n-butyl acetate, 2-(methylammonio)ethyl acetate, 2- (N,N-diethyl-N-n-butylammonio)ethyl acetate (DEBAAc) and 2-(methylsulfonyl)ethyl acetate. For the N-trimethyl quaternary ions related to AcCh, tetramethylammonium ion, choline and choline ethyl ether, noncompetitive inhibition (Ki(nonc) is more favorable with the slower substrates than with AcCh, i.e., when E.S greater than EA, and is attributed to formation of enzyme-substrate-inhibitor complexes, E.S.I'. Noncompetitive inhibition by tetraethyl-, tert-butyl- and isopropylammonium ions, and acetamidocholine and its lower dimethyl analogue, is also attributed to E.S.I' complexes. Peripheral binding of these inhibitors decreases acylation more than deacylation. Some tertiary dimethylamonio ions have more favorable Ki(nonc) values with AcCh, decreasing deacylation more than acylation. The substrate DEBAAc is a more effective noncompetitive than competitive inhibitor in hydrolysis of AcCh, indicating that it binds more strongly in a peripheral site than in the active site of the free enzyme. In its hydrolysis by AcChE, it acts as its own noncompetitive inhibitor, by this non-productive binding. Formation of E.S.I' complexes is a general characteristic of hydrolysis by AcChE and decrease in rates at high concentrations of AcCh and related substrates is attributed to peripheral regulatory site binding, formation of E.S.S' complexes, rather than to binding to the acetylenzyme.

People with genetic variants of cholinesterase respond abnormally to succinylcholine, experiencing substantial prolongation of muscle paralysis with apnea rather than the usual 2-6 min. The structure of usual cholinesterase has been determined including the complete amino acid and nucleotide sequence. This has allowed identification of altered amino acids and nucleotides. The variant most frequently found in patients who respond abnormally to succinylcholine is atypical cholinesterase, which occurs in homozygous form in 1 out of 3500 Caucasians. Atypical cholinesterase has a single substitution at nucleotide 209 which changes aspartic acid 70 to glycine. This suggests that Asp 70 is part of the anionic site, and that the absence of this negatively charged amino acid explains the reduced affinity of atypical cholinesterase for positively charged substrates and inhibitors. The clinical consequence of reduced affinity for succinylcholine is that none of the succinylcholine is hydrolyzed in blood and a large overdose reaches the nerve-muscle junction where it causes prolonged muscle paralysis. Silent cholinesterase has a frame shift mutation at glycine 117 which prematurely terminates protein synthesis and yields no active enzyme. The K variant, named in honor of W. Kalow, has threonine in place of alanine 539. The K variant is associated with 33% lower activity. All variants arise from a single locus as there is only one gene for human cholinesterase (EC 3.1.1.8). Comparison of amino acid sequences of esterases and proteases shows that cholinesterase belongs to a new family of serine esterases which is different from the serine proteases.

Acetylcholinesterase (AChE) inhibited by the organophosphate soman (1,2,2-trimethyl-propylmethylphosphonofluoridate) rapidly becomes resistant to reactivation by oximes due to dealkylation of the soman-enzyme complex. This reaction is called aging. The effect of the four mono- and bisquaternary ammonium compounds tetramethylammonium (TMA), hexamethonium, decamethonium and suxamethonium on the reactivatability of soman-inhibited, solubilized AChE from human erythrocytes was investigated in vitro. All compounds were reversible inhibitors of AChE; the respective dissociation constants and the type of inhibition exhibited considerable differences. The affinities to both the active and the allosteric site were considerably higher for suxamethonium (Kii 81.3 microM; Ki 15.9 microM) and decamethonium (Kii 15.4 microM; Ki 4.4 microM) than for TMA (Kii 1 mM; Ki 289.6 microM) and hexamethonium (Kii 4.5 mM; Ki 331.8 microM). The reactivation experiments were performed in a four-step procedure (soman-inhibition at 0 degree and pH 10, aging at 37 degrees and pH 7.3, reactivation by the oxime HI 6 at 37 degrees and pH 7.3 followed by AChE assay). After these four steps (total duration 55 min), AChE was inhibited by soman to 95-100%. HI 6 could reactivate about 20% of the inhibited enzyme. All effectors increased the AChE reactivatability by HI 6 when added before aging was started. The maximal increase in reactivatability was higher in the presence of 1.6 mM suxamethonium (+35.8%) and 150 microM decamethonium (+40%) than of 22 mM TMA (+22.5%) and 8.3 mM hexamethonium (+19.2%). If the effectors were added after 5 min of aging they increased the activity of soman-inhibited AChE, but to a considerably smaller extent than HI 6. A good correlation of the respective Kii values and the effective concentrations of these drugs was observed, indicating that an allosteric binding site of AChE might be involved in the protective effect of these drugs.

Studies have been made of the effect of three groups of ammonia reversible inhibitors on the activity of erythrocyte acetylcholinesterase, serum butyrylcholinesterase, cholinesterase from frog brain, as well as cholinesterases from the optical ganglia of the Pacific and three populations of the commander squids. Determination of kinetic parameters of the reversible inhibition of these enzymes revealed differences resulting from the specific structure of their catalytic centers. Tetramethylammonium assay confirmed different properties of cholinesterases in individuals of the commander squid from various habitats in the Bering Sea; this finding may be taken as an indication of intraspecific differentiation of these cephalopods. Certain similarity was noted in the inhibitory specificity of cholinesterases from the Pacific and "southern" commander squids with the overlapping habitats.

The inhibitory effect of paraquat on cholinesterase activity was investigated in comparison with four paraquat derivatives, six monoquaternary ammoniums and six anticholinergic drugs. Inhibitor concentrations to cause 50% inhibition (I50) and Hill coefficients for three enzymes, human erythrocyte acetylcholinesterase (AChE), Electrophorus electricus AChE and human plasma butyrylcholinesterase (BCHE) were measured. The results obtained were as follows. The I50 for erythrocyte AChE was similar to the I50 for eel AChE. Secondary to edrophonium, diethylparaquat, paraquat, morfamquat and monoquat showed lower I50 for AChE, and possessed higher inhibition selectivity (IS), expressed as the ratio of I50 for BCHE to I50 for erythrocyte AChE. However, diquat showed higher I50 for AChE and lower IS, similar to the other monoquaternary ammoniums. A negative correlation was observed between log [I50 for erythrocyte AChE] and log [IS], among paraquat and its derivatives, monoquaternary ammoniums and anticholinergic drugs, respectively. With respect to Hill coefficients, these inhibitors could be classified into four groups, [1] competitive inhibitors: diquat, edrophonium, choline, tetramethylammonium and trimethylphenylammonium, [2] inhibitors showing negative cooperativity: paraquat, diethylparaquat, morfamquat, d-tubocurarine, atropine, gallamine and nicotine, [3] moderate type inhibitors: monoquat, hexamethonium and decamethonium. [4] the other type inhibitors showing positive cooperativity for erythrocyte AChE: tetraethylammonium and ethyltrimethylammonium.

The organophosphorus pesticide bromophos and the tetramethylammonium and sodium salts of demethylbromophos were tested for cytogenetic and embryotoxic activity on mice of different strains. Single intraperitoneal (ip) doses of 183.0 mg/kg (0.5 mmol/kg) and 73.2 mg/kg (0.2 mmol/kg) bromophos caused a significant enhancement in the percentage of chromosome aberrations in bone marrow cells of CFLP mice; similar effects were produced by a single dose of 0.2 mmol/kg demethylbromophos tetramethylammonium salt and demethylbromophos sodium salt trihydrate, respectively, indicating that the cytogenetic activity of bromophos is not connected with its alkylating properties. After repeated ip or oral administration to pregnant mice of strains AB Jena/Halle and DBA, none of the tested compounds showed a marked influence on the total implantation losses, although in some cases the postimplantation losses were significantly increased.

The binding of D-tubocurarine (TC) to acetylcholinesterase (AChE) was studied using different methods of enzyme kinetics. The main results are as follows. TC reversibly inhibits the hydrolysis of different substrates of AChE with three different inhibition constants (Ki1 = 7.0 +/- 0.8 X 10(-5) M, Ki2 = 3.1 +/- 1.0 X 10(-4) M, and Ki3 = 4.2 +/- 0.5 X 10(-3) M). Reference inhibitors tetramethylammonium (TMA), tetraethylammonium (TEA), and decamethonium (C-10) inhibit the hydrolysis of different substrates with constants, which are the same for each individual inhibitor. These three inhibitors compete with TC in the inhibition of enzymatic hydrolysis of acetylthiocholine (ASCh); all three of them affect the noncompetitive component of the inhibition of the hydrolysis of ASCh by TC, which arises from the binding of TC to the peripheral anionic site of AChE, but TEA and C-10 affect also the competitive component of this inhibition, which arises from the binding of TC at the catalytic anionic site. TC partially inhibits the methanesulfonylation of AChE; dissociation constant for TC in this process is KA = 4.5 X 10(-4) M. All our results lead to the conclusion that TC binds to three regions on the active surface of AChE. The first region is at the peripheral anionic site; the other two regions are situated in the vicinity of the catalytic anionic site and the esteratic site.

Interaction between spin-labeled methacyne (I) and butyrylcholinesterase (BChE) was studied by ESR and enzyme kinetic methods. The compound (I) was shown to be a competitive reversible inhibitor, the value of Ki appeared to be 1.3 X 10(-5) M. Insertion of nitroxyl fragment in the methacyne molecule results in a two-fold increase of its inhibitory activity. The ESR spectrum of the enzyme-inhibitor complex was registered. This complex dissociates under the action of eserine, tetramethylammonium and hexamethonium. Scatchard plot reveals two different types of binding sites with Kdiss values 1.5 X 10(-5) M and 2.6 X 10(-4) M. One type of binding sites is identified as the enzyme active centre. The restricted motion of (I) in complex with BChE proves the assumption that the enzyme active centre is located in the split of macromolecule surface.

VIP immunoreactivity was identified in nerve fibers and in 10-13% of the neurons in pelvic and bladder ganglia of the cat. Ninety percent of the VIP positive neurons contained acetylcholinesterase. VIP immunoreactivity was not altered in decentralized ganglia 1 week to 8 months after transection of the pelvic and hypogastric nerves indicating that VIP fibers arose from neurons within the peripheral nervous system. The intra-arterial administration of VIP (1-50 micrograms/kg) enhanced the postganglionic discharge elicited by the muscarinic agonist, acetyl-beta-methylcholine, but did not alter the postganglionic firing elicited by the nicotinic agonist, tetramethylammonium or by electrical stimulation of preganglionic axons in the pelvic nerve. VIP did not elicit a postganglionic discharge in untreated ganglia, but did evoke a prolonged discharge in ganglia treated with an irreversible anticholinesterase agent, 217AO. This discharge was not affected by hexamethonium but was blocked by atropine. VIP suppressed the muscarinic inhibition of ganglionic transmission produced by acetyl-beta-methylcholine without altering the response to other inhibitory agents (norepinephrine, leucine-enkephalin and gamma-aminobutyric acid (GABA). VIP (0.1-0.3 micrograms/kg) also had a direct inhibitory effect on bladder smooth muscle. These findings raise the possibility that intraganglionic pathways containing VIP may exert a selective modulatory influence on muscarinic transmission in vesical parasympathetic ganglia.

A steroid, the dibutyrate analogue of pancuronium bromide (9.8 X 10(-8)M), has been used as differential inhibitor in the study of the plasma cholinesterase variants. Pancuronium dibutyrate numbers have been measured for 190 individuals, and the mean values for six of the known genotypes, E1uE1u, E1uE1f, E1uE1a, E1fE1a, E1aE1a, and E1fE1f, have been calculated. Evidence is presented that a combination of the pancuronium dibutyrate number and the fluoride number give better resolution of the six genotypes than the combination of the pancuronium dibutyrate and the dibucaine number. This new differential inhibitor has real potential for revealing the probable existence of new genotypes.

The reversible binding constant (Ki) for tetramethylammonium ion (TMA) was determined from the decrease in the bimolecular rate constant (ki) observed with each of 21 organophosphate or carbamate inhibitors of acetylcholinesterase (EC 3.1.1.7). The Ki values obtained were reasonably constant (5.8 X 10(-4) +/- 0.38 M), and this is consistent with reports indicating that TMA binds to a single site on the enzyme.

A bis-quaternary fluorescence probe, propidium diiodide, has been found to exhibit a tenfold enhancement of fluorescence when bound to acetylcholinesterase from Torpedo california. The complex is characterized by a high affinity, KD = 3.0 times 10-7 M, and 1:1 stoichiometry with the 82,000 molecular weight subunit of acetylcholinesterase. A wide variety of other quaternary ammonium ligands such as decamethonium, gallamine, d-tubocurarine, tetraethylammonium, and tetramethylammonium will completely dissociate propidium from the enzyme as will monovalent and divalent inorganic cations. The competitive dissociation does not show cooperative behavior or a distinct, requirement for occupation of multiple sites of different affinity to produce displacement. While a directly competitive relationship can be illustrated macroscopically, the various quaternary ligands show a different susceptibility toward inorganic cation displacement. The affinity of propidium relative to gallamine increases with ionic strength. This finding indicates that there is not complete equivalence in the negative subsites to which quaternary groups bind. Although edrophoniumwill also displace propidium from the enzyme, the dissociation constant obtained from this competitive relationship is 3.5 orders of magnitude greater than the constants obtained for inhibition of catalysis. By competitive displacement titrations it is shown that the primary binding site of edrophonium is distinct from that of propidium and a ternary complex with the two ligands can form on each subunit. In contrast to edrophonium, the binding of propidium is unaffected by methanesulfonylation of the active center serine and is uncompetitive with the carbamylating substrate, N-methyl-7-dimethylcarbamoxyquinolinium. Thus, it appears that propidium associates with a peripheral anionic center on the enzyme. Although propidium and edrophonium associate at separate sites on acetylcholinesterase, bis-quaternary ligands where the quaternary nitrogens are separated by 14 A displace both ligands from the enzyme with equal effectiveness.