Mammalian Acetylcholinesterase Research Articles

Cardiotonic drugs inhibit purified mammalian acetylcholinesterase

Oxime- and non-oxime-related drugs, as well as cardiotonic drugs (CDs), have been used to treat the effects of organophosphorus (OP) poisoning. We conducted our experiments to determine what effects CDs may have on acetylcholinesterase (AChE), and how CDs interact with other treatment drugs as well as with OP-inhibited AChE. True AChE (EC 3.1.1.7) was purified from fetal bovine serum, and enzyme activity was measured according to Ellman et al. The CDs coumingine, cassaine, proscillaridin and convallatoxin were incubated with AChE at 550 microM at pH 7.6 and 25 degrees C. The CD ouabain was incubated with AChE at 500 microM. The CDs inhibited AChE by 97%, 89%, 10%, 7% and 6%, respectively. The mean AChE activities for these experiments, except for ouabain, were significantly different (P = 0.05) from their controls, as determined by the two-tailed Student's t-test. In a separate experiment, the oxime TMB-4.2Br (100 microM), which did not inhibit AChE, increased the inhibitory effect of proscillaridin from 4% to 11% (a 3.7-fold increase). When AChE was inhibited 39% with 37 nM VX, the addition of proscillaridin increased the inhibition to 51% (a 1.3-fold increase). When TMB-4 was added to the proscillaridin- and VX-inhibited AChE mixture, the inhibition decreased from 50% to 32% (a 0.37-fold decrease), whereas TMB-4 alone added to VX-inhibited AChE decreased the inhibition from 39% to 24% (a 0.38-fold decrease). The results show that TMB-4 increases the inhibition of AChE by proscillaridin. However, TMB-4 decreases the inhibition of AChE by VX and proscillaridin combined.(ABSTRACT TRUNCATED AT 250 WORDS)

Detoxification of organophosphate pesticides using an immobilized phosphotriesterase from Pseudomonas diminuta

A purified phosphotriesterase was successfully immobilized onto trityl agarose in a fixed bed reactor. A total of up to 9200 units of enzyme activity was immobilized onto 2.0 mL of trityl agarose (65 micromol trityl groups/mL agarose), where one unit is the amount of enzyme required to catalyze the hydrolysis of one micromole of paraoxon in one min. The immobilized enzyme was shown to behave chemically and kinetically similar to the free enzyme when paraoxon was utilized as a substrate. Several organophosphate pesticides, methyl parathion, ethyl parathion, diazinon, and coumaphos were also hydrolyzed by the immobilized phosphotriesterase. However, all substrates exhibited an affinity for the trityl agarose matrix. For increased solubility and reduction in the affinity of these pesticides for the trityl agarose matrix, methanol/water mixtures were utilized. The effect of methanol was not deleterious when concentrations of less than 20% were present. However, higher concentrations resulted in elution of enzyme from the reactor. With a 10-unit reactor, a 1.0 mM paraoxon solution was hydrolyzed completely at a flow rate of 45 mL/h. Kinetic parameters were measured with a 0.1-unit reactor with paraoxon as a substrate at a flow rate of 22 mL/h. The apparent K(m) for the immobilized enzyme was 3-4 times greater than the K(m) (0.1 mM) for the soluble enzyme. Immobilization limited the maximum rate of substrate hydrolysis to 40% of the value observed for the soluble enzyme. The pH-rate profiles of the soluble and immobilized enzymes were very similar. The immobilization of phosphotriesterase onto trityl agarose provides an effective method esterase onto trityl agarose provides an effective method for hydrolyzing and thus detoxifyuing organophosphate pesticides and mammalian acetylcholinesterase inhinbitors.

Complete amino acid sequence of fetal bovine serum acetylcholinesterase and its comparison in various regions with other cholinesterases

The complete amino acid sequence of a mammalian acetylcholinesterase from fetal bovine serum (FBS AChE) is presented. This enzyme has a high degree of sequence identity with other cholinesterases, liver carboxyesterases, esterase-6, lysophospholipase, and thyroglobulin. The locations of 191 amino acids in 10 regions of the FBS enzyme were compared with corresponding sequences of Torpedo, human, and Drosophila AChEs and human serum butyrylcholinesterase (BChE). In one region there is a marked difference in both the number of amino acids and their sequence between mammalian AChE and other AChEs and the human serum BChE. The amino acid sequence of FBS AChE showed overall homologies of 90% with human AChE, 60% with T. californica AChE, 50% with human serum BChE, and 39% with Drosophila AChE in these regions.

Inactivation of organophosphorus nerve agents by the phosphotriesterase from Pseudomonas diminuta

The phosphotriesterase from Pseudomonas diminuta was tested as a catalyst for the hydrolysis of phosphofluoridates. The purified enzyme has been shown to hydrolyze the phosphorus-fluorine bond of diisopropyl fluorophosphate, isopropyl methylphosphonofluoridate, and 1,2,2-trimethylpropylmethylphosphono-fluoridate at pH 7.0, 25 °C, with turnover numbers of 41, 56, and 5 s −1, respectively. The enzymatic rate enhancement for the hydrolysis of sarin at pH 7.0 is 2.2 × 10 7. The turnover number for paraoxon hydrolysis is 2100 s −1. The enzyme does not hydrolyze methanesulfonyl fluoride, phenylmethylsulfonyl fluoride, or O-p-nitrophenyl phenylsulfonate nor do these compounds inactivate or inhibit the ability of the enzyme to hydrolyze diethyl p-nitrophenyl phosphate. The breadth of substrate utility and the efficiency of the hydrolytic reaction exceed the more limited abilities of other prokaryotic and eukaryotic enzymes that catalyze similar reactions. The substantial rate enhancement exhibited by this enzyme for the hydrolysis of a wide variety of organophosphorus nerve agents make this enzyme the prime candidate for the biological detoxification of insecticide and mammalian acetylcholinesterase inhibitors.

Purification and Properties of E-Caprolactone Hydrolases from Acinetobacter NCIB 9871 and Nocardia globerula CL1

The ε-caprolactone hydrolases (EC 3.1.1-) induced by growth of Acinetobacter NCIB 9871 and Nocardia globerula CL1 with cyclohexanol were purified to homogeneity. Both enzymes constituted approximately 1% of the soluble protein of the bacteria. Each was formed from two electrophoretically indistinguishable subunits and each had a closely similar M r value (≈60000). Both enzymes had high turnover numbers typical of carboxyesterases, broad pH-activity spectra and very restricted substrate specificities. In contrast to other bacterial lactone hydrolases they catalysed irreversible lactone hydrolysis and were not inhibited by thiol-reactive compounds. Their sensitivity to Paraoxon (diethyl p-nitrophenylphosphate) suggested that they, in common with mammalian acetylcholinesterase and carboxyesterases, have a functional catalytic centre serine.

Isolation and characterization of acetylcholinesterase from Drosophila.

The purification and characterization of acetylcholinesterase from heads of the fruit fly Drosophila are described. Sequential extraction procedures indicated that approximately 40% of the activity was soluble and 60% membrane-bound and that virtually none (less than 4%) corresponded to collagen-tailed forms. The membrane-bound enzyme was extracted with Triton X-100 and purified over 4000-fold by affinity chromatography on acridinium resin. Hydrodynamic analysis by both sucrose gradient centrifugation and chromatography on Sepharose CL-4B revealed an Mr of 165,000 similar to that observed for dimeric (G2) forms of the enzyme in mammalian tissues. In contrast, the purified enzyme gave predominant bands of about 100 kDa prior to disulfied reduction and 55 kDa after reduction on polyacrylamide gel electrophoresis in sodium dodecyl sulfate, values that are significantly lower than those reported for purified G2 enzymes from other species. However, the presence of a faint band at 70 kDa which could be labeled by [3H]diisopropyl fluorophosphate prior to denaturation suggested that the 55-kDa band as well as a 16-kDa species arose from proteolysis. This was confirmed by reductive radiomethylation and amine analysis of the 70-, 55-, and 16-kDa bands. All three contained ethanolamine and glucosamine residues that are characteristic of a C-terminal glycolipid anchor in other G2 acetylcholinesterases. The catalytic properties of the enzyme were examined by titration with a fluorogenic reagent which revealed a turnover number for acetylthiocholine that was 6-fold lower than eel and 3-fold lower than human erythrocyte acetylcholinesterase. Furthermore, the Drosophila enzyme hydrolyzed butyrylthiocholine much more efficiently than these eel or human enzymes, an indication that the fly head enzyme has a substrate specificity intermediate between mammalian acetylcholinesterases and butyrylcholinesterases.

Kinetic investigations into the interactions of aprophen with cholinesterases and a carboxylesterase

Acetylcholinesterases, butyrylcholinesterases, and carboxylesterases appear to form kinetically a homologous enzyme series with respect to many substrates and inhibitors. The present paper evaluates the interaction of aprophen with acetylcholinesterases, butyrylcholinesterases, and carboxylesterases with respect to (1) protecting the enzyme from organophosphate and carbamate inhibition, (2) accelerating pralidoxime iodide (2-PAM) regeneration of the diisopropylphospho-enzyme, and (3) comparing the inhibition and regeneration kinetics of a soluble mammalian acetylcholinesterase with that of bovine erythrocyte acetylcholinesterase. The irreversible inhibition kinetics of diisopropyl fluorophosphate (DFP) and eserine inhibition of fetal bovine serum acetylcholinesterase were typical of other acetylcholinesterases as indicated by the bimolecular inhibition rate constants, k i , of 7.7 ± 1.3 × 10 4 M −1 and 2.9 ± 1.7 × 10 6 M −1, respectively. Similarly, the bimolecular regeneration rate constant, k r , for 2-PAM regeneration of the diisopropylphospho-acetylcholinesterase was 14.7 M −1 min −1. The bimolecular rate constants, k i and k r , were not statisticaly perturbed when the reaction was monitored in the presence of aprophen with the fetal bovine serum acetylcholinesterase. Human serum butyrylcholinesterase was partially protected from DFP inhibition by aprophen with no detectable change in the bimolecular inhibition rate constant, k i . The regeneration of the diisopropylphospho-butyrylcholinesterase by 2-PAM was accelerated in the presence of aprophen by a factor of 2.7 over that of 2-PAM alone (8.4 ± 2.2 M −1 to 23.1 ± 2.6 M −1 min −1respectively). Neither the inhibition (DFP) nor the regeneration (2-PAM) kinetics observed for the carboxylesterase was perturbed by the presence of aprophen.

In vivo and in vitro assays for organophosphate poisoning therapeutic chemicals using the house fly, Musca domestica L.

1. 1. Thoracic injection of 31 μg of N-methyl pyridinium-2-aldoxime chloride (2-PAM) per fly (1.55 g/kg) 1 hr before or after diisopropylfluorophosphate (DFP) elevated the baseline LD 50, of DFP (14 ng/fly; 0.7 mg/kg) by 50 and 162 times, respectively. 2. 2. The net increase in the acetylcholinesterase (AChE) activity (“regeneration”) by the addition of 2-PAM (1.55 × 10 −4 M) 3 min before or after the addition of DFP (8.43 × 10 −7 M) to the fly homogenate ranged from 48.5 to 50.5%. Toxogonin was comparable to 2-PAM. HI-6 and HS-4 were less effective than 2-PAM. 3. 3. The toxic syndrome casued by injection of acetylcholine chloride, edrophonium chloride, atropine sulfate and gallamine triethiodide showed that the cholinergic nervous system was readily accessible. 4. 4. Brain AChE showed typical substrate-inhibition and a K m of 141 μM with acetylthiocholine. Fly brain AChE was comparable electrophoretically with mammalian AChE (fetal bovine serum) but was immunochemically distinct from several mammalian AChE.

Fasciculin, a powerful anticholinesterase polypeptide from Dendroaspis angusticeps venom

A powerful inhibition of mammalian acetylcholinesterase was detected in the venom of the snake Dendroaspis angusticeps (green mamba). The substances responsible for such inhibition were isolated and purified by gel filtration on Sephadex G-50 and ion exchange chromatography on Bio-Rex 70 and SP Sephadex C-25. These substances were polypeptides and were named, fasciculins. Upon intraperitoneal injection into mice fasciculins elicited severe, generalized, long-lasting muscle fasciculations with complete clinical recovery. In vitro preincubation with fasciculins at concentrations of 0.01 μg ml −1 inhibited brain and muscle acetylcholinesterases up to 80%. Histochemical assay for acetylcholinesterase showed an almost complete disappearance of the black-brown precipitate at the neuromuscular end-plate after in vitro incubation with fasciculins. Fasciculins represent a new type of acetylcholinesterase inhibitors provoking muscle fasciculations through a powerful inhibition of enzyme activity at the neuromuscular end-plate, interfering with the normal degradative activity of the acetylcholine molecule. Fasciculins are also powerful inhibitors of brain acetylcholinesterases.

Propionylcholinesterase in Hirudo medicinalis: Purification, partial characterization and comparative study with a mammalian acetylcholinesterase

1. The authors carried out the purification of a cholinesterase from Hirudo medicinalis by homogenization, ultracentrifugation, (NH 4) 2SO 4 or DEAE-cellulose fractionation and Sephadex G-200 chromatography. 2. Certain molecular properties (mol. wt, electrophoretic pattern) were studied. 3. A comparative study of the kinetic parameters of cholinesterase from H. medicinalis and acetylcholinesterase from rabbit brain was performed, using substrates with a different composition; the enzymes show signs of marked differences in the active site conformation and/or in the catalytic mechanism. 4. The enzyme from H. medicinalis is a propionylcholinesterase, particularly active on propionylthiocholine, and, to a lower extent, on butyrylthiocholine and acetylthiocholine.

Inactivation of Electrophorus electricus acetylcholinesterase by benzenemethane sulfonylfluoride

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.

Crystal and molecular structure of organophosphorus insecticides. 10. Chlorpyrifos

The crystal and molecular structure of chlorpyrifos (O,O-diethyl O-3,5,6-trichloro-2-pyridyl thiophosphate, (H/sub 5/C/sub 2/O)/sub 2/P(S)OC/sub 5/NHCl/sub 3/, monoclinic, C2/c, a = 22.06 (1), b = 9.485 (2), c = 15.990 (6) A, ..beta.. = 114.63 (4)/sup 0/, Z = 8, Mo K..cap alpha.. radiation) has been determined by three-dimensional x-ray analysis. The structure was solved by conventional Patterson and Fourier techniques to a final discrepancy index R = 0.066 for 1421 observed reflections (absolute value F/sub 0/ > 2.5 sigma (F/sub 0/)). The phosphorus-meta hydrogen distance of 5.78 A is within the range of literature values cited for insect acetylcholinesterase (AChE), yet is well outside that for mammalian AChE. CNDO molecular orbital charge density calculations and van der Waals arguments are presented to correlate the solid state structure to a probable in vivo model. 3 figures, 1 table.

Crystal and molecular structure of organophosphorus insecticides. 8. Ronnel oxon

The crystal and molecular structure of ronnel oxon (0,0-dimethyl 0-2-,4-5-trichlorophenyl phosphate, (H/sub 3/CO)/sub 2/P(O)OC/sub 6/H/sub 2/Cl/sub 3/, monoclinic, P2/sub 1//c, a = 9.659 (5), b = 11.388 (2), c = 14.465 (9) A, ..beta.. = 130.09 (4)/sup 0/, Z = 4, Mo K..cap alpha.. radiation) has been determined by three-dimensional x-ray analysis. The structure was solved by direct methods and refined by full-matrix least-squares procedures to a final discrepancy index R = 0.060 for 1842 observed reflections (F/sub 0/ < 2.5sigma (F/sub 0/)). The phosphorus-meta hydrogen distance of 5.49 A is well within the range of literature values cited for the intramoleculr site separaton distance for insect acetylcholinesterase (AChE), yet is well outside that for mammalian AChE. CNDO molecular orbital calculations are presented to show the charge density distribution in ronnel oxon.

Crystal and molecular structure of organophosphorus insecticides. 6. Amidithion.

The crystal and molecular structure of amidithion (O,O-dimethyl S-(N-2-methoxyethylcarbamoylmethyl) phosphorodithioate), (CH/sub 3/O)/sub 2/P(S)SCH/sub 2/C(O)NHC/sub 2/H/sub 4/OCH/sub 3/, has been determined by three-dimensional x-ray analysis. The compound crystallizes in the monoclinic space group P2/sub 1//c with a = 10.975 (3), b = 9.335 (2), c = 13.702 (4) A, and ..beta.. = 102.84 (4)/sup 0/ with Z = 4. Graphite-monochromated Mo K..cap alpha.. radiation (lambda..gamma.. = 0.70954 A) was used for measurement of diffraction data. The structure was solved via direct methods and refined by a full-matrix least-squares procedure to a final discrepancy index of 0.066. The atoms with greatest residual positive charge, as determined by a CNDO molecular orbital calculation are, in addition to the phosphorus, the carbonyl carbon and the amide hydrogen. The distance from phosphorus to either atom, 3.91 (1) or 4.24 (2) A, respectively, is significantly shorter than the accepted anionic-esteratic site separation in mammalian acetylcholinesterase and may account for the lower toxicity of amidithion as compared with azinphosmethyl. The partial charge on phosphorus was found to be similar to that in azinphos-methyl, +1.063 vs. +1.041 e, respectively. Intramolecular interactions appeared to be slight.

Crystal and molecular structure of organophosphorus insecticides. 5. Fospirate.

The crystal and molecular structure of fospirate (dimethyl 3,5,6-trichloro-2-pyridyl phosphate, (H/sub 3/CO)/sub 2/P(O)OC/sub 5/NHCl/sub 3/, monoclinic, P2/sub 1//c, a = 12.267 (5), b = 8.685 (1), c = 14.102 (6) A, ..beta.. = 126.62 (3)/sup 0/, Z = 4, Mo K..cap alpha.. radiation) has been determined by three-dimensional x-ray analysis. The structure was solved by conventional Patterson and Fourier techniques to a final discrepancy index R = 0.062 for 1104 observed reflections (F/sub 0/ greater than 2.5 sigma(F/sub 0/)). The phosphorus-meta hydrogen distance of 5.79 A is within the range of literature values cited for the intramolecular active site-separation distance for insect acetylcholinesterase (AChE), yet is well outside that for mammalian AChE. CNDO molecular orbital calculations are presented to correlate the solid state structure to a probable in vivo model.

Crystal and molecular structure of organophosphorus insecticides. 4. Bromophos.

The crystal and molecular structure of bromophos (O-(4-bromo-2,5-dichlorophenyl) O,O-dimethyl phosphorothioate, (H/sub 3/CO)/sub 2/P(S)OC/sub 6/H/sub 2/Cl/sub 2/Br, orthorhombic, P2/sub 1/2/sub 1/2/sub 1/, a = 7.307 (5), b = 27.19 (3), and c = 6.440 (4) A, Z = 4, Mo K..cap alpha.. radiation) has been determined by three-dimensional x-ray analysis. The structure was solved by conventional Patterson and Fourier techniques to a final discrepancy index R = 0.048 for 1217 observed reflections (F/sub 0/ > 2.5 sigma (F/sub 0/)). The structure features an intramolecular hydrogen bond which restricts rotation about the phenolic C--O bond, thus giving a very probable in vivo model. The phosphorus-meta hydrogen distance of 5.52 A falls well within the range of literature values cited for the intramolecular active site-separation distance for acetylcholinesterase (AChE), yet is well outside that for mammalian AChE.

The effects of inhibitors on the hydrolysis of acetylcholine by four species of schistosoma

1. The response to various acetylcholinesterase (E.C.3.1.1.7.) and cholinesterase (E.C.3.1.1.8) inhibitors on the hydrolysis of acetyl-1 14C-choline iodide by extracts of adult Schistosoma haematobium, S. mansoni, S. mattheei and S. bovis was studied. 2. Eserine (physostigmine) produced total inhibition at concns between 6 × 10 −4 M and 1 × 10 −15 M. 3. The cholinesterase inhibitors, Lysivane, Tiguvon and iso-OMPA produced a slight inhibition. 4. The enzymes were partially inhibited by the mammalian acetylcholinesterase inhibitors 62C47 and 284C51. 5. Female S. haematobium enzyme was more sensitive and male S. haematobium enzyme more refractory to metrifonate (Bilarcil) than other species.

Mechanism of inhibition in vitro of mammalian acetylcholinesterase and cholinesterase in solutions of O, O-dimethyl 2,2,2-trichloro-1-hydroxyethyl phosphonate (trichlorphon)

The rate of decomposition of trichlorphon into DDVP was measured polarographically at pH 7.4. The first order rate constants of decomposition at 25° are 7.27 × 10 −4 and 6.05 × 10 −4 min −1 for trichlorphon concentrations of 0.150 and 15.0 mM respectively; at 37° the corresponding rate constants are 53.1 × 10 −4 and 37.1 × 10 −4 min −1. The rate of decomposition of trichlorphon was also calculated from the kinetics of inhibition of acetylcholinesterase (EC 3.1.1.7) and cholinesterase (EC 3.1.1.8) in trichlorphon solutions at 25° and 37° (pH 7.4). The following enzyme sources were used: bovine erythrocytes and rat brain acetylcholinesterase, and human, horse and rat plasma cholinesterase. The rate of decomposition of trichlorphon was calculated by assuming that only DDVP formed from trichlorphon is the enzyme inhibitor, while trichlorphon itself does not act as an inhibitor. The calculated rate constants for the decomposition of trichlorphon are lower or just within the range of the rate constants obtained by the polarographic method. This agreement was taken as kinetic evidence that trichlorphon is not an inhibitor of mammalian cholinesterases. The effect of pH on enzyme inhibition supports this conclusion. The rate of inhibition of bovine erythrocyte acetylcholinesterase by DDVP is the same at pH 7.4 and pH 6.0 (37°). However, the rate of enzyme inhibition in trichlorphon solutions is 30 times faster at pH 7.4 than at pH 6.0, and this agrees with the greater stability of trichlorphon at the lower pH value. The rate of spontaneous reactivation of the enzyme was measured (37°, pH 7.4) after inhibition in trichlorphon solutions of acetylcholinesterase (human and bovine erythrocytes) and cholinesterase (human plasma). For all three enzyme preparations, the rate of spontaneous reactivation was the same as that obtained after inhibition by DDVP. All results point to the conclusion that trichlorphon in vitro is not an inhibitor of mammalian cholinesterases.

Some components of the cholinergic system in the prawn Palaemonetes varians (Leach).

1. An enzyme similar to mammalian acetylcholinesterase is found in high activity in the nervous tissue of Palaemonetes varians, i.e. eyes plus stalks, brain, suboesophageal ganglion and ventral cord. Acetylcholinesterase is also found associated with the abdominal muscles. Multiple enzyme forms are found in extracts of nervous tissues and muscles by electrophoresis and isoelectric focusing. 2. Cholinesterase is present in high activity in the stomatogastric system of P. varians. Three electrophoretically separable forms are found, having isoelectric points at pH4.2, 4.5 and 5.4. 3. Approx. 50% of the total acetylcholinesterase activity, approx. 80% of the choline acetyltransferase activity and 100% of the acetylcholine equivalents are found associated with the nervous tissue. Among the tissues examined, eyes plus stalks were the richest source of both choline acetyltransferase and acetylcholine equivalents. Suboesophageal ganglion and brain also contained large amounts of these components. 4. The distribution of these components could support the function of acetylcholine as a central and/or sensory transmitter in P. varians.

The influence of ionic strength on the interaction of quaternary drugs with mammalian acetylcholinesterase in relation to possible regulatory effects.

Abstract— The effects of inorganic salts, gallamine triethiodide and (+)‐tubocurarine chloride on mammalian acetylcholinesterase (AChE) were examined. The results were obtained mainly from soluble erythrocyte AChE; particle‐bound and detergent‐solubilized rat brain enzymes were also used. Three aspects of AChE were examined, namely direct effects on activity, the recovery of activity of the diethylphosphorylated enzyme and thirdly the aggregation of the enzyme at low ionic strength as shown by chromatography on columns of Sepharose 6B. The action of gallamine on AChE was controlled by the substrate concentration and the ionic strength of the medium. Both inhibition and activation by gallamine could be observed, depending on the particular conditions used. All effects of gallamine disappeared when the ionic strength was raised to 015. The action of gallamine closely resembled the result of increasing ionic strength by adding NaCl, for in both cases the apparent affinity of AChE for substrate decreased and concomitantly the maximum velocity of hydrolysis increased. The phosphorylated enzyme recovered activity more rapidly when gallamine or tubocurarine were present, or when the ionic strength was increased. Aggregation of all enzyme forms was observed at low ionic strength; an increase to I= 015 dissociated AChE to the single molecular form. It was concluded that the mammalian enzymes closely resembled electric eel AChE. The possible methods by which regulation of AChE activity could occur are discussed in relation to these results.