Inactivation Of Acetylcholinesterase Research Articles

The inactivation of acetylcholinesterase by alpha-terthienyl and ultraviolet light Studies in vitro and in larvae of the mosquito Aedes aegypti

Acetylcholinesterase (EC 3.1.1.7) was inactivated photochemically in solution, in the presence of dissolved terthiophene sensitizers. Alpha-terthienyl (2,2′:5,2″-terthiophene) and its isomers 3,2′:5′,2″- and 3,2′:5′,3″-terthiophenes showed very similar sensitizing properties. With all three terthiophenes, the photosensitization was completely suppressed under anaerobic conditions, and therefore the inactivation process required the presence of oxygen. The enzyme was inactivated in vivo when fourth instar larvae of the mosquito Aedes aegypti were treated with alpha-terthienyl in the presence of long-wavelength ultraviolet light. No inactivation was observed when the organisms were treated with the ultraviolet light alone, with the chemical alone, or with a previously irradiated sample of the chemical. This paper represents the first example of acetylcholinesterase inactivation in vivo by a photoactive insecticide.

A comparison of the muscarinic cholinoceptors and benzodiazepine receptors of guinea-pig brain and rat brain

Some properties of muscarinic cholinoceptors and benzodiazepine receptors in selected brain regions of guinea-pigs and rats were compared under identical experimental conditions. The regions investigated were striatum, hippocampus and pons-medulla, and the properties examined were the concentrations of receptors; apparent dissociation constants of the ligands [ 3H]quinuclidinyl benzilate (for muscarinic receptors) and [ 3H]flunitrazepam (for benzodiazepine receptors); Hill coefficients for the interactions of the antagonist atropine and the agonist acetylcholine with the muscarinic receptors; the affinities of these compounds for the muscarinic receptors; and the effects of chronic administration of an organophosphate cholinesterase inhibitor (di-isopropylfluorophosphate) on the concentrations of receptors. Rat striatal and hippocampal muscarinic receptors were found to have a slightly higher affinity for acetylcholine than the corresponding guinea-pig receptors. Administration of di-isopropylfluorophosphate reduced the concentration of muscarinic receptors in rat brain by 30%, but had no significant effect on the concentration of receptors in guinea-pig brain. In all other aspects, the properties of the brain receptors of the two species were very similar. For both species, the affinities of the muscarinic receptors for acetylcholine were higher in the pons-medulla than in the striatum and hippocampus. This was found to be the result of differences in the values of the association constants of the high- and low-affinity states of the receptors, rather than because of varying proportions of two states which have the same association constant in all regions. The insensitivity of guinea-pig brain muscarinic receptors to chronic administration of an organophosphate confirms the results of a previous study on the guinea-pig alone, and makes this system unique. Many other studies on various species have all indicated that prolonged activation of a receptor by an agonist (caused in the present work by inactivation of acetyl-cholinesterase) leads to a decrease in the concentration of the receptor.

Activation and inactivation of acetylcholinesterase by metal ions.

The kinetic consequences of acetylcholinesterase peripheral site occupation by metal ions were examined using three substrates; acetylthiocholine, p-nitrophenylacetate, and 7-(dimethylcarbamoyloxy)-N-methylquinolinium iodide. Two classes of metal ion effects were noted: activation by a group including Mg2+, Ca2+, Mn2+, and Na+, and inactivation by a second group which to date includes Zn2+, Cd2+, Hg2+, Ni2+, Cu2+, and Pb2+. Activation is demonstrable only in solutions of low ionic strength whereas inactivation can be readily observed in solutions of both low and high ionic strength. Activation appears to be due to a combination of metal ion binding and ionic strength effects and involves binding to peripheral sites which are distinct from those which bind organic cationic activators such as gallamine, propidium, and 7-(dimethylcarbamoyloxy)-N-methylquinolinium. The principal activating effect is on the deacylation phase of the enzyme-substrate reaction. Inactivators effect a slow conversion of the enzyme to an unreactive form. The kinetics of inactivation are biphasic at low ionic strength but become essentially monophasic at high ionic strength. More than 80% of the enzyme activity can be recovered upon addition of EDTA provided the chelating agent is added immediately following completion of the inactivation process. Prolonged exposure to inactivators results in a progressive decrease in the amount of recoverable activity, Although peripheral ligand interactions may result in a variety of catalytic site conformations, the macroscopic properties can be accounted for in terms of three ligand-dependent states of the enzyme in which catalytic ability (actual or potential) is retained, and a fourth denatured state.

Photosuicide inactivation of acetylcholinesterase by nitrosamine derivatives.

Methyl(acetoxymethyl)nitrosamine and methyl-(butyroxymethyl)nitrosamine are respectively substrate (KM - 10(-2) M) and competitive inhibitor (Ki = 2 x 10(-3) M) of electric eel acetylcholinesterase (EC 3.1.1.7). Irradiation of an incubation mixture of this enzyme with either nitrosamine leads to an irreversible loss of enzyme activity. The inactivation rates are dependent on photolysis wavelength, light intensity, and inhibitor concentration. Experiments where acetylcholinesterase was radioactively labeled by [14C]-methyl(acetoxymethyl)nitrosamine show that the incorporation of 1 mol of radioactive label per active site is sufficient to cause complete enzyme inactivation irrespective of the reaction conditions used. Methyl(acetoxymethyl)nitrosamine shows no affinity for horse serum butyrylcholinesterase (EC 3.1.1.8) while methyl(butyroxymethyl)nitrosamine is a competitive inhibitor (Ki = 2 x 10(-3) M), but no irreversible inhibition is induced by the action of light. We propose that a suicide type of inhibition [Bloch, K. (1969) Acc. Chem. Res. 2, 193-198] is responsible for the inactivation of acetylcholinesterase, based on photoactivation of nitrosamines only when associated with an acidic hydrogen of the active site.

STEADY STATE AND REGENERATING LEVELS OF ACETYLCHOLINESTERASE IN THE SUPERIOR CERVICAL GANGLION OF THE RAT FOLLOWING SELECTIVE INACTIVATION OF PROPIONYLCHOLINESTERASE1

Abstract— The effects of selective inactivation of propionylcholinesterase (PrChE) by tetramonoisopropylpyrophosphortetramide (iso‐OMPA) on the steady state and regenerating levels of acetylcholinesterase (AChE) were investigated on the superior cervical ganglion (SCG) of the rat. Over the dosage range of 1.5‐40.0 μmol iso‐OMPA/kg intraperitoneally, which produced nearly total inactivation of ganglionic PrChE and 0‐35% inactivation of AChE, there was no subsequent increase in AChE activity above the control level. Single or repeated injections of iso‐OMPA at total doses of 5.0‐40.0 μmol/kg intraperitoneally caused no reduction in the rate of regeneration of ganglionic AChE during the 24 h following its inactivation by sarin, 2.0 μmol/kg intravenously. Both sets of findings differ from those obtained previously in a similar study of ganglionic AChE and butyrylcholinesterase (BuChE) in the cat. Possible reasons for this distinct species difference are discussed.

Labeling of human erythrocyte membranes with eosin probes used for protein diffusion measurements. Inhibition of anion transport and photo-oxidative inactivation of acetylcholinesterase

The binding of eosin-isothiocyanate (eosin-NCS) and iodoacetamido-eosin (IA-eosin) to band 3 proteins in the membrane of human erythrocytes is characterized by studying the effect of these probes on the anion transport system. Although the unbrominated fluorescein precursors do not affect anion transport, both eosin labels are strong inhibitors of sulphate exchange in intact erythrocytes. 50% inhibition is obtained by binding 4.7 · 10 5 or 6.0 · 10 5 molecules/cell for eosin-NCS and IA-eosin, respectively. Both eosin probes are irreversibly bound and occupy common binding sites with 4,4′-diisothiocyano-1,2-diphenyl-ethane-2,2′-disulfonic acid (H 2DIDS), although other sites are labeled as well. The inhibition of anion transport is light independent and can therefore not be attributed to a photosensitizing action of the eosin probes. Both eosin derivatives, however, inactivate acetylcholinesterase upon illumination of air-equilibrated samples of hemoglobin-free labeled ghosts. The inactivation of the enzyme is accompanied by the formation of protein aggregates as visualized by polyacrylamide gel electrophoresis. These effects are not observed when intact erythrocytes are illuminated in the presence of eosin probes suggesting a protective effect of hemoglobin during the labeling procedure. Protection of ghosts from photo-oxidation is achieved by displacing air with argon. These results are discussed in relation to the use of these and similar probes to measure protein diffusion in membranes.

Diffusion barrier for acetylcholine at the frog neuromuscular junction

INACTIVATION of acetylcholinesterase (AChE) causes the half-decay times of the endplate currents (e.p.cs) and miniature e.p.cs (m.e.p.cs) to increase from about 1.5–2.0ms to 3.0–10.00 ms, times far too long to be accounted for by removal of the transmitter by free diffusion1–3. To explain this, Katz and Miledi1 postulated that, when the AChE is blocked, each acetylcholine (ACh) molecule undergoes numerous collisions with the post-synaptic membrane, leading to several attachments with the ACh receptors and a consequent delay in transmitter removal. This was supported by the observation that ( + )-tubocurarine, which reduces the number of free receptors, markedly decreases the half-decay of m.e.p.cs and e.p.cs after treatment with neostigmine1,3. In addition to this delaying mechanism, due to interaction with the ACh receptors the results described below suggest the existence of a second diffusion barrier for the transmitter at the frog neuromuscular junction. This barrier seems to be formed by non-depolarising sites which bind the ester moiety of ACh. Saturation of these sites with substances which possess –CO–O– or –CO–O–CO– groups accelerates the diffusion of externally applied ACh towards the ACh receptor, enhancing its depolarising effects. The barrier also hinders the outward diffusion of neurally released ACh in neostigmine-treated muscles, but this effect can only be demonstrated if the motor nerve is stimulated at high frequencies (200 Hz).

Inactivation of acetylcholinesterase by propanol and sodium dodecyl sulphate

Departure from first-order kinetics was observed for the inactivation of bovine erythrocyte and electric eel acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) by n- propanol . This was attributed to the presence of isoenzymes in the commercial preparations, although inactivation via a two-step process cannot be eliminated. The rate of inactivation of bovine erythrocyte enzyme by propanol or sodium dodecyl sulphate was decreased by the presence of added protein and increased by Triton X-100. The presence of a substrate (acetylthiocholine) decreased the rate of inactivation of bovine erythrocyte acetylcholinesterase by sodium dodecyl sulphate, but increased the rate of inactivation by propanol. Re-interpretation of earlier data on the inactivation and reversible inhibition of acetylcholinesterase by simple aliphatic alcohols indicated that they do not bind to hydrophobic regions of the active site.

Acetylcholinesterase inactivation in young and old human red blood cells

A comparison is made of the effects of certain enzyme-inactivating agents on the acetylcholinesterase (ACHE) of young and old human erythrocytes. Normal red cells and ACHE-deficient red cells are separated in accordance with their density and then exposed to trypsin, cephalothin and tannic acid. The ACHE activity of young and old cells is affected to the same extent, indicating that inactivation is independent of cullular age and the initial enzyme activity.

Stability of membrane acetylcholinesterase in human erythrocytes treated with tetraphenylboron.

Treatment of human erythrocytes with non-hemolytic concentrations of tetraphenylboron causes irreversible inactivation of acetylcholinesterase, an enzyme located at or near the outer cell surface. By contrast to other agents which also inactivate acetylcholinesterase activity such as 1,5-difluoro-2,4-dinitrobenzene and glutaraldehyde, tetraphenylboron does not affect the thermostability of the enzyme, alter its susceptibility to denaturation by urea or hinder the proteolytic digestion of the erythrocyte membrane.

Chemical modification of electric eel acetylcholinesterase by tetranitromethane

The reaction of acetylcholinesterase (acetylcholinehydrolase, EC 3.1.1.7) with tetranitromethane has been studied. The reaction caused a decrease in enzyme activity as measured with the substrate acetylthiocholine under conditions where hydrolysis of the neutral substrate indophenyl acetate was unaltered. The inactivation of acetylcholinesterase by tetranitromethane was greatly accelerated by the quaternary oximes pyridine-2-aldoxime methyl nitrate or toxogonin, though not by other quaternary inhibitors tested and not by an aliphatic oxime. The enhanced inactivation by tetranitromethane in the presence of pyridine-2-aldoxime methyl nitrate was blocked by the enzyme inhibitor decamethonium. The oxime-induced inactivation of acetylcholinesterase by tetranitromethane was accompanied by significant changes in the immunological properties of the enzyme as demonstrated by complement fixation. The reaction also resulted in the disappearance of tyrosine and appearance of nitrotyrosine.

Methanesulfonyl fluoride inactivation of acetylcholin-esterase in the presence of substrates and reversible inhibitors

Reaction of acetylcholinesterase with methanesulfonyl fluoride is accelerated in the presence of those N-alkyl analogs of acetylcholine that are relatively poor substrates (i.e. slowly hydrolyzed compared to acetylcholine), but is greatly retarded by those that are good substrates, such as acetylcholine itself. The observations are not explained by acetyl enzyme formation. They may be understood if such substrates are first bound at the anionic site and then intermittently contact the esteratic site; attenuation of this second interaction accounts for slow hydrolysis as well as failure to protect the enzyme against methanesulfonyl fluoride, which attacks the esteratic site. Ligands which contain a phenyl ring, including both poor and good substrates, generally protect the enzyme. A phenyl-binding region may therefore be closely associated with the esteratic site. Acceleration of methanesulfonyl fluoride reaction by cationic substrates apparently results from non-specific and normally inhibitory modes of substrate attachment. Like these substrates, reversible cationic inhibitors (which also accelerate the reaction) are now known to be bound in non-specific regions adjacent to the active center. It follows that the activation mechanism is unlikely to involve specific, and normally functional, conformational changes in acetylcholinesterase.

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

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

Selective, near-total, irreversible inactivation of peripheral pseudocholin-esterase and acetylcholinesterase in cats In vivo

Tetramonoisopropyl pyrophosphortetramide (Iso-OMPA) was confirmed to be a highly selective inactivator of cat pseudocholinesterase (BuChE) over wide ranges of concentration and temperature in vitro; intravenous doses of 3.0 or 6.0 μmoles (approximately 1.0 or 2.0 mg)/kg produced nearly total inactivation of peripheral BuChE, with no detectable inactivation of acetylcholinesterase (AChE). Selective inactivation of AChE in vivo was obtained as follows: after a dose of mephentermine, 2.0 to 3.0 mg/kg, i.v., to sustain blood pressure, cats received an intravenous dose of 10-(α-diethylaminopropionyl) phenothiazine HCl (Astra 1397) up to 200 μmoles (73 mg)/kg, which produces selective reversible inhibition of BuChE; they were then given 1.0 μmole (0.332 mg) 2-diethyoxyphosphinylthioethyldimethylamine acid oxalate (217 AO)/kg, which is sufficient when given alone to produce essentially total, irreversible inactivation of AChE and BuChE. Under these conditions, approximately one-third of the autonomic ganglionic BuChE, but none of the AChE, was protected and restored to activity. Quantitative results, obtained by a spectrophotometric method, were confirmed histochemically.

Acetylcholinesterase activity in En(a-) red cells.

SummaryEn(a–) red cells show surface alterations reminiscent of those seen in normal cells exposed to proteolytic enzymes. Since this kind of treatment causes inactivation of acetylcholinesterase, a comparison of the activity and properties of this surface‐located enzyme in En(a–) and in normal red cells was made. No significant differences in regard to specific activity, Km, heat stability, inactivation by exogenous agents and density (age)‐dependent distribution were observed.

Modifications of the erythrocyte membrane by glutaraldehyde: effect on acetylcholinesterase.

SummaryWhen erythrocytes are treated with glutaraldehyde they become resistant to hemolytic agents (water) and to mechanical stresses (sonication, freeze-thawing). These alterations are accompanied by the irreversible inactivation of acetylcholinesterase, an integral part of the erythrocyte membrane. Enzyme inactivation is dependent on concentration, pH, time and temperature. The reduction in activity is not associated with changes in substrate specificity, Km and pH profile. However, the residual acetylcholinesterase activity is more labile to denaturation by heat and urea than the normal enzyme.

The action of trypsin and neuraminidase on the synaptic membranes of brain cortex

The population of intracellular membranes of rat cerebral cortex which exhibited the highest relative specific concentration of bound N- acetylneuraminic acid (NANA) and the highest relative specific activity of acetylcholinesterase (EC 3.1.1.7) (synaptic membranes) was incubated with bacterial neuraminidase and with trypsin under a number of experimental conditions. The electrokinetic profile displayed by the membrane preparation upon zonal density gradient electrophoresis changed as a result of the removal of bound NANA by neuraminidase, while its acetylcholinesterase activity remained unaffected; conversely, the action of trypsin led to the inactivation of acetylcholinesterase before any significant alterations of the electrophoretic mobility of the membranes became apparent. Prolonged incubation alone or in the presence of either neuraminidase or trypsin, resulted in the loss of membrane electrophoretic homogeneity, a circumstance which, most likely, reflects a disaggregation of the structural matrix of the membrane.

On the effects of tannic acid on erythrocyte membrane acetylcholinesterase.

SummaryTreatment of human erythrocytes with tannic acid caused irreversible inactivation of the surface located acetylcholinesterase (ACHE). This effect was dependent on concentration, time, temperature, and pH. The ACHE inactivation was prevented by albumin but not by coating the cells with blood-group specific antibodies. The enzyme was not affected by the other phenolic compounds tested.

Effects of tannic acid on erythrocyte enzymes.

MANY properties of the red cell have been studied with the aid of tannic acid since the original observations by Roberts1. Treatment with this acid causes reduction in anion permeability2, agglutination3, alterations in osmotic resistance4 and renders erythrocytes capable of adsorbing proteins from solution5. The latter characteristic serves as a basis for the widely used procedures of passive haemagglutination. We have shown that the exposure of human erythrocytes to tannic acid, in conditions commonly employed in passive haemagglutination techniques, results in irreversible inactivation of acetylcholinesterase (AChE)6. The effect on this membrane enzyme7 was dependent on concentration, pH, time and temperature. Although the mechanism by which tannic acid exerts its action on the red cell has not been elucidated, it has been suggested that in low concentrations this agent traverses the erythrocyte membrane4. To test this hypothesis we have investigated the effect of tannic acid on two intracellularly located enzymes, glucose-6-phosphate dehydrogenase (G6PD)8 and acid phosphatase7.

Normal human red cells treated with 2-aminoethylisothiouroniumbromide (AET). Acetylcholinesterase inactivation and susceptibility to acid lysis in vitro

Emazie umane normali trattate con AET mostrano, similmente alle emazie dell'emoglobinuria parossistica notturna, una diminuzione della loro attivita acetilcolinesterasica ed una suscettibilita all'eniolisi acida. Nel presente lavoro vengono studiati i rapporti esistenti tra questi due parametri e viene concluso che essi non sono interdipendenti.