Gallamine and tubocurarine as possible allosteric modifiers of soluble acetylcholinesterase activity at physiological ionic strength

Esters of dimethylcarbamic acid are known to be poor substrates of acetylcholinesterase. They carbamylate the active catalytic site of the enzyme and the subsequent decarbamylation is a slow but measurable process. Similarly, acetylcholinesterase can be phosphonylated, and the dephosphonylation is extremely slow. Rapid hydrolysis of phosphonylated acetylcholinesterase can be brought about by oximes, but dealkylation of the phosphonyl group on the enzyme (known as ageing) renders the inhibited enzyme insensitive to oximes.

In the present work, decarbamylation of dimethylcarbamyl-acetylcholinesterase and ageing of isopropylmethylphosphonyl-acetylcholinesterase were studied at a physiological ionic strength (154 mM). Gallamine, d-tubocurarine and alcuronium accelerated reactivation of dimethylcarbamyl-acetylcholinesterase. Gallamine and tubocurarine enhanced the effect of the nucleophile 3,3-dimethyl-1-butanol on decarbamylation, and the interaction was synergistic in the case of gallamine. Gallamine and tubocurarine retarded ageing of isopropylmethylphosphonyl-acetylcholinesterase, whereas 3,3-dimethyl-1-butanol had no effect. Nevertheless 3,3-dimethyl-1-butanol enhanced the retarding effects of gallamine and tubocurarine.

All these effects, except the effects of 3,3-dimethyl-1-butanol on ageing, had been previously observed at low ionic strength, in which case the effects were more marked and were observed at lower concentrations of the drugs. The effects at low ionic strength have been attributed to binding of the drugs to a peripheral site on the enzyme with a consequent change in conformation at the active site, leading to altered kinetic properties. The present results suggest that such allosteric effects may persist at physiological ionic strength. There have been few indications previously that this is so, particularly in the case of solubilised acetylcholinesterase.     

Comparative study of enzymatic activity of cholinesterases of different origin in thionaphthylacetate hydrolysis reactions

A comparative determination of kinetic parameters V and Km in the reaction of hydrolysis thionaphthylacetate and well known substrate acetylthiocholine by choline esterases from different sources was conducted. It is shown that butyrylcholine esterases hydrolyze thionaphthylacetate with velocity comparable with that of hydrolysis of acetylthiocholine, while acetylcholine esterases and propionylcholine esterases hydrolyze this substrate several times slower than acetylthiocholine. The values of Km in the reactions of hydrolysis of thionaphthylacetate for all studied cholinesterases is an order higher than for acetylthiocholine except cholinesterase of blood serum of fish. This value for the latter enzyme is practically equal.     

Effect of the substrate structure on the mechanism of reversible inhibition of cholinesterases of different origin

The mechanism of reversible inhibition of human erythrocyte acetylcholinesterase, horse blood serum butyrylcholinesterase, cholinesterase from optical ganglia of the squids, PacificTodarodes pacificus and CommodoreBerryteuthis magister, from different zones of habitation area is studied in the presence of substrates of various structures (acetylcholine, butyrylcholine, acetylthiocholine, butyrylthiocholine, phenylacetate, indophenylacetate, 2,6-dichlorophenylindophenylacetate). Tested as reversible inhibitors were tetramethylammonium iodide, tetraethylammonium iodide, choline iodide, and two derivatives of α,ω-bis(trimethylammoniommethyl)oligodimethylsiloxane dichloride. It has been revealed that the mechanism of the reversible anticholinesterase action depends essentially both on the enzyme nature and on the structures of substrate and inhibitor. The transfer from cation-containing to hydrophobic substrates increased essentially the contribution of uncompetitive component of the inhibitory constant. In the presence of butyric acid esters (butyrylcholine, butyrylthiocholine), the potency of inhibitors was lower than at hydrolysis of the corresponding acetates. The effect of the substrate structure on the mechanism of reversible inhibition was revealed to a greater extent in reactions with participation of squid cholinesterases.     

Internal standard method for the measurement of choline and acetylcholine by capillary electrophoresis with electrochemical detection

An internal standard method has been developed for the determination of the neurotransmitter acetylcholine and/or its metabolic precursor choline. This approach couples the high separation efficiency of capillary electrophoresis with the sensitivity and selectivity of electrochemical detection at an enzyme-modified electrode. Indirect electrochemical detection is accomplished at a 25 microm platinum electrode modified by cross-linking the enzymes choline oxidase and acetylcholinesterase with glutaraldehyde. Although in this simple form of electrode fabrication there is a gradual loss of response from the electrochemical detector with time, accurate quantitation is achieved by the addition of butyrylcholine, which is also a substrate for acetylcholinesterase, as an internal standard. A linear response is achieved between 0 and 125 microM with a limit of detection of 2 microM (25 fmol). The utility of this method was demonstrated by monitoring the kinetics of choline uptake in synaptosomal preparations.     

Muscarinic Mobilization of Choline in Rat Brain In Vivo as Shown by the Cerebral Arterio-Venous Difference of Choline

In anesthetized rats, the choline levels of cerebrospinal fluid and plasma obtained from blood collected from peripheral vessels (carotid artery, cardiac vessels) and from the transverse sinus were determined with a radioenzymatic assay. Cortical release of choline was studied using the "cup technique." The plasma choline level of the peripheral blood (11.5 mumol/L) was lower than that of the sinus blood. The resulting cerebral arterio-venous difference of choline was negative (3.2 mumol/L) and reflected the net release of choline from the whole brain. The plasma choline levels were not different irrespective of whether the rats were anesthetized with ether, urethane, or pentobarbital. However, the choline level of the cerebrospinal fluid, which normally was lower than the plasma choline levels, was increased by urethane anesthesia to a level between the arterial and venous plasma concentrations of the brain. In old rats (24 months), the choline level of the cerebrospinal fluid was significantly lowered, when compared with the results obtained with younger rats (2-4 months). In rats kept on a low-choline diet for 2 weeks, the plasma choline level of the peripheral blood was reduced to 51% of the control. The effect on the choline level of the sinus blood was smaller; the cerebral arterio-venous difference of choline was not reduced (it was even slightly enhanced). Likewise, the choline level of the cerebrospinal fluid and the cortical release of choline were not altered. Intraperitoneal administration of oxotremorine in pentobarbital-anesthetized rats kept on a low-choline diet increased the plasma levels of choline.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of a galactose-free 20-kDa fragment exhibiting butyrylcholine esterase and aryl acylamidase activity from human serum butyrylcholine esterase by limited alpha-chymotrypsin digestion

Purified human serum butyrylcholine esterase (approximately 90-kDa subunit), which also exhibits aryl acylamidase activity, was subjected to limited alpha-chymotrypsin digestion. Three major protein fragments of approximately 50 kDa, approximately 21 kDa and approximately 20 kDa were found to be produced, as observed by SDS-gel electrophoresis of the chymotryptic digest. The purified butyrylcholine esterase could fully bind to a Ricinus-communis-agglutinin-Sepharose column but after chymotryptic digestion about 15-20% of the enzyme activity remained unbound and was recovered in the run-through fractions. Sephadex G-75 chromatography of the chymotryptic digest showed an enzymatically active fragment eluted at an approximate molecular mass of 20 kDa, apart from the undigested butyrylcholine esterase eluted at the void volume. The butyrylcholine esterase fragment that did not bind to Ricinus communis agglutinin also was eluted at an approximate molecular mass of 20 kDa from a Sephadex G-75 column. This enzymatically active low-molecular-mass fragment from Sephadex G-75 chromatography showed a single protein band of approximately 20 kDa on SDS-gel electrophoresis. Neutral sugar analysis of the approximately 20 kDa fragment showed the presence of mannose only, whereas the undigested butyrylcholine esterase showed the presence of both mannose and galactose. Amino-terminal-sequence analysis of the approximately 20 kDa fragment showed the sequence Arg-Val-Gly-Ala-Leu, which agrees with amino acid residues 147-151 reported for human serum butyrylcholine esterase [Lockridge et al. (1987) J. Biol. Chem. 262, 549-557]. Both cholinesterase and aryl acylamidase activities were co-eluted in all chromatographic procedures. The results suggested that limited alpha-chymotrypsin digestion of human serum butyrylcholine esterase resulted in the formation of a approximately 20-kDa enzymatically active fragment with Arg147 as its N-terminal residue and which was devoid of galactose.     

Procaine as a substrate and possible allosteric effector of cholinesterases

The local anaesthetic procaine showed the properties of an allosteric effector of bovine erythrocyte acetylcholinesterase at low ionic strength; it antagonised inhibition of substrate hydrolysis caused by decamethonium, decreased the rate of ageing of isopropylmethylphosphonyl-acetylcholinesterase, increased the rate of decarbamylation of dimethylcarbamyl-acetylcholinesterase, and interacted synergistically with the nucleophilic alcohol 3,3-dimethyl-1-butanol in the acceleration of decarbamylation. These allosteric effects almost completely disappeared as the ionic strength was increased to a physiological level, and they could not be demonstrated at the physiological ionic strength with membrane-bound human erythrocyte acetylcholinesterase. There was no evidence of significant cooperativity in the binding of procaine to the enzyme, nor in the binding of the substrate acetylthiocholine in the presence of procaine, contrary to reports in the literature for other sources of acetylcholinesterase. Procaine was not hydrolysed by acetylcholinesterase (EC 3.1.1.7) although it is a substrate for serum cholinesterase (EC 3.1.1.8).The possibility that the results at low ionic strength can be explained on the basis of procaine binding to the active site of acetylcholinesterase (at low concentrations) and also to a peripheral allosteric site (at higher concentrations) is discussed. The results confirm the complexity of the kinetics of acetylcholinesterase, and extend the range of compounds with the ability to modify rates of decarbamylation and ageing.     

Oxime-induced decarbamylation and atropine/oxime therapy of guinea pigs intoxicated with pyridostigmine

The generally accepted explanation for the effects of oximes in countering organophosphorus (OP) anticholinesterase is reactivation of the inhibited acetylcholinesterase (AChE). With soman, the inhibited AChE rapidly becomes resistant to oxime reactivation due to a phenomenon called aging. Thus, pretreatment with pyridostigmine (Py) or physostigmine (Ph) followed by atropine sulfate therapy is required to achieve significant protection against soman; the effectiveness of a pretreatment/therapy (P/T) regimen can be further increased against certain OPs (e.g. sarin and VX) by including an oxime in the therapy regimen. The P/T regimen is clouded by a controversy concerning the use of oximes in the treatment of carbamate intoxication, because 2-PAM has been reported to exacerbate intoxication by some carbamates and to have no effect on decarbamylation rates. To better understand the role of oxime therapy in the theory of pretreatment of OP intoxication we examined the effects of 2-PAM and HI-6 on the rate of decarbamylation of Py-inhibited erythrocyte AChE in vitro and in vivo, and studied the effects of atropine plus 2-PAM or HI-6 on Py toxicity. In decarbamylation experiments, Py-inhibited guinea pig erythrocytes were washed free of excess Py and incubated with vehicle or oxime (2 X 10(-4) M, pH 7.3 and 37 degrees C). Aliquots were assayed for AChE activity at various times during a 60 min incubation period. Rate constants were calculated and compared to determine whether the presence of oxime affected decarbamylation. The data from in vitro and in vivo experiments revealed that oximes accelerated the decarbamylation (p less than 0.05) of inhibited AChE. Lethality data for Py-treated guinea pigs showed that treatment with atropine (23 mumoles/kg, im) plus 2-PAM or HI-6 (145 mumoles/kg, im) at one min after injection of Py increased the protective ratio from 4.2 (atropine only) to 5.1 and 12.2, respectively. It is suggested that the enhanced therapeutic efficacy of atropine by oximes against Py intoxication is related to oxime-induced reactivation.     

Differential inhibition of soluble and membrane-bound acetylcholinesterase forms from mouse brain by choline esters with an acyl moiety of an intermediate size

Differential inhibitions of soluble and membrane-bound acetylcholinesterase forms purified from mouse brain were examined by the comparison of kinetic constants such as a K _m value, a K_ss value (substrate inhibition constant), and IC₅₀ values of active site-selective ligands including choline esters. Membrane-bound acetylcholinesterase form (solubilized only in the presence of detergent) showed lower K_m and K_ss values than soluble acetylcholinesterase form (easily solubilized without detergent). Edrophonium expressed a slightly but significantly (p<0.01) higher inhibition of detergent-soluble acetylcholinesterase form than aqueous-soluble acetylcholinesterase form, while physostigmine inhibited both forms with a similar potency. A remarkable difference in inhibition was observed using choline esters; although choline esters with acyl chain of a short size (acetyl-to butyrylcholine) or a long size (heptanoyl- to decanoylcholine) showed a similar inhibitory potency for two forms of acetylcholinesterase, pentanoylcholine and hexanoylcholine inhibited more strongly aqueous-soluble acetylcholinesterase than detergent-soluble acetylcholinesterase. Thus, it is suggested that the two forms of AChE may be distinguished kinetically by pentanoyl- or hexanoylcholine.This work was supported in part by Agency for Defense Development.     

GAS CHROMATOGRAPHIC ESTIMATION OF ACETYLCHOLINE IN THE RABBIT HEART USING A NITROGEN SELECTIVE DETECTOR

The distribution of ACh in the rabbit heart was investigated by a modified gas chromatographic estimation method. ACh was extracted with perchloric acid, precipitated as reineckate and demethylated with sodium benzenethiolate. The tertiary amines derived from ACh and other choline esters were concentrated by a microdistillation procedure. Gas chromatography was performed using a nitrogen selective detector. In the range of concentrations between 0.4 and 2.5 nmol ACh per tissue sample the coefficient of variation was 5.2 per cent. The recovery of ACh added to heart extracts was 101 per cent. Evidence for the identity of the choline ester isolated from rabbit hearts and authentic ACh was obtained by equal retention times and by correspondence of the ratio N/C of the respective tertiary amines. Parallel measurements using gas chromatography and bioassay on the rat blood pressure yielded closely corresponding values of ACh levels in the rabbit heart. The concentration of ACh was much higher in the atria than in the ventricles. In both atria, and ventricles the ACh concentration was higher in the right than in the left part of the rabbit heart. Endogenous propionylcholine or butyrylcholine were not detected.     

Phosphorylation of choline and ethanolamine in Ehrlich ascites-carcinoma cells

1. Ehrlich ascites-cell extracts convert choline and ethanolamine approximately equally well into their respective phosphoryl derivatives. 2. Choline is a potent inhibitor of ethanolamine phosphorylation, but ethanolamine has little effect on choline phosphorylation. 3. 2,3-Dimercaptopropanol, cysteine and Ca(2+) inhibit ethanolamine phosphorylation, but have no detectable effect on choline phosphorylation. 4. Choline-phosphorylating activity in Ehrlich ascites-cell extracts is more stable during storage than ethanolamine-phosphorylating activity. 5. Choline phosphorylation is stimulated in the presence of benzoylcholine, succinylcholine, butyrylcholine and propionylcholine, whereas ethanolamine phosphorylation is inhibited. This relationship is reciprocal: the compounds causing the greatest stimulation of choline phosphorylation bring about the greatest inhibition of ethanolamine phosphorylation.     

CARBAMYLATION AND DECARBAMYLATION OF ACETYLCHOLINESTERASE: EFFECT OF CHOLINE, 3,3-DIMETHYL-l-BUTANOL AND SOME ALLOSTERIC EFFECTORS

Abstract— Choline is more effective than 3,3-dimethyl-l-butanol (DMB) in protecting bovine erythrocyte AChE against carbamylation by physostigmine sulphate (an ester of methylcarbamic acid). Both of these alcohols bind to dimethylcarbamyl-AChE and accelerate its decarbamylation. Choline has a higher affinity for the inhibited enzyme, and causes a more rapid reactivation than does OMB. At low ionic strength, various allosteric effectors also bind to the dimethylcarbamylenzyme and accelerate its reactivation, but not to the same extent as choline and DMB. The rate of reactivation in the presence of an allosteric effector and choline is less than the sum of the individual rates. However the rate of reactivation in the presence of an allosteric effector and DMB exceeds the sum of the individual rates. The results provide further support for a previous proposal that choline and DMB bind to different sites on the enzyme, despite their structural similarity.     

Formation of unesterified choline by rat brain

Two preparations of rat brain (ischemic intact brain and homogenized whole brain) formed large amounts of unesterified (free) choline when incubated at 37 degrees C. The accumulation of choline was inhibited by microwave irradiation of brain, or by heating of brain to 50 degrees C, and was maximal at 37 degrees C at pH 7.4-8.5. Choline formation was only observed in subcellular fractions of brain that contained membranes. In homogenates of brain, choline accumulated at a rate exceeding 10 nmol/mg protein per h. There was a significant decrease in brain phosphatidylcholine concentration (of 50 nmol/mg protein) during incubation for 1 h at 37 degrees C. Concentrations of phosphocholine rose (by 2.3 nmol/mg protein), and concentrations of glycerophosphocholine and sphingomyelin did not change during this period. We used radiolabeled phospholipids to trace the fate of phosphatidylcholine and sphingomyelin during incubations of homogenates of brain. Phosphatidylcholine was degraded to form phosphocholine, glycerophosphocholine and free choline. No lysophosphatidylcholine accumulated. Sphingomyelin was degraded to form phosphocholine and a small amount of free choline. Magnesium ions stimulated choline production, while zinc ions were a potent inhibitor. Other divalent cations (calcium, manganese) had little effect on choline accumulation. ATP concentrations in brain homogenates were less than 5 nmol/mg protein (rapidly microwaved brain contained 27 nmol/mg protein). Addition of ATP or ADP to brain homogenates increased ATP concentrations and significantly inhibited choline accumulation. ATP diminished the formation of choline from added phosphatidylcholine, lysophosphatidylcholine, phosphocholine and glycerophosphocholine. The effects of ATP, zinc ion, or magnesium ion upon choline accumulation were not mediated by changes in the rates of utilization of choline for formation of phosphocholine or phosphatidylcholine. In summary, we showed that there was enhanced formation of choline when ATP concentrations within brain were low. This choline was derived, in part, from the degradation of phosphatidylcholine, and we suggest that phospholipase A activity was the primary initiator of choline release from this phospholipid.     

Substrate preferences of wild and a mutant house fly acetylcholinesterase and a comparison with the bovine erythrocyte enzyme

Comparisons were made of purified acetylcholinesterase from the heads of wild type house flies with a mutant form (which bound organophosphates and carbamates less tightly). Using 12 substrates and 6 quaternary inhibitors, the only substantial difference was that the Km for butyrylcholine was 25 times greater for the mutant enzyme, suggesting that butyrylcholine and the organophosphates and carbamates shared a common binding site. The pure enzyme from the wild type house fly was also compared with bovine erythrocyte acetylcholinesterase. The major difference was again with butyrylcholine as substrate: the ability to acylate or deacylate was 30 times greater in the fly enzyme (the Km values differed by a factor of 4).     

Small Rises in Plasma Choline Reverse the Negative Arteriovenous Difference of Brain Choline

The concentrations of free choline in blood plasma from a peripheral artery and from the transverse sinus, in the CSF, and in total brain homogenate, have been measured in untreated rats and in rats after acute intraperitoneal administration of choline chloride. In untreated rats, the arteriovenous difference of brain choline was related to the arterial choline level. At low arterial blood levels (less than 10 microM) as observed under fasting conditions, the arteriovenous difference was negative (about -2 microM), indicating a net release of choline from the brain of about 1.6 nmol/g/min. In rats with spontaneously high arterial blood levels (greater than 15 microM), the arteriovenous difference was positive, implying a marked net uptake of choline by the brain (3.1 nmol/g/min). The CSF choline concentration, which reflects changes in the extracellular choline concentration, also increased with increasing plasma levels and closely paralleled the gradually rising net uptake. Acute administration of 6, 20, or 60 mg of choline chloride/kg caused, in a dose-dependent manner, a sharp rise of the arterial blood levels and the CSF choline, and reversed the arteriovenous difference of choline to markedly positive values. The total free choline in the brain rose only initially and to a quantitatively negligible extent. Thus, the amount of choline taken up by the brain within 30 min was stored almost completely in a metabolized form and was sufficient to sustain the release of choline from the brain as long as the plasma level remained low. We conclude that the extracellular choline concentration of the brain closely parallels fluctuations in the plasma level of choline.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemical modification of the bifunctional human serum pseudocholinesterase. Effect on the pseudocholinesterase and aryl acylamidase activities

The effect of chemical modification on the pseudocholinesterase and aryl acylamidase activities of purified human serum pseudocholinesterase was examined in the absence and presence of butyrylcholine iodide, the substrate of pseudocholinesterase. Modification by 2-hydroxy-5-nitrobenzyl bromide, N-bromosuccinimide, diethylpyrocarbonate and trinitrobenzenesulfonic acid caused a parallel inactivation of both pseudocholinesterase and aryl acylamidase activities that could be prevented by butyrylcholine iodide. With phenylglyoxal and 2,4-pentanedione as modifiers there was a selective activation of pseudocholinesterase alone with no effect on aryl acylamidase. This activation could be prevented by butyrylcholine iodide. N-Ethylmaleimide and p-hydroxy-mercuribenzoate when used for modification did not have any effect on the enzyme activities. The results suggested essential tryptophan, lysine and histidine residues at a common catalytic site for pseudocholinesterase and aryl acylamidase and an arginine residue (or residues) exclusively for pseudocholinesterase. The use of N-acetylimidazole, tetranitromethane and acetic anhydride as modifiers indicated a biphasic change in both pseudocholinesterase and aryl acylamidase activities. At low concentrations of the modifiers a stimulation in activities and at high concentrations an inactivation was observed. Butyrylcholine iodide or propionylcholine chloride selectively protected the inactivation phase without affecting the activation phase. Protection by the substrates at the inactivation phase resulted in not only a reversal of the enzyme inactivation but also an activation. Spectral studies and hydroxylamine treatment showed that tyrosine residues were modified during the activation phase. The results suggested that the modified tyrosine residues responsible for the activation were not involved in the active site of pseudocholinesterase or aryl acylamidase and that they were more amenable for modification in comparison to the residues responsible for inactivation. Two reversible inhibitors of pseudocholinesterase, namely ethopropazine and imipramine, were used as protectors during modification. Unlike the substrate butyrylcholine iodide, these inhibitors could not protect against the inactivation resulting from modification by 2-hydroxy-5-nitrobenzyl bromide, N-bromosuccinimide and trinitrobenzenesulfonic acid. But they could protect against the activation of pseudocholinesterase and aryl acylamidase by low concentrations of N-acetylimidazole and acetic anhydride thereby suggesting that the binding site of these inhibitors involves the non-active-site tyrosine residues.     

Effect of external high potassium and pH on the uptake of choline in glial and neuronal cells in culture

TheV _max of the uptake of choline was increased in nerve cell cultures by lowering (from 7.4 to 6.5) or increasing (from 7.4 to 8.1) the pH. In neurons no effect was observed on the value of theK _m's of the uptake of either the apparent high or low affinity components. In glial cells only a low affinity component was measured at pH 6.5 and diffusion was observed at pH 8.1. An excess of K⁺ ions in the incubation medium reproduced the increase inV _max observed with changes in pH suggesting a possible dependence of the uptake of choline upon the H⁺ and OH^– gradients. Taking into account the characteristics already known of the transport of choline into nerve cells, such a dependence adds new insight in the mechanisms underlying the transport and indicates another possible regulation of choline entry, eventually directed towards the synthesis of acetylcholine.     

Peripheral Nerve Phospholipid Composition: Development in Normal Nerve and Age-Dependent Changes in Wallerian Degenerated Nerve

Abstract: The phospholipid composition of normal peripheral nerve as a function of developmental age as well as that of Wallerian-degenerated nerve as a function of age at nerve transection and duration of Wallerian degeneration have been quantitated in rabbit sciatic nerve. During development, increases in the proportions of ethanolamine plasmalogen, sphingomyelin, and combined phosphatidyl serine plus phosphatidyl inositol and decreases in the proportions of phosphatidyl choline and phosphatidyl ethanolamine correlated well with the concurrent myelin accretion. During Wallerian degeneration, age-dependent changes in phospholipid composition were observed. The large and statistically significant increase in the proportion of phosphatidyl choline and decrease in the proportion of ethanolamine plasmalogen were manifest promptly in nerves transected at 2 weeks of age but in a delayed manner in nerves transected at 8, 12, and 20 weeks of age. The rate of loss of individual phospholipids was greater in nerves transected at younger ages. The findings from normal developing peripheral nerve may well serve as baseline data for subsequent studies of phospholipid composition in pathological peripheral nerve. The Findings from Wallerian-degenerated peripheral nerve provide additional evidence for age-dependent chemical changes occurring in Wallerian-degenerated peripheral nerve that may be of significance in explaining the superior functional recovery from peripheral nerve injury observed in younger compared with older subjects.     

Phenyldichlorophosphate as an aid in studies of decarbamylation of carbamylated acetylcholinesterase

An improved method for assaying carbamylated acetylcholinesterase is described which has substantial benefits over current methods. Acetylcholinesterase was carbamylated with neostigmine and diluted extensively into buffer to allow decarbamylation to occur. At various times, phenyldichlorophosphate was added to the mixture of free and carbamylated enzyme, whereupon two very rapid, simultaneous reactions occurred: near total, and permanent, inactivation of free acetylcholinesterase by the organophosphate, and inactivation of phenyldichlorophosphate by hydrolysis. The carbamylated acetylcholinesterase was allowed to reactivate fully and then assayed for enzyme activity. The assay provided a measure of the amount of carbamylated enzyme present at the time of addition of phenyldichlorophosphate, thereby enabling the first-order rate constant for decarbamylation to be calculated. This new method of studying decarbamylation was applied to two systems of soluble acetylcholinesterase, where the half-life for decarbamylation was approximately 1/2 h or 4 min, respectively, and to membrane-bound acetylcholinesterase. The results agreed well with those determined by a conventional method; moreover, the standard error of the mean was lower for the new method. The advantages of the method using phenyldichlorophosphate over conventional methods are particularly evident when decarbamylation is rapid or when in vivo studies are being performed and it is not practical or desirable to run assays immediately on isolation of the tissue. The new method also has advantages over a published related technique using the organophosphate anticholinesterase soman.