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Tetrameric cholinesterase from horse serum undergoes concentration-dependent dissociation. The dimer is highly stable so that even on SDS polyacrylamide gels subunit dissociation to the 80-kDa polypeptide chains is incomplete. Glutaraldehyde cross-linking confirms this finding, giving rise to a tetramer: dimer ratio of approximately 1:1. The beta-adrenergic agent isoproterenol acts as an inhibitor of the enzyme with respect to butyrylthiocholine hydrolysis; inhibition kinetics point to a dissociative effect of the ligand as the underlying mechanism (Soylemez, Z. & Ozer, I. (1985) Comp. Biochem. Physiol. 81c, 433-437). Evidence from sedimentation analysis confirms this hypothetical mechanism: the sedimentation coefficient in the presence of saturating concentrations of both the substrate butyrylthiocholine and the inhibitor isoproterenol shows a 35 +/- 5% decrease; in high speed sedimentation equilibria the weight average molecular mass is shifted from the tetramer (Mr = 312 +/- 12 kDa) to the dimer (Mr = 160 +/- 10 kDa). The transition is complete at isoproterenol concentrations below saturation. Applying glutaraldehyde cross-linking to monitor the particle distribution at varying isoproterenol concentrations confirms the change in quaternary structure in a qualitative way. Enzyme concentrations applied in the present experiments are in the range of the concentration of cholinesterase in horse serum. Therefore the dissociative mechanism of isoproterenol on the enzyme may be of biological significance.

Four methods for measuring serum cholinesterase activity have been applied to sera of normal individuals and of patients shown to be sensitive to short-acting muscle relaxants of the succinyldicholine type. They have been assessed according to their ability to differentiate between sensitive and insensitive individuals on the basis of enzyme activity measurements alone. The method described, based upon that of Dietz et al. [Clin. Chem. 19, 1309 (1973)], in which propionylthiocholine is used as substrate, is best for this purpose, being capable of identifying over 90% of affected individuals with no false positives. Acetylcholine and butyrylthiocholine are slightly inferior substrates in this respect, and benzoylcholine gives little useful information.

Considerable acetylcholinesterase (AChE) activity was detected in an Aedes aegypti established cell line. The enzyme is blocked by 10(-6) M eserine sulfate, displays excess substrate inhibition and slowly hydrolyzes butyrylthiocholine. A 2-fold stimulation of AChE activity was shown after 2 days exposure to 3 x 10(-7) M beta-ecdysone. AChE activity found in the fresh medium is the contribution of the fetal calf serum portion. A direct relationship between levels of serum and the AChE activity in the cultured cells was demonstrated.

Human serum butyrylcholinesterase (EC 3.1.1.8) loses 100% of its activity toward butyrylthiocholine in 60 min at pH 3.0. This deactivation is retarded by 1.37 M ammonium sulfate to a loss of 40% after 60 min at pH 3.0. Reneutralization experiments suggest that the mechanism for this acid inactivation does not exclusively involve hydrolysis of peptide bonds or protonation of the enzyme's active site. Studies with different anions and cations demonstrate that the order of their effectiveness as protective agents against acid inactivation closely follows the Hofmeister series. No relationship was found between catalytic activation or inhibition by salt and protection from acid inactivation. Ultraviolet difference studies at 288 nm with enzyme brought to pH 2.7 from pH 8.0 in the presence and absence of 1.37 M ammonium sulfate demonstrated no change in UV absorbance with ammonium sulfate present and approximately a 0.15 ODU rise in absorbance in the absence of ammonium sulfate. These results suggest that acidic pH conditions result in deactivating stereochemical changes in the active site of butyrylcholinesterase and that certain anions and cations, according to the Hofmeister series, are able to protect the enzyme from acid inactivation by stabilizing the active conformation of its active site.

Experiments with butyrylcholine and benzoylcholine as substrates revealed that inhibition of horse serum cholinesterase by d -tubocurarine is linearly mixed. The ratio between k i and K ′ i is practically the same for both substrates, K ′ i being five times as large as K i . The difference is in the degree of inhibition: d -tubocurarine is a more potent inhibitor of butyrylcholine than benzoylcholine hydrolysis. The mixed inhibition of serum cholinesterase by d -tubocurarine can be explained by the allosteric binding of d -tubocurarine to the enzyme protein and is not due to the presence of isoenzymes displaying different affinities for substrates and inhibitors.

An acetylcholine hydrolyzing enzyme was prepared and purified (40 times) from dwarf bean hypocotyl hooks. The purity of the enzyme was proved by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was determined to be 65,000 daltons. Enzyme activity was the highest at pH 8.0 and between 30 and 36 C. The enzyme had an apparent affinity constant (K(m)) for acetylcholine of 460/micromolar. The affinity for substrate analogs increased from butyrylthiocholine to propionylthiocholine to acetylthiocholine. The enzyme activity was inhibited by choline, neostigmine, physostigmine, manganese, and calcium. Magnesium had no influence on the enzyme activity. We conclude that the enzyme from dwarf beans is an acetylcholinesterase (EC 3.1.1.7).

1. 1. Pseudocholinesterase activity of sweat, lacrimal fluid, urine, parotid saliva, whole saliva, whole blood and plasma was estimated using butyrylthiocholine iodide and propionylthiocholine iodide as substrates. 2. 2. There was similarity in activity between erythrocytes and plasma, but considerable differences between whole blood and other body fluids. The level of enzyme activity was approximately the same in sweat, lacrimal fluid, urine, parotid fluid and whole saliva, but it was only about 1 1500 of that of plasma. 3. 3. The enzyme activity in either whole blood or plasma is a good measure of organophosphate exposure but in other fluids activity can hardly give more than an approximate indication of acute poisoning.

We report the evaluation of a new commercially available assay system for the determination of serum pseudocholinesterase (EC 3.1.1.8) catalytic activity, and its application to a kinetic analyser. The assay is based on the colorimetric method of Okabe et al. (Clin. Chim. Acta 80, 87-94 (1977]: choline, liberated from benzoylcholine by pseudocholinesterase, is oxidized by choline-oxidase (EC 1.1.3.17) to betaine with the simultaneous production of hydrogen peroxide, which oxidatively couples with 4-aminoantipyrine and phenol in the presence of peroxidase to yield a coloured compound with maximal absorbance at 500 nm. The procedure not only has the advantage of being continuous, colorimetric and totally enzymatic but also appears to be precise (between-day analysis gives coefficient of variation between 3.5 and 5.6%) and accurate; the results obtained from normal and pathological sera show excellent correlation with those obtained by the alternative procedures employing propionylthiocholine, acetylthiocholine and butyrylthiocholine as substrates.

The steady-state kinetics of the butyrylcholinesterase-catalysed hydrolysis of butyrylthiocholine and thiophenyl acetate were shown to deviate from Michaelis–Menten kinetics. The ‘best’ empirical rate law was selected by fitting different rate equations to the experimental data by non-linear regression methods. The results were analysed in view of two alternative interpretations: (1) the reaction is catalysed by a mixture of enzymes, or (2) the activity is due to a single enzyme displaying deviations from Michaelis–Menten kinetics. It was concluded that the second alternative applies, and this conclusion was further supported by experiments involving simultaneous hydrolysis of alternative thiol ester substrates (butyrylthiocholine/thiophenyl acetate) as well as alternative thiol ester and oxygen ester substrates (butyrylthiocholine/phenyl acetate; thiophenyl acetate/butyrylcholine; acetylthiocholine/phenyl acetate). On the basis of the conclusion that a single enzyme is responsible for the activity, a molecular model is proposed. This model involves an acylated enzyme, and implies binding to the enzyme of one acyl group and one ester molecule, but not two ester molecules at the same time. Thus butyrylcholinesterase, which is structurally a tetramer, behaves functionally as a co-operative dimer, an interpretation in accordance with available data from active-site titrations.

The cholinesterase equipment of cerebral microvessels was studied in some rodents and carnivores using the Koelle-Friedenwald histochemical method with 3 artificial substrates and specific inhibitors for butyrylcholinesterase or acetylcholinesterase. Our observations reveal a great heterogeneity in cholinesterase types and their distribution in each of the different species studied. Only in the rat, butyrylcholinesterase appears to be a marker for the microvessels provided with a blood-brain barrier.

Acetylcholinesterase (AchE: EC 3.1.1.7) was identified and purified from the hemolymph of the scorpion Heterometrus bengalensis. The purity of the enzyme was determined by polyacrylamide gel electrophoresis (PAGE). The molecular weight of the enzyme, determined by sodium dodecyl sulfate-PAGE, was 80,000. The purified AchE hydrolysed acetylthiocholine iodide, but it did not react with butyrylthiocholine iodide. BW284C51, a specific inhibitor of AchE, strongly inhibited the enzyme. The known inhibitor (tetramonoisopropylpyrophosphortetramide) of pseudocholinesterase did not produce any inhibition of the enzyme activity. The purified AchE of scorpion hemolymph was vulnerable to high substrate concentration. The presence of Cu2+ and Ni2+ reduced the enzyme activity, whereas the metal ion, Sn2+, enhanced AchE activity. Ca2+ produced neither inhibition nor activation. (Na+, K+)-ATPase and Mg2+-ATPase activities were greatly enhanced by the purified AchE.

Spin–lattice relaxation times (T1) of protons in polycrystalline acetylthiocholine chloride, acetylthiocholine iodide, butyrylcholine chloride, butyrylthiocholine iodide and carbamylcholine chloride have been measured over a range of temperatures by pulsed proton magnetic resonance. For all the compounds double minima were observed in T1, and the results have been interpreted by considering the methyl-group reorientations about the C3 axis and the role of spin diffusion of other protons to the methyl protons in the compounds. Except for carbamylcholine chloride the double minima have been shown to be due to the two types of methyl groups present in the compounds. The double minima in T1 for carbamylcholine chloride have been attributed to reorientations of methyl groups bonded to the N atom about the C′3 axis and reorientations of each methyl group about its C3 axis.

Activity and properties of cholinesterase fromAphelenchoides ritzema-boosi, a plant feeding nematode, were investigated by testing the reaction of the enzyme with different substrates and inhibitors. Butyrylthiocholine was a better substrate than propionyl- and acetylthiocholine. When compared with mammalian erythrocyte and plasma cholinesterase, the nematode enzyme was found to be extremely insensitive towards a number of well-known organophosphorus and carbamate inhibitors.

Referance values are reported for the assay of cholinesterase (substratae: butyrylthiocholine iodide) in serum at 25 degrees C. There was no evidence of any age-dependency or sex-specific distinction in children (1 to 15 years, N = 309), nor were there any age-dependent changes observed in males (16 to 94 years, N = 718). Compared with the reference range for children, there was no detectable difference in the location and distribution of catalytic cholinesterase concentrations. In females (16 to 99 years, N = 861), an age-dependent difference in cholinesterase values became apparent: Regardless of pregnancy or use of hormonal contraceptives, the catalytic concentrations were found to be lower in younger females (16 to 39 years) than in older ones (greater than or equal to 40 years); the reference values in the older age group (40 to 99 years) did not differ from those of males and children. Therefore the following reference values are proposed (in each case 2.5 to 97.5 percentile: Children, males and females above 40 Years: 3.5 to 8.5 kU/l, Females (16-39 years, nonpregnant, not taking hormonal contraceptives): 2.8 to 7.4 kU/l, Females (18-41 years, pregnant or taking hormonal contraceptives): 2.4 to 6.0 kU/l.

Highly purified human plasma butyrylcholinesterase was inhibited by reversible inhibitors of esterase activity and modified by active-site-directed irreversible inhibitors of esterases and proteases. Peptidase and esterase activities of inhibited enzyme were simultaneously essayed from rates of hydrolysis of substance P (first cleavage) and butyrylthiocholine respectively. Inhibition parameters values and rates of inactivation of the two activities provide evidence that the peptidasic site is distinct from the esteratic site.

In the present work pseudocholinesterase (PCE) activity in human whole saliva was determined using butyrylthiocholine iodide as substrate. Samples of both unstimulated and stimulated saliva of 31 male and 24 female subjects were studied. Males had about two times greater salivary PCE activities than females. The activity was not much affected by salivary flow rate. In men, however, it was a little higher in stimulated than in unstimulated saliva. In women no significant difference was found. Salivary PCE activity showed diurnal variation so that activities were significantly greater at four o'clock after midnight than at four o'clock in the afternoon both in males and females. No correlation was found between PCE activities in saliva and serum.

Etomidate (Hypnomidate), a safe and effective short-acting hypnotic drug of special value for induction of anesthesia, seems to be a noncompetitive activator, as well as a competitive inhibitor, of pseudocholinesterase E.C. 3.1.1.8). This is analogous to the effects of morphine and its derivatives with which etomidate has a structural similarity. The analysis of enzyme kinetics in vitro gave a Km of 10.4 (+/- 1.1) microM and a Vmax of 2.4 (+/- 0.3) mmoles/1 min for the hydrolysis of S-butyrylthiocholine by human pseudocholinesterase. Etomidate does not show any influence on the reaction rate of pseudocholinesterase on S-butyrylthiocholine in the standard test. From the experimental results it is derived that even under in vivo conditions of induction of anesthesia by etomidate, no influence is to be expected on the turnover of succinylcholine by the normal pseudocholinesterase. There is no explanation for the occasionally prolonged postnarcotic apnea following administration of etomidate as far as competition of etomidate with succinylcholine for the pseudocholinesterase is concerned and as long as enzyme variants of higher affinity for etomidate can be excluded.

The activity and type of cholinesterases in various tissues of the frog (Rana esculenta) and possible seasonal differences in the activity were investigated. The activity is highest in the sympathetic chain, the brain and spinal ganglia, somewhat lower in Bidder's ganglia of the heart, and low in cardiac muscle, skeletal muscle, blood and liver. Cholinesterases in frog tissues are acetylcholinesterases; their sensitivity to eserine is lower than that of mammalian cholinesterases, which might be responsible for the failure of eserine to increase the negative inotropic effect of acetylcholine in the frog heart.

1. 1. Isoproterenol inhibits the hydrolysis of butyrylthiocholine by horse plasma cholinesterase, while it stimulates the hydrolysis of p-nitrophenyl butyrate. 2. 2. The inhibition pattern obtained for butyrylthiocholine is consistent with a dimeric model for the enzyme showing negative cooperativity. 3. 3. The kinetics of inhibition point to a dissociative effect of isoproterenol, superimposed on its competitive inhibitory action. The hydrolysis of p-nitrophenyl butyrate is not sensitive to changes in the subunit composition of the enzyme.

We report the existence, in Torpedo marmorata tissues, of a cholinesterase species (sensitive to 10−5M eserine) that differs from acetylcholinesterase (AChE, EC 3.1.1.7) in several respects: (a) The enzyme hydrolyzes butyrylthiocholine (BuSCh) at about 30% of the rate at which it hydrolyzes acetylthiocholine (AcSCh), whereas Torpedo AChE does not show any activity on BuSCh. (b) It is not inhibited by 10−5M BW 284C51, but rapidly inactivated by 10−8M diisopropyl-fluorophosphonate. (c) It does not exhibit inhibition by excess substrate up to 5 × 10−3M AcSCh. (d) It does not cross-react with anti-AChE antibodies raised against purified Torpedo AChE. This enzyme is obviously homologous to the “nonspecific” or pseudocholinesterase (pseudo-ChE, EC 3.1.1.8) that exists in other species, although it is closer to “true” AChE than classic pseudo-ChE in several respects. Thus, it shows the highest Vmax with acetyl-, and not propionyl- or butyrylthiocholine, and it is not specifically sensitive to ethopropazine. Pseudo-ChE is apparently absent from the electric organs, but represents the only cholinesterase species in the heart ventricle. Pseudo-ChE and AChE coexist in the spinal cord and in blood plasma, where they contribute to AcSCh hydrolysis in comparable proportions. Pseudo- ChE exists in several molecular forms, including collagen-tailed forms, which can be considered as homologous to those of AChE. In the heart the major component of pseudo-ChE appears to be a soluble monomeric form (G1). This form is inactivated by Triton X-100 within days. In addition, it is converted, on storage of the extracts, into more rapidly sedimenting, Triton X-100-resistant forms (G2 and G4). A G4 form exists in spinal cord and plasma. Collagen-tailed forms, A12 and A8, were characterized in high-salt extracts from spinal cord and heart ventricle. Pseudo-ChE molecular forms sediment slightly slower than the corresponding AChE forms. The two polymorphic enzyme systems, AChE and pseudo-ChE, therefore arose as early as elasmobranchs during the evolution of vertebrates.

One of the previously described five purified monoglyceride-cleaving carboxylesterases from rat liver microsomes proved to be a carnitine ester hydrolase. This esterase, with an isoelectric point of 5.2, is most active with medium-chain acyl- l -carnitines (C 12 -C 14 ). The esterase is also remarkably active with 1,3-diglycerides, especially 1,3-dioctanoylglycerol, that are hydrolyzed faster than the corresponding 1-monoglycerides and triglycerides. Only one of the other four purified carboxylesterases has moderate acylcarnitine-hydrolyzing activity. An altered procedure for the separation of the two microsomal acylcarnitine-cleaving enzymes is described. Both enzymes hydrolyze carnitine esters optimally at pH 8 and both are inactive with acetylcarnitine, palmitoyl-CoA, and butyrylthiocholine. The possible natural functions of the hydrolases are discussed. Besides their detoxifying action on natural membrane-lysing detergents (like carnitine esters and lysophospholipids), these enzymes could be involved in the transport of carnitine out of the liver.

In acute severe anticholinesterase poisoning by organophosphate compounds, pralidoxime (P-2-AM, pyridine-2-aldoxime methiodide) used in the recommended doses, intravenously, has not been shown to reactivate the inhibited cholinesterase, as evidenced both clinically and biochemically. In vitro studies using pralidoxime iodide up to ten times the recommended concentrations, produced insignificant reactivation of cholinesterases inhibited by the organophosphate insecticide Bidrin (di-methyl-3-hydroxyl-N, N-dimethyl-crotonamide phosphate). This was even so despite prolonged exposure of the inhibited cholinesterases to the oxime. The value of pralidoxime as a reactivator of phosphorylated cholinesterases is therefore in doubt, and should not be used in preference to large doses of atropine and other supportive treatment in poisoning by organophosphate insecticides.

A histomorphological and histochemical study was made on the nerve supply to the apocrine sweat glands in the general hairy skin of the goat. In harmony with the previous report that the sweat glands in the goat are functionally under the control of sympathetic nervous system, the present study clearly demonstrates cholinesterase-reactive nerve fibers that closely surround the secretory portion of these glands in most of the hairy skin area, though the nerve network is fairly coarse. Analysis with cholinesterase inhibitors indicated that the sudomotor nerves in the goat contain both specific and non-specific cholinesterase.

Human serum cleaves two dipeptides from the N-terminus of the neurohormone substance P. It has been suggested that this degrading activity is inherent to serum cholinesterase. We oppose this, because it turned out that highly purified serum cholinesterase contains traces of dipeptidyl peptidase IV, an enzyme known to attack the N-terminus of substance P. The peptidase is incompletely separated from cholinesterase during the procainamide-gel affinity chromatography as the last step of the usual purification procedure. Physostigmine completely inhibits the hydrolysis of butyrylthiocholine by such purified cholinesterase preparations, but not their substance P-degrading activity. Vice versa, epsilon-carbobenzoxy-lysylproline, an inhibitor of dipeptidyl peptidase IV, inhibits the peptidase activity of these preparations more than their esterase activity. After rechromatography on procainamide gel the peptidase is completely separated and the remaining cholinesterase has lost its substance P-degrading activity. We conclude that the N-terminal region of substance P is not degraded by cholinesterase but by the contaminating dipeptidyl peptidase IV, a different serine enzyme.

1. 1. The cholinesterase of the nematode parasite Metastrongylus apri (M. apri) was studied at 25°C in 0.1 M phosphate buffer pH 7.4. 2. 2. The activity vs substrate concn curves for acetylthiocholine (ATCh) and butyrylthiocholine (BTCh) are both bell-shaped, and each has an optimum at 10 mM substrate. The Michaelis constants for ATCh and BTCh are 0.1 and 0.8 mM, resp. The substrate inhibition constants for ATCh and BTCh are > 100 and a 30 mM, resp. At optimum substrate concentration, ATCh is hydrolysed 15 times faster than BTCh. 3. 3. The enzyme is soluble (97%) in 0.15 M NaCl. 4. 4. DDVP (0,0-dimethyl 2,2-dichlorovinyl phosphate) and haloxon (di-(2-chloroethyl) 3-chloro-4- methylcoumarin-7-yl phosphate) are progressive inhibitors with rate constants of inhibition 7.2 × 104 and 2.5 × 105 M−1min−1, resp. Metrifonate (0,0-dimethyl 2,2,2-trichloro-1-hydroxyethyl phosphate) is not an inhibitor. 5. 5. The results indicate that M. apri has only one cholinestrease, but it could not be established. whether it is the E.C. 3.1.1.7 or E.C. 3.1.1.8 enzyme.

Plasma cholinesterase activity, determined using either benzoyl choline or butyrylthiocholine as substrate, is stable for prolonged periods (> 12 months) when stored at −20°C. Likewise, repeated freezing and thawing of plasma did not markedly affect cholinesterase activity.

Choline ester hydrolysis has been studied in vitro employing routine and unbuffered variations of the histochemical medium described by Karnovsky and Roots (1964) in their direct coloring thiocholine method for the localization of cholinesterase activity in situ. The substrates acetylcholine and acetylthiocholine did not hydrolyze spontaneously in either buffered or unbuffered media. However, hydrolysis of both choline esters proceeded immediately when exogenous acetylcholinesterase was present in enzyme to substrate ratios of 0.1 or less. All reactions were slower at higher enzyme to substrate ratios. Hydrolysis was prevented by including 2×10−4M eserine in the medium while 2×10−4M tetraisopropylpyrophosphoramide had no detectable effect. Butyrylthiocholine was not hydrolyzed by exogenous acetylcholinesterase in this system. Homogenates of fresh brains or brains fixed in aldehyde were equally effective in hydrolyzing acetylthiocholine in the unbuffered medium. Results of the light and electron microscopic observations confirmed other reports that acetylthiocholine in combination with the appropriate inhibitor results in a staining reaction representing cholinesterase distribution.

Native horse serum butyrylcholinesterase (acylcholine acylhydrolase; EC 3.1.1.8) is a tetrameric enzyme which can dissociate after a limited proteolysis by trypsin into three additional molecular forms, including the monomeric entity. The trypsin-generated monomer of butyrylcholinesterase, isolated by ultracentrifugation on sucrose gradient, is stable and allows the relations between the polymeric structure of butyrylcholinesterase and its kinetic characteristics to be approached, e.g., substrate activation and complex thermal denaturation curves. The trypsin-generated monomer of butyrylcholinesterase behaves with identical kinetic parameter values as the native tetrameric enzyme. On the other hand, the thermal denaturation of the native tetrameric butyrylcholinesterase does not follow first-order kinetics, but may be described by a sum of exponential terms. This behavior is not due to the polymeric nature of butyrylcholinesterase but seems to be related to a structural heterogeneity induced by the heat treatment.

Aprophen, α-methyl-α-phenylbenzeneacetic acid-2-(diethylamino) ethyl ester, is a potent reversible inhibitor and a poor substrate of human serum butyrylcholinesterase (BuChE). Complex mixed competitive noncompetitive inhibition kinetics were observed; an apparent competitive inhibition constant was estimated to be 3.7 × 10 −7 M. BuChE hydrolysis of aprophen to diphenylpropionic acid and diethylaminoethanol did not appear to follow Michaelis-Menten kinetics. The BuChE turnover number for aprophen was 2.0 × 10 −3 sec −1 . Rabbit liver oligomeric and monomeric carboxylesterases (CE) also hydrolyzed aprophen with a similar turnover number that varied from 1.4 × 10 −3 sec −1 to 4.3 × 10 −4 sec −1 respectively. Comparison of the catalytic rate of aprophen hydrolysis with butyrylthiocholine (BTC) and the neutral aromatic substrate, phenylthiobutyrate (φTB), indicated that BuChE hydrolyzed BTC and φTB 3.2 × 10 5 and 3.1 × 10 5 times more rapidly than aprophen respectively. Similarly, the CEs also hydrolyzed BTC and φTB 17.6 and 1.9 × 10 5 times more rapidly than aprophen. Acetylcholinesterases from bovine erythrocyte and electric eel were not inhibited by aprophen nor was aprophen hydrolyzed by these enzymes. The hydrolysis and inhibition reactions may best be described by a complex reaction scheme involving multiple binding sites for both the substrate and the inhibitor as well as positive cooperative ligand binding.

Pseudocholinesterase (PCE) activity in oral fluid of 31 male and 24 female subjects was determined using butyrylthiocholine iodide as substrate. Males had approximately twice as much salivary PCE activity as did females [4.8 ± 2.4 (S.D.) U/1 and 2.2 ± 1.5 (S.D.) U/1, respectively]. The activity was not much affected by salivary flow rate, although in men it was a little higher in stimulated than in unstimulated saliva. Salivary PCE activity showed diurnal variation. Accordingly, activities were about three times greater at four a.m. than at four p.m. Parotid PCE activity correlated with that of whole saliva in both men and women. PCE activity in crevicular fluid (four subjects) was 120 ± 48 (S.D.) U/1. An elevation of PCE activity in oral fluid was found after experimental induction of gingival inflammation. However, the mean PCE activity of patients with clinical gingivitis was not significantly higher than that of healthy subjects, although some exceptionally high values were found. Sonicated samples of plaque did not contain any PCE activity. No correlation existed between PCE activities in saliva and serum.

1. 1. The musculature of isolated tube feet of several species of sea urchins was found to contract in response to GABA and to acetylcholine (ACh). The action of GABA but not that of ACh is blocked by bicuculline and picrotoxin. 2. 2. The action of GABA but not that of ACh requires the presence of Na + ; it is reduced when the external Cl concentration is diminished. 3. 3. The following cholinomimetics were found to be effective: acetyl-β-methylcholine, carbamylcholine, butyrylcholine and nicotine. The contractile effect of these agents, but also that of GABA was prevented by the cholinolytics atropine, banthine, decamethonium, flaxedil, hexamethonium and mytolon. 4. 4. The action of ACh and of acetyl-β-methylcholine, but also that of GABA, was greatly enhanced by anticholinesterases (eserine, neostigmine). 5. 5. Both ACh and GABA were detected in tube foot extracts. ACh is specifically localized in those parts that contain nerve tissue, with concentrations up to 75 μg/g wet wt. 6. 6. It is concluded that GABA causes excitation of cholinergic motoneurones and does not affect the muscle fibers directly. The cholinoreceptors of the muscle fibers have both muscarinic and nicotinic properties.

Ten distinct immunoprecipitates with esterase activity were identified in the reaction between rat liver microsomes and rabbit anti-rat liver microsomal antisera in crossed immunoelectrophoresis. These esterases were further characterized by the use of inhibitors, different substrates, hormone induction experiments and cross-reactivity studies with microsomes from other organs. Two of the esterase active antigens were found to be present in 2–3-fold higher concentrations in microsomes from female than male liver. Administration of the female sex hormone estradiol to male rats caused an induction of these esterases to the female level. The microsomal esterases showed a preference to hydrolyze substrates with short fatty-acid chains. Only one esterase could attack acetyl- and butyrylthiocholine. It was also inhibited by 10−3M eserine and was thus considered to be a cholinesterase. All esterases except one were inhibited by 10−3M DFP. In cross-reactivity experiments it was shown that this DFP insensitive esterase was also present in lung and testes microsomes. Kidney microsomes had two other esterases in common with liver, while microsomes from intestine, ovaries and heart gave one weak and diffuse precipitate, not further characterized, with the rabbit anti-rat liver microsomal antiserum.

We investigated the spontaneous reactivation of mouse plasma cholinesterase (ChE) after inhibition by various organophosphorus compounds. The remarkable spontaneous reactivations during storage at 24°C were observed in plasma ChE prepared 30 min after oral administration of three O, O-dimethyl organophosphorus compounds, i.e. malathion, methyl-parathion and cyanox ; while the spontaneous reactivation did not occur after inhibition by tolclofos-methyl, one of O, O-dimethyl organophosphorus compounds. The plasma ChEs inhibited by surecide, salithion and leptophos, which contain no O, O-dimethyl moiety, were not reactivated or only slightly so. These results suggest that a more sufficient attention should be paid in the determination of activity of plasma ChE inhibited by O, O-dimethyl organophosphorus compounds than that of plasma ChE inhibited by organophosphorus compounds without O, O-dimethyl moiety, since plasma ChE inhibited by the organophosphorus compounds with O, O-dimethyl moiety, except for tolclofos-methyl, was more easily reactivated, and the correct activity of inhibited plasma ChE can not be obtained without attention to the spontaneous reactivation. Furthermore, these spontaneous reactivations were examined by using butyrylthiocholine as well as acetylthiocholine as a substrate, and results showed that there was little difference between the spontaneous reactivations observed in using acetylthiocholine and butyrylthiocholine. So, it is concluded that these spontaneous reactivations take place only in pseudo ChE.

Cholinesterase activity of the tapeworm was studied histochemically with the Koelle method, using acetylthiocholine and butyrylthiocholine as substrates, and biochemically with the Warburg technique, using acetylcholine, acetyl-β-methylcholine and butyrylcholine as substrates.Eserine.tetra-isopropylpyrophosphoramide (iso-OMPA), 1,5-bis (4-trimethylammonium-phenyl-phntan-3-one diiodide (BW 62 C 47) and 1,5-bis(4-allylmethyl-ammonium-phenyl) pentan-3-one diiodide (BW 284 C 51) were used as inhibitors.Both the histochemically and the biochemically demonstrable tapeworm cholinesterase activity was readily inhibited by low concentrations of eserine but resistant to the other inhibitors employed, whatever substrate was used.In this respect the tapeworm cholinesterase activity markedly differed from that in the human serum, the rat brain homogenate or the duodenum of the cat, which were used as reference sources of cholinesterase.The histochemical cholinesterase reactions obtained with acetylthiocholine and butyrylthiocholine, with or without iso-OMPA or BW 62 C 47, showed identical distributions, selectively limited to nervous ganglia, nerve trunks and nerve fibres of the worm, including those innervating the suckers.It is concluded that the tapeworm cholinesterase is distinct from mammalian acetylcholinesterase and non-specific cholinesterase, even if it is, like acetylcholinesterase, a selectively neuronal enzyme and inhibited by excess substrate.

Publisher Summary This chapter discusses the ultrastructural localization of cholinesterase activity in the dental pulp. Cholinesterases are divided into specific—those that degrade acetylcholine—and nonspecific, such as butyrylcholinesterases (BuChE). Provided that specific inhibitors are used as controls, the thiocholine method is one of the most specific histochemical methods for localizing acetylcholinesterase (AChE) and BuChE. The content of cholinesterase in sympathetic ganglia and neurons varies between different species. A high cholinesterase activity in ganglia also corresponds to a high activity in the involved axons. The occurrence of an AChE activity that do not vanish on inhibition with BW284C51 may possibly depend on an incomplete inhibition. Incubation with butyrylthiocholine (BuThCh), with and without the different inhibitors, do not show any enzyme activity in the axonal membranes of the myelinated nerves.

A method is described for the separation and quantitation of cholinesterase isoenzymes using disc electrophoresis and a thiocholine histochemical staining technique followed by transmission densitometry. The method is specific and reproducible with a high resolution. By means of this method 12 cholinesterase isoenzymes can be demonstrated in normal human plasma, one fraction (ChE 7 ) constituting approximately 80% of total activity. Identical patterns are obtained by the use of acetylthiocholine and butyrylthiocholine as substrates, all cholinesterase components, however, showing less activity against acetyl- than against butyrylthiocholine. Diisopropyl phosphorofluoridate 10 −6 M and physostigmine 10 −5 M inhibit all bands except for one (ChE 5 ) which is inhibited only 75%. Extra bands were observed in 3 of 6 normal subjects and in 5 of 7 patients with chronic simple glaucoma.

1. 1. Electrophoresis of human plasma in polyacrylamide gel separates the cholinesterase (acylcholine acyl-hydrolase, EC 3.1.1.8 (pseudocholinesterase)) activity into five isoenzyme bands but the origin of these are unknown. 2. 2. The slowest moving band (cholinesterase)5 contained most of the activity. Purified preparations of this isoenzyme when treated with trypsin, α-chymotrypsin, papain, ficin and plasmin gave rise to bands which had the mobilities of each of the other isoenzymes. 3. 3. The isoenzyme bands obtained in this way showed the normal cholinesterase characteristics, they hydrolyzed butyrylthiocholine and were inhibited by 1 · 10−5 M eserine. 4. 4. The isoenzyme bands of plasma cholinesterase may thus correspond to parts of the molecules of the enzyme of cholinesterase5 type.

Cholinesterase activities of homogenates of rat brain and superior cervical ganglion were measured by automatic titration at pH 8. Acetylcholine, acetylthiocholine, acetyl-β-methylcholine, propionylcholine, propionylthiocholine, α-naphthyl acetate, butyrylcholine, butyrylthiocholine and benzoylcholine were used as substrates. As selective inhibitors of specific and nonspecific cholinesterases, 1: 5-bis-(4-allyl dirnethylammoniumphenyl) pentan-3-one diiodide (284 C 51) and tetra-isopropylpyrophosphoramide (iso-OMPA) were used, respectively. Acetyl-β-methylcholine and α-naphthyl acetate were mainly split by specific cholinesterase, butyrylcholine was predominantly hydrolyzed by non-specific cholinesterase, while acetylcholine, acetylthiocholine, propionylcholine and propionylthiocholine were readily split by both enzymes. Michaelis—Menten constants, determined by the Lineweaver-Burk procedure, were equal for acetylcholine and propionylcholine, on the one hand, and for acetylthiocholine and propionylthiocholine, on the other. The specific (284 C 51-sensitive) components of the cholinesterase activities of these 4 substrates showed strong inhibition by excess substrate, while the non-specific activity towards the same substrates increased with increasing substrate concentration. When 2 of these substrates were in turn used together as substrates, no additive increase of activity was observed. It is concluded that propionylcholine and propionylthiocholine serve, like acetylcholine and acetylthiocholine, as substrates of both specific and non-specific cholinesterases in rat brain and sympathetic ganglion.

Human teeth have been studied by treatment with copper thio-choline, the method developed by Koelle for demonstrating activity of both specific and nonspecific cholinesterases. Freshly extracted teeth were collected and immediately sectioned on a cutting machine designed for calcified tissues. One series of teeth was sectioned sufficiently thin for microscopic study. Another series of teeth was bisected to expose the pulp chambers to the reagents. These teeth were divided into 5 experimental groups. The first group was treated with 10-6M di-isopropylfluorophosphate (DFP) for 30 min at 37°C and then incubated with acetylthiocholine (AThCh) for 16 to 20 hr at 37°C in order to reveal the sites of activity of the specific enzyme, AChEase. The second group was incubated in a substrate of butyrylthiocholine (BuThCh) for 12 to 16 hr at 37°C to indicate the sites of the nonspecific ChEase. The third group was incubated in AThCh for 16 to 20 hr at 37°C without previous treatment by an inhibitor in order to reveal ...

1. Starch gel electrophoresis of rat brain homogenates has demonstrated 9 esterases which hydrolyze α-naphthyl acetate and naphthol AS-acetate. One of these has been identified as acetylcholinesterase. The nature of the others is undefined. In addition an enzyme (presumably pseudocholinesterase) acting against butyrylthiocholine iodide has been located at the site of sample insertion. 2. Homogenates of human white matter have yielded 9 esterases active against the same naphthol substrates. None of these is a cholinesterase. However, with acetylthiocholine iodide as substrate, acetylcholinesterase is demonstrable in human material. Enzymic activity against butyrylthiocholine iodide is demonstrable at the origin in human material, also. 3. Best results are obtained by use of homogenates centrifuged at 20,000 x g for 60 minutes.

Four isozymes of horse serum butyrylcholinesterase have been resolved by means of polyacrylamide gel electrophoresis combined with specific substrate staining and gel-scanning procedures. They differ 5-fold in their Km for butyrylthiocholine and 5 and 7-fold in reactivity with two organophosphates, malaoxon and Tetram.

Summary. Based upon the biochemical controls of the thiocholine method (Holmstedt 1957) the various steps of the histochemical procedure have been investigated. The inhibitors Mipafox and BW 284c51 and the substrates acetylthiocholine and butyrylthiocholine were combined as shown in Table 3 with the object of producing a reliable method of separating the two types of cholin-esterase. Sufficient controls are included in the table to allow a distinct separation of the two types of cholinesterase. The technique of the modified thiocholine method is given in detail. The following modifications have been made. 1. Inclusion of new inhibitors as above. (Mipafox and BW 284c51.) 2. No presaturation with copper thiocholine. 3. No development with ammonium sulphide. With the modified method, stellate ganglion, striated muscle and stomach wall have been stained in order to produce confirmation of the biochemical controls, not with the purpose of studying the morphology in detail. Comparison of sympathetic ganglia and motor end-plates shows that the ganglion cells contain only acetylcholinesterase but that the motor end-plates also contain a considerable amount of butyrylcholinesterase. The reasons for the alterations of the original thiocholine method are discussed. The exclusion of the presaturation with copper thiocholine produces no effect on the staining of tissue sections. The exclusion of development with ammonium sulphide diminishes diffusion artefacts to a considerable degree. Only fresh frozen sections can be used at present, if the purpose is to distinguish between the two types of cholinesterase.

Summary. Minute amounts of a number of choline esters, injected into the carotid artery, elicited increased chemoceptor activity of the sinus nerve. The effect was especially pronounced for acetyl-, acetylthio- and butyrylthiocholine; propionyl- and butyrylcholine were slightly less active, whereas for benzoyl-, acetyl-β-methyl-and succinyl-choline somewhat larger doses were required. After local treatment of the carotid body with the anticholinesterase mintacol the responses to acetyl-, propionyl-, butyryl-and benzoyl-choline were greatly increased, but the sensitivity to the thiocholines was lowered. Oxygen lack led to a stronger stimulation after mintacol than before. DFP, applied locally, also led to a sensitization of the cheinoceptors to acetyl-, butyryl-, propionyl- and benzoylcholine as well as for oxygen lack. Acetylcholine was again an exception. The results support the view that acetylcholine or related substances are involved in the initiation of the chemoceptive discharge from the carotid body.

1. 1. Cholinesterase activity in insect head extracts was investigated by disc electrophoresis followed by spectrophotometric analysis. The supernatant of 100 000 × g , 1 h, revealed the presence of four isozymes in the housefly, two in the American cockroach and only one in the southern army worm. 2. 2. The four isozymes, present in the head extract of houseflies, were totally inhibited by 10 μM guthoxon or eserine but were unaffected by 10 μ M p- chloromercuribenzoate (PCMB) or tri -o- cresylphosphate . All four isozymes had higher activity with acetylthiocholine than butyrylthiocholine as substrates; indicating they are isozymes of acetylcholinesterase. 3. 3. Centrifugation at 200 000 × g for 4 h precipitated 66% of the acetylcholin-esterase originally present in the supernatant of 100 000 × g for 1 h but the four isozymes remained soluble and were estimated to have molecular weights of less than 500 000. 4. 4. Two methods of gel staining were used to detect esterase activity, the direct-coloring thiocholine method using acetylthiocholine as a substrated and the modified Gomori method using α-naphthylacetate. The first proved to be effective for detecting cholinesterase activity; but at least two of the eserine-sensitive bands found by the technique of Gomori 20 were not cholinesterase.

The distribution of acetylcholinesterase (E.C. 3.1.1.7.) and other cholinesterases (E.C. 3.1.1.8.) in the fine structure of the myoneural junction of the rat diaphragma was studied, using acetylthiocholine and butyrylthiocholine as substrates. p]Acetylcholinesterase was located in the muscle sarcoplasm closely related to the postsynaptic membrane. Other cholinesterases are observed in the primary and secondary synaptic clefts and between the axon and the teloglial cells.