Total Cholinesterase Activity Research Articles

Acetylcholinesterase activity in ventricular and cisternal CSF of dogs: effect of chlorpromazine.

Acetylcholinesterase (AChE) activity in dog plasma is significantly higher than in either ventricular or cisternal CSF. However, since protein levels in plasma are about 100-fold higher than in CSF, the specific activity of AChE is lower in plasma than in CSF. Acetylcholinesterase activity in plasma represents only 22% of total cholinesterase (ChE) activity, while preliminary findings indicate that in ventricular CSF it is 50-60%. Acetylcholinesterase activity in ventricular CSF is significantly lower than in cisternal CSF. Chlorpromazine (10 mg/kg, intravenous), a drug which increases acetylcholine turnover, increased AChE-specific activity in all dogs. Our results support the hypothesis of a neuronal origin of AChE activity in CSF.

Multiple forms of acetylcholinesterase in the ciliary ganglion and iris of the pigeon

Different methods of extraction and analysis have been used to investigate the cholinesterase activity of the ciliary ganglion and iris from the adult pigeon. The molecular forms of acetylcholinesterase in the extracts have also been separated either by gel electrophoresis or by sedimentation velocity in a density gradient. The activity and molecular forms in (i) a ‘soluble fraction’ (the high-speed supernatant of an isotonic sucrose homogenate) and (ii) a ‘Triton extract’ (as above, but with 0.2% Triton X-100 in the homogenate) were examined. The cholinesterase activity in whole homogenates and in the two extracts was characterized by enzyme kinetics and use of different substrates and inhibitors. In the iris, as distinct from the ganglion, the total cholinesterase activity involved a small but definite proportion attributable to a butyrylcholinesterase. In the soluble fraction from both ganglion and iris. which contained 13 and 29% of the total activity, respectively, either method of separation revealed three main molecular forms of acetylcholinesterase; the evidence suggests that these are size isomers. In addition, Triton X-100 extracts from ganglia were shown by electrophoresis to contain a fourth slow migrating component. This form contained most of the enzymic activity and appears to be a membrane-bound form of acetylcholinesterase; it was not detected in the iris.

Acetylcholinesterase turnover in brain, cerebrospinal fluid and plasma.

Abstract—By assay of acetylcholine hydrolysis to measure total cholinesterase activity and acetyl‐β‐methylcholine hydrolysis to measure acetylcholinesterase (E.C 3.1.1.7) activity, patterns of regeneration of enzyme activity were measured in seven areas of brain, cerebrospinal fluid and plasma of cats after administration of an irreversible inhibitor. Halftimes of recovery of total cholinesterase in the brain tissues ranged from 0·9 to 3·8 days (av = 2·5 days) and acetylcholinesterase recovery halftimes ranged from 1·2 to 5·3 days (av = 3·6 days).Regeneration of total cholinesterase was also followed in subcellular fractions of guinea‐pig and rat brains after similar inhibition. In both species, the fastest recovery occurred in the soluble fraction with halftimes of 1·8 and 1·6 days, while the synaptosomal fractions exhibited the slowest recoveries with halftimes of 8·3 and 4·1 days. Regeneration of activity in plasma and CSF most nearly resembled that of the soluble brain fraction.

The Effects of Germination on the Subcellular Distribution of Cholinesterase in Cotyledons of Phaseolus vulgaris

AbstractHomogenates of Phaseolus vulgaris cotyledons have been found capable of hydrolyzing acetylthiocholine. The hydrolysis occurs optimally at pH 8.0, and is inhibited by neostigmine but not eserine. Total activity of the enzyme increases about three‐fold between the second and third days of germination, and remains high until day 6 before dropping coincident with the appearance of visible morphological symptoms of senescence in the tissue.Fractionation studies have revealed that the enzyme is enriched in preparations of purified cell wall and plasma membrane and is also present in a soluble fraction. The soluble enzyme accounts for more than 70% of the total cholinesterase activity two days after planting but by the fourth day of germination only about 30% of the total activity in the tissue is soluble. During the same period there is a large increase in the specific activities of both the cell wall and plasma membrane enzymes. By the seventh day of germination the particulate and soluble forms of the enzyme both show much reduced activities, but the specific activities of the cell wall and plasma membrane enzymes subsequently increase again. This is thought to reflect breakdown of protein other than cholinesterase in these structures as they in turn become subject to the increasing pressures of senescence.Cholinesterase in plant tissue presumably serves to regulate the endogenous titre of acetylcholine. The behaviour of this enzyme in bean cotyledons has been interpreted in terms of patterns of physiological and ultrastructural change known to characterize this tissue during germination.

The Development and Distribution of Cholinesterases in Cultured Skeletal Muscle with and without Nerve

Cholinesterase development was studied biochemically and histochemically in monolayer cultures of muscle cells from 10 day chick embryo leg muscle. Cholinesterase activity was low in myoblasts but increased rapidly after cell fusion and myotube formation; activity was distributed unevenly throughout myotubes, older fibres having some small areas of intense activity. Total Cholinesterase activity increased over the 10 day culture period while soluble activity reached a plateau after 4 days and was between 30–40% of the total. Butyrylcholines-terase activity did not change significantly during the period of study, comprising about 12% of the total Cholinesterase activity at 10 days. The possible effects on Cholinesterase development of combining muscle with dissociated spinal cord cells and with spinal cord explants, and of growing muscle in medium from which non-specific neural factors had been removed, was examined. No evidence was found for a nerve trophic effect on the amount or on the localization of muscle Cholinesterases.

Prolonged suxamethonium apnoea—the signal for what?

Most cases of prolonged apnoea following suxamethonium administration are associated with pseudo-cholinesterase variant enzymes, yet an appreciable number cannot be explained on this basis. Over 50 cases of prolonged apnoea have been referred for pseudocholinesterase typing, total cholinesterase activity against benzoylcholine and the determination of dibucaine and fluoride numbers, and some 25 families have been genotyped. From this work it can be shown that: 1.Genotyping of pseudocholinesterase is important to establish whether a variant enzyme was responsible for the prolonged suxamethonium apnoea. 2.Subjects should not be told they are ‘allergic’ to suxamethonium unless supported by appropriate laboratory investigation. 3.When an enzyme variant is indicated, the proband's sibship should always be examined since similar variants are most likely to occur in the siblings. 4.Seven of the ten possible genotypes at the E1 locus have been identified including 5 cases of the homozygous state of the silent gene E18. E18. Most cases of prolonged apnoea following suxamethonium administration are associated with pseudo-cholinesterase variant enzymes, yet an appreciable number cannot be explained on this basis. Over 50 cases of prolonged apnoea have been referred for pseudocholinesterase typing, total cholinesterase activity against benzoylcholine and the determination of dibucaine and fluoride numbers, and some 25 families have been genotyped. From this work it can be shown that: 1.Genotyping of pseudocholinesterase is important to establish whether a variant enzyme was responsible for the prolonged suxamethonium apnoea. 2.Subjects should not be told they are ‘allergic’ to suxamethonium unless supported by appropriate laboratory investigation. 3.When an enzyme variant is indicated, the proband's sibship should always be examined since similar variants are most likely to occur in the siblings. 4.Seven of the ten possible genotypes at the E1 locus have been identified including 5 cases of the homozygous state of the silent gene E18. E18.

Various enzymes involved with putative neurotransmitters. Regional distribution in the brain of deceased mentally normal, chronic schizophrenics or organic brain syndrome patients.

Levels of protein and several enzymes involved with neurotransmitters Were determined in 15 brain areas of subjects diagnosed as chronic schizophrenics (CS), organic brain syndrome (OBS, an older age group), or mentally normal (N) prior to death. Enzymes studied were total cholinesterase (TChE), acetylcholinesterase (AChE), pseudocholinesterase (ChE), choline acetyltransferase (ChAc), monoamine oxidase (MAO), and N-methyltransferase (NMT). The only significant decrease in protein was in the temporal cortex of the OBS group. The septal area of CS patients ( [ill] <.05) and the cingulate gyrus of the OBS group ( [ill] <.01) showed significantly less TChE activity than the mentally normal group. The septal area of the CS also had less AChE activity ( [ill] <.05). ChE activity was greater than normal in the CS patients previously on neuroleptic drugs. ChAc activity was significantly increased ( [ill] <.05) in the medial amygdala of CS patients. MAO activity did not differ significantly between groups. NMT activity was extremely low throughout and showed no statistically significant differences among the diagnostic catagories. The majority of brain areas sampled showed no significant differences in enzyme activity among the three groups. The differences that were observed must be considered preliminary and subject to further validation under better controlled conditions.

Biochemical and histochemical observations on the postnatal development of cholinesterases in the sympathetic ganglion of the rat

The activities of acetylcholinesterase and non-specific cholinesterase were measured by automatic titration in the superior cervical ganglia of albino rats aged o (newborn), 3, 6, 12, 24 and 90 days using acetylcholine and butyrylcholine as substrates and BW 284 C51 for distinguishing acetylcholinesterase from non-specific cholinesterase. The total cholinesterase activity was 29 μmol/g protein/min at birth but increased to 185 μmol/g protein/min on the 90th postnatal day. The acetylcholinesterase activity was 24 μmol/g protein/min at birth; it increased mainly between the 24th and 90th postnatal day, an increase being 44 to 131 μmol/g protein/min. The non-specific cholinesterase activity was still low on the 6th day (18 μmol/g protein/min) but on the 12th postnatal day reached almost the adult level of 57 μmol/g protein/min. Acetylcholinesterase was demonstrated histochemically with acetylthiocholine as substrate used in conjunction with iso-OMPA. Throughout postnatal development acetylcholinesterase was present in the cytoplasm of the sympathetic nerve cells and there was great variation in its activity in individual cells. The average activity increased with age up to the 24th day but showed little change thereafter. Fibres between the nerve cells first exhibited acetylcholinesterase activity on the 12th day. The number of acetylcholinesterase-positive fibres increased until the animals were adult. Non-specific cholinesterase activity, demonstrated using butyrylthiocholine with BW 284 C51 as inhibitor, was observed soon after birth in the cytoplasm of young nerve cells. Its activity increased until the 12th day but decreased thereafter in most nerve cells and increased in satellite and Schwann cells. However, some nerve cells showed a strong non-specific cholinesterase activity from the 12th to 90th day. It is concluded that (1) acetylcholinesterase activity is connected with both the differentiation and the prospective function of the sympathetic nerve cells; (2) non-specific cholinesterase plays an active role in the maturation of young nerve cells but is permanently present in some, presumably non-adrenergic, mature nerve cells and glial cells; and (3) the cholinesterases shown an adult pattern about 3 weeks after birth.

Effect of visible light on phospholipid and cholinesterase of the chicken retina.

Chickens were adapted to dark and light for 30 days and their retinas were studied in comparison to the chickens kept in normal environments. Total phospholipid content and cholinesterase activity were determined biochemically in the retinas of each group of animals. An increase of approximately 22% was observed in the phospholipid content of the dark adapted retinas, whereas the light adapted retinas showed no significant change in comparison with the normal counterparts. Retinal cholinesterase activity decreases by about 64% on dark adaptation and increases by about 14% on light adaptation.

Cholinesterase activity in rough and smooth brain microsomal membranes.

AbstractRat brain cortex was homogenized in 0.25 M or 0.32 M sucrose and sedimented at 10,000 Xg for 20 min. The supernatant was further separated into two subtractions by density gradient centrifugation in Cs+‐containing sucrose. Electron microscopy, RNA/protein and phospholipid/ protein indices showed that one of the subfractions was similar to the rough‐surfaced (R) and the other to the smooth‐surfaced (S) microsomal fractions separated from liver tissue by Dallner (1963). Cholinesterase (ChE) activity was studied by titration. Only 8–13 % of the total ChE activity was recovered in the 10,000 × g supernatant and about 3–6 % in the subfractions. Both the R and S microsomal subfractions displayed ChE activity; however, the S sub‐fraction had about three times as much activity per mg protein as the R subfraction. Activity due to nonspecific cholinesterase was 13 % of the total ChE activity in the R fraction and 7 % in the S fraction when the homogenate was prepared by homogenization with a clearance of 250 urn at 840 rpm. More vigorous homogenization increased the activities. Histochemical staining for AChE and ns. ChE activities revealed reaction product adjacent to both R and S vesicles.

Cholinesterase activity of muscle fibers and motor end plates: Comparative studies

Cholinesterase activity of skeletal muscle and its subcellular fractions, including motor end plates, was compared chemically and histochemically in man, mouse, guinea pig, rat, dog, and chicken. The total cholinesterase activity of muscle per milligram of nitrogen was highest in man (4,135 × 10 −9 moles of acetylcholine hydrolyzed in 30 min) and in descending order, decreased in mouse, chicken (red muscle), rat, and dog, to a value of 245 in chicken (white muscle). Cholinesterase was present in all subcellular components fractionated by differential centrifugation, and was greatest in the microsome fraction followed generally, in descending order, by the mitochondria, myofibril, and supernatant fractions. Each of these fractions had greater cholinesterase activity in human muscle than in mouse muscle, and in mouse muscle than in muscle of other species. The ratio of the activities of the microsome fraction and total muscle ranged from 9.5 in man to 2 in guinea pig. Greater concentration of cholinesterase activity was also demonstrated electron microscopically in the sarcotubular system in rat. Because of its relatively greater proportion, the myofibril fraction appears to contribute most to the total cholinesterase activity of muscle. Isolated muscle membrane contained high cholinesterase activity of motor end plates, and the activity was greater than the activity of the microsome fraction in guinea pig, rat, and chicken. The cholinesterase activity per motor end plate was in descending order from 0.805 in the rat, through man, guinea pig, and mouse, to 0.180 in dog, and the variation is less than the variation of the total muscle cholinesterase activity among these species.

Developmental changes of cholinesterases and monoamine oxidase in chick embryo spinal and sympathetic ganglia.

Abstract— Total cholinesterase, acetylcholinesterase, (AChE) and monoamine oxidase (MAO) activity and protein content were determined throughout the embryonic life of the chick in spinal and sympathetic ganglia. The greatest part of total cholinesterase activity was due to AChE.AChE and MAO activity increased in both spinal and sympathetic ganglia very similarly from the 6th to the 12th day of incubation; from this day on a significant divergence occurred, mainly owing to a steady fall in spinal ganglion AChE, which decreased to approximately one tenth of the maximum value. The ratio of MAO activity in sympathetic and spinal ganglia increased from the 8th day onwards and approached 5·0 at hatching. The ratio between sympathetic and spinal ganglia, for AChE, choline acetylase (ChAc) and MAO activity, suggests a relationship between the maturation of the synapse in the sympathetic ganglia and the maximal activity of these enzymes.

Cholinesterase activity of motor end plate in human skeletal muscle.

The activity and properties of cholinesterase of the motor end plate in human intercostal muscle were studied in the isolated muscle membrane. This preparation was used because cholinesterase activity of the membrane preparation was localized in the motor end plate without contamination of cholinesterase of other muscle components. Under the experimental conditions, cholinesterase in a human end plate hydrolyzed 1.21 x 10(8) molecules of acetylcholine per msec, which is smaller than hydrolysis of 2.69 x 10(8) by a motor end plate of rat intercostal muscle. Studies with cholinesterase inhibitors and specific substrates indicated that about 90% of cholinesterase of human motor endplates is acetylcholinesterase, and about 10% is pseudocholinesterase. The end plate cholinesterase had an optimal pH of 7.8 and a Michaelis-Menten constant of 4.15 mmoles/liter, and was stable at 4 degrees C for at least 4 wk. Motor end plates were estimated to contain only about 2% of the total cholinesterase activity of human intercostal muscle, compared with about 20% in rat tibialis anterior muscle. The difference is due to the lower cholinesterase activity of the motor end plate and higher cholinesterase activity of non-end plate components in human muscle than in rat muscle. The isolated muscle membrane provides a useful preparation for the study of the properties of motor end plate in human skeletal muscle.

Cholinesterase activity of the motor endplate in isolated muscle membrane.

Abstract— The cholinesterase activity of motor endplates in tibialis anterior muscle of rats accounted for about 20 per cent of the total cholinesterase activity of the muscle. In the isolated muscle membrane preparation of rat intercostal muscle, the cholinesterase activity was localized solely in the motor endplate, as shown by cholinesterase staining. The cholinesterase activity of the membrane per unit of nitrogen was 26·9 times that of the muscle homogenate. The membrane (endplate) cholinesterase had an optimal pH of 8, Km value of 3·1 mm, and was stable at 4° for at least 13 days. Cholinesterase of a motor endplate hydrolysed 2·69 × 108 acetylcholine molecules in 1 msec. Since it is estimated that 108 cholinesterase active sites are present in a motor endplate, the turnover time (time necessary for one enzyme site to hydrolyse one acetylcholine molecule) is calculated to be 372 μsec, and the turnover number (molecules of acetylcholine hydrolysed by one enzyme site/min) to be 1·61 × 105. From studies with cholinesterase inhibitors, cholinesterase activity was estimated to be due mostly to acetylcholinesterase, and only a minor part to pseudocholinesterase. The muscle membrane preparation seems to be useful for the study of other properties of the motor endplate.

Acetyl- and pseudocholinesterase activities in sympathetic ganglia of rats.

Abstract—The quantitative method of Ellman, Courtney, Andres and Featherstone (1961) was adapted to a differential assay for the determination of acetyl‐ and pseudocholinesterase activities of sympathetic ganglia of rats. The activities of the cholinesterases of superior cervical, stellate and thoracic chain ganglia and of the abdominal ganglionic complexes in apposition to the superior mesenteric and coeliac arteries (superior mesenteric, coeliac and cardiac ganglia) were measured. B.W.284C51 dibromide, 5 × 10−5m, and ethopropazine hydrochloride, 3·15 × 10−5m, were employed to inhibit selectively acetyl‐ and pseudocholinesterases, respectively. Linearity was shown to be maintained with enzyme concentrations corresponding to 0·12‐0·5 mg of ganglion (wet wt.)/incubation. Under the experimental conditions of this assay, the rates of the reaction of ganglionic acetyl‐ and pseudocholinesterases were linear for time periods greater than those employed for calculating the rates of hydrolysis in the homogenates of sympathetic ganglia. Several experimental approaches were used to ascertain the specificity of the inhibitors and of the reaction.Of the total cholinesterase activity of sympathetic ganglia of rats, 55‐63 per cent was due to acetylcholinesterase and 31‐39 per cent to pseudocholinesterase. On the basis of the specific enzyme activity, superior cervical, stellate and superior mesenteric ganglia contained higher acetyl‐ and pseudocholinesterase activities than did thoracic chain, coeliac and cardiac (abdominal) ganglia. The specific activity of acetylcholinesterase was similar in rat and cat superior cervical ganglia and sympathetic cervical trunks while the pseudocholinesterase activity of these two tissues was somewhat lower in cats than in rats.

House fly age as a factor in their response to certain carbamates.

Adult house flies, Musca domestica L., treated topically with Baygon® ( o -isopropoxyphenyl methyicarbamate), Banol® (6-chloro-3,4-xylyl methyicarbamate), and carbaryl were most resistant to these chemicals when 5 days old. Susceptibility was greatest in adults 1 and 15 days old. Neither total cholinesterase activity nor its sensitivity to the insecticides, varied sufficiently in flies of different ages to explain the mortality data. Rates of penetration and excretion of carbaryl-C14 were not related to the mortality changes. However, in vivo inhibition of cholinesterase did correlate with the observed toxicity of the carbonates to adult house flies, suggesting that accumulation of the toxicant at the site of action varied in insects of different ages.

Vaccarezza's Test. Its Use in the Presumptive Diagnosis of Cancer

This new presumptive diagnostic test for cancer is based on the marked diminution in pseudocholinesterase, acetylcholinesterase and total cholinesterase activity in patients with malignant tumors. In 50 cases of pulmonary cancer and 50 of extrapulmonary cancer the difference with respect to normal subjects was highly significant (P In tuberculosis the positive (pathologic) reactions of this test are neither so pronounced nor so frequent. This fact is useful for a differential diagnosis with cancer of the lung. Benign tumors studied gave negative (normal) reactions. To increase sensitivity of the test it is necessary to carry out the three different enzymatic determinations simultaneously. Positive reactions were found in all of the patients suffering from cancer of the lung, 18 per cent showing (++) and 78 per cent (+++). Only 5 per cent of the controls showed positive reactions (+).

Micro-colorimetric determination of cholinesterase activity of motor end plates in the rat diaphragm

Abstract A spectrophotometric method is described for the determination of total cholinesterase activity in groups of 3–30 end plates, after staining with copper thiocholine and dissecting from the rat diaphragm, by continuous recording of the yellow colour (412 mμ) produced by reaction between dithiobisnitrobenzoate (1mm) and thiol groups liberated enzymatically from acetylthiocholine (5mm) at pH 7·0. Cholinesterase activity of end plates after correction for muscle ranged from 3·3–22·3 times 10−11m/end plate/hr and muscle cholinesterase activity ranged from 8·8–29·4 times 10−11 μg/hr. 2·9–26·7% of measured end plate cholinesterase activity was attributable to muscle. The results for end plate cholinesterase agreed with those obtained by other workers using microgasometric techniques. It was calculated that there were approximately 3·6 times 106 cholinesterase active sites/end plate which compared closely with 6 times 106 molecules acetylcholine released/ nerve ending/impulse and 2·6 times 106 cholinergic receptors/end plate given in the literature. It is suggested that each acetylcholine molecule after liberation from a nerve ending may interact with one receptor and be destroyed by one cholinesterase active site.

Increased cholinesterase activity of intact cells caused by snake venoms

Cottonmouth moccasin and Eastern diamondback rattlesnake venom increase the cholinesterase (ChE) activity observed in intact squid and lobster nerves and single electroplax. Cottonmouth was more effective than rattlesnake venom in lobster nerve and electroplax. Neither venom increased the activity of rabbit cerebral cortex slices or of a partially purified preparation of electroplax ChE. Both venoms are free of ChE activity. Strong permeability barriers are present in homogenized electroplax: they can be most effectively reduced by the use of the venoms. The venoms cause a reduction in the wet and dry weights of electroplax cells. They also depolarize and block electrical activity in concentrations of 50–100 μg/ml. Venoms may be useful in studies where it is necessary to measure the total ChE activity in intact cell preparations containing permeability barriers to the substrate normally employed in this enzyme assay.

Acetylcholine metabolism in the brain of rats treated with vanadyl sulfate and certain phenylacetic acid derivatives

The present work describes the effect produced by phenylacetic acid, phenylethylacetic and diphenylacetic acids (sodium salts) separated and together with vanadyl sulfate on some “indices,” of acetylcholine metabolism in the rat's brain. Total cholinesterase activity and the “free” and “bound” (conditionally) acetylcholine levels served as indices. As shown experimentally the use of phenylacetic acid derivatives is accompanied by reduction of the “bound” acetylcholine, content, whereas that of “free” acetylcholine and the total tissue cholinesterase activity remain unchanged. Vanadyl sulfate provokes a significant reduction of the total tissue cholinesterasic activity, but does not change the content of “free” and “bound” acetylcholine therein. In conjoint action of the above-mentioned substances, the effects of phenylacetic acid erivatives are supplemented by the anticholinesterase effect of vanadyl sulfate.