Membrane-bound Acetylcholinesterase Research Articles

Membrane-bound acetylcholinesterase: an early differentiation marker for skeletal myoblasts

Cell-bound acetylcholinesterase (AChE) was found to be an early differentiation marker on embryonic chick skeletal myoblasts in mixed primary cell cultures. AChE biosynthesis was detected and characterized by (a) a sensitive microtiter assay, (b) use of selective inhibitors, and (c) with mono- and polyclonal antibodies. Both secreted and cell-bound AChE appeared on the first day in culture, at a time when no muscle cell fusion was observed. Characterization of this enzyme revealed that true AChE was bound and secreted by myoblasts. BW284c51, which permeates cell membranes poorly, inhibited all the cell-associated AChE activity on myoblasts, suggesting that the activity measured was on the outer cell surface. On the other hand, fibroblasts appeared to have no or very little bound enzyme and the low level of secreted enzyme activity had the characteristics of pseudo-, or butyrylcholinesterase. Polyclonal anti- Torpedo californica electroplax AChE antibody and several monoclonal antibodies were found to bind specifically to chick myoblasts. Since the cells had not been made permeable before antibody binding, a membrane-bound form of the enzyme was most likely being detected. The cell-bound true AChE was present in identifiable quantities from the first day of culture. Membrane-bound AChE can thus serve as an early differentiation marker for embryonic chick myoblasts in mixed primary cultures.

Acetylcholine receptor-enriched membrane vesicles in response to ethanol: Activity and microcalorimetric studies

The activity of acetylcholinesterase (AChE) in acetylcholine receptor (AChR)-enriched membrane vesicles isolated from electric organ of Torpedo californica exhibited a biphasic response to ethanol action. Below an ethanol concentration of 35 m M, AChE activity increased with increasing concentration of ethanol. At ethanol concentrations greater than 35 m M, the activity was found to decrease montonically. In contrast, ethanol (35–400 m M) increased the activity of soluble AChE. This biphasic behavior was consistent with the proposed important role of ethanol-membrane interaction. Microcalorimetric measurements revealed that the enthalpy change in acetylcholine (ACh) hydrolysis reaction was 586 J/mol in association with membrane-bound AChE in AChR-enriched membrane vesicles, as compared to −544 J/mol with the isolated soluble AChE. This discrepancy was attributed to the presence of membranes. Unlike its action on the enzyme activity, ethanol did not affect the enthalpy change in ACh hydrolysis reaction catalysed by either membrane-bound or soluble AChE. Comparison of results on activity and heat measurements suggested that the interaction of ethanol with membrane vesicles was nonspecific with no ethanol-induced membrane structural or conformational change.

A study of human erythrocyte acetylcholinesterase inhibition by chlorpromazine.

Membrane-bound acetylcholinesterase (AChE) from the human erythrocyte is inhibited by chlorpromazine (CPZ) in a concentration range within this amphiphilic drug has been demonstrated to interact with erythrocyte membranes, causing a large spectrum of physical and structural effects; membrane solubilization with 0.5% Triton X-100 results in a complete loss of CPZ inhibitory potency. Although these observations might suggest a role of membrane lipid environment in mediating human erythrocyte AChE inhibition, we observed that CPZ retains its full inhibitory effect on the fraction of enzyme (5-6% of total) that is solubilized from erythrocytes upon treatment with phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis; furthermore, Triton X-100 is able to reverse the CPZ effect also in the case of PI-PLC-solubilized enzyme. These results demonstrate unequivocally that CPZ inhibits human erythrocyte AChE through direct molecular interaction. The inhibition kinetics displayed by CPZ on human erythrocyte AChE are dependent on drug concentration: evidence is provided that this phenomenon may be related to formation of CPZ micellar aggregates.

Size and charge isomers of acetylcholinesterase in the cerebral cortex of young and aged rats

Previous studies in this laboratory showed an age-related decline of acetylcholinesterase (AChE) activity in the cerebral cortex of rats. In the present study the age-related differences in enzymatic activity were evaluated in terms of individual molecular forms. Extracts containing total, soluble and membrane-bound AChE were analyzed both by ultracentrifugation in sucrose gradient and by non-denaturing gradient polyacrylamide gel electrophoresis. By ultracentrifugation two molecular forms, namely 10S and 4S (corresponding to tetrameric-G4 and monomeric-G1 forms, respectively) were separated in extracts of total and soluble AChE, while only 10S forms were present in extracts of membrane-bound AChE. Electrophoresis of soluble AChE extracts revealed slowly- and fast-migrating bands, grouped in two clusters of at least three bands each; membrane-bound AChE contained only a single slowly-migrating band. Electrophoresis of the single forms isolated by ultracentrifugation showed that slowly- and fast-migrating bands corresponded to G4 and G1 forms, respectively. Therefore, in soluble AChE no one-to-one relationship between charge- and size-isomers was observed; on the contrary, such relationship has been shown for membrane-bound AChE. This implies that soluble G4 forms and membrane-bound-G4 forms are electrophoretically different, being heterogeneous the former and homogeneous the latter. The age-related decline of total AChE, accompanied by a decrease of G4/G1 ratio, depended mainly on a decrease of membrane-bound AChE while soluble AChE and its G4/G1 ratio was unchanged. The qualitative pattern of charge isomers was not modified by aging.

Bovine brain acetylcholinesterase primary sequence involved in intersubunit disulfide linkages.

Three distinct classes of membrane-bound acetylcholinesterases (AChEs) have been identified. A12 AChE is composed of 12 catalytic subunits that are linked to noncatalytic collagen-like subunits through intersubunit disulfide bonds. G2 AChE is localized in membranes by a glycoinositol phospholipid covalently linked to the C-terminal amino acid. Brain G4 AChE involves two catalytic subunits linked by a direct intersubunit disulfide bond while the other two are disulfide-linked to a membrane-binding 20-kDa noncatalytic subunit. Molecular cloning studies have so far failed to find evidence of more than one AChE gene in any organism although alternative splicing of torpedo AChE mRNA results in different C-terminal sequences for the A12 and G2 AChE forms. Support for a single bovine AChE gene is provided in this report by amino acid sequencing of the N-terminal domains from the G2 erythrocyte, G4 fetal serum, and G4 brain AChE. Comparison of the 38-amino acid sequences reveals virtually complete identity among the three AChE forms. Additional extensive identity between the fetal serum and brain AChEs was demonstrated by sequencing several brain AChE peptides isolated by high performance liquid chromatography after trypsin digestion of nitrocellulose blots of brain AChE catalytic subunits. Cysteines involved in intersubunit disulfide linkages in brain AChE were reduced selectively with dithiothreitol in the absence of denaturants and radioalkylated with iodoacetamide. The observed sequence of the major radiolabeled tryptic peptide was C*SDL, where C* was the radioalkylated cysteine residue. This sequence is precisely the same as that observed at the C terminus of fetal bovine serum AChE and shows close homology to the C-terminal sequence of torpedo A12 AChE. We conclude that the mammalian brain G4 AChEs utilize the same exon splicing pattern as the A12 AChEs and that factors other than the primary sequence of the AChE catalytic subunits dictate assembly with either the collagen-like or the 20-kDa noncatalytic subunits.

Biochemical studies of the actions of ethanol on acetylcholinesterase activity: Ethnol-enzyme-solvent interaction

1. 1. Biochemical studies of the actions of ethanol on the activity of acetylcholinesterase (AChE), isolated from electric eel ( Electrophorus electricus) and purified by affinity chromatography, were performed to elucidate ethanol-enzyme-solvent interactions. 2. 2. Ethanol at a low concentration ([EtOH] = 2.7–200 mM) was found to enhance AChE activity slightly and systematically. 3. 3. This observation was consistent with the result from enzyme-kinetic studies that ethanol might noncompetitively activate AChE activity at this lower concentration range. 4. 4. If ethanol alters the hydrophobic site interaction on the enzyme and subsequently induces a favorable conformation for the active center of the enzyme, then a slight increase in the AChE activity in the presence of a low concentration of ethanol will be observed. 5. 5. This speculation was supported by the finding ofethanol's ability to perturb the inhibition of AChE activity by tetrabutylammonium bromide and to affect hydrophobic interaction between this salt and AChE, as investigated by enzyme activity and microcalorimetric measurements. 6. 6. The ethanol effect on the activity of this soluble AChE was found to be distinguishable from that on a membrane-bound AChE. 7. 7. Furthermore, to elucidate the effect ofethanol-solvent interaction on AChE activity, enzyme activity in the presence of much higher concentrations of ethanol was also examined. 8. 8. At [EtOH] > 800 mM, ethanol can perturb the structure of water around hydrophobic areas of AChE, causing an instability in the enzyme conformation and subsequently decreasing AChE activity.

Erythrocyte membrane-bound acetylcholinesterase in vitamin E deficient rabbits

1. The specific activities of erythrocyte membrane-bound acetylcholinesterase (EC 3.1.1.7.) and soluble hexokinase (EC 2.7.1.1.) in vitamin E deficient and vitamin E sufficient rabbits were investigated. 2. Acetylcholinesterase specific activities values of 43.4 in deficient and 57.4 in sufficient vitamin E rabbits were obtained. Hexokinase specific activity was not modified, and values of 3.31 in deficient and 3.6 in controls were found. 3. No peroxidation process was detected by us on vitamin E deficient diets. 4. These observations would suggest that the membrane stabilizing effect of vitamin E may be accomplished by a mode of action not necessarily related to its ability to prevent lipid peroxidation.

Subcellular Distribution and Characterization of Acetylcholinesterase Activities From Sheep Platelets: Relationships Between Temperature-Dependence and Environment

Acetylcholinesterase is a key enzyme in cholinergic neurotransmission for hydrolyzing acetylcholine and has been shown to possess arylacylamidase activity in addition to esterase activity. The enzyme is found at various loci, where its functional significance remains to be clarified, and it exists in multiple molecular forms. Sheep platelets have been shown to exhibit acetylcholinesterase activity associated with plasma membrane (Bp), endoplasmic reticulum (Cp), mitochondria granules (Dp), and soluble (As) fractions. These activities show differences in some physicochemical and kinetic properties. The soluble acetylcholinesterase is the most thermostable, and the enzyme from the Cp fractions shows the lowest affinity for the acetylthiocholine substrate and the strongest inhibition by fluoride. In all cases a noncompetitive inhibition of the enzyme by this ion is found. When membrane-bound acetylcholinesterases were assayed at temperatures between 12 degrees C and 33 degrees C, the Arrhenius plots of all activities exhibited a break point at about 17 degrees C. This discontinuity was abolished by addition of detergent to the assay medium (0.02% Triton X-100, final concentration). Their Hill coefficients were calculated in the presence of fluoride, showing unitary values in all cases, which points to a noncooperative effect and nonallosteric behavior in the particulate enzyme. These results suggest that the sheep platelet acetylcholinesterase associated with membrane-bound systems is modulated by the physical state of its environment, despite the fact that the enzyme might be lipid- or nonlipid-dependent.

Subcellular distribution and characterization of acetylcholinesterase activities from sheep platelets: relationships between temperature- dependence and environment

Acetylcholinesterase is a key enzyme in cholinergic neurotransmission for hydrolyzing acetylcholine and has been shown to possess arylacylamidase activity in addition to esterase activity. The enzyme is found at various loci, where its functional significance remains to be clarified, and it exists in multiple molecular forms. Sheep platelets have been shown to exhibit acetylcholinesterase activity associated with plasma membrane (Bp), endoplasmic reticulum (Cp), mitochondria granules (Dp), and soluble (As) fractions. These activities show differences in some physicochemical and kinetic properties. The soluble acetylcholinesterase is the most thermostable, and the enzyme from the Cp fractions shows the lowest affinity for the acetylthiocholine substrate and the strongest inhibition by fluoride. In all cases a noncompetitive inhibition of the enzyme by this ion is found. When membrane-bound acetylcholinesterases were assayed at temperatures between 12 degrees C and 33 degrees C, the Arrhenius plots of all activities exhibited a break point at about 17 degrees C. This discontinuity was abolished by addition of detergent to the assay medium (0.02% Triton X-100, final concentration). Their Hill coefficients were calculated in the presence of fluoride, showing unitary values in all cases, which points to a noncooperative effect and nonallosteric behavior in the particulate enzyme. These results suggest that the sheep platelet acetylcholinesterase associated with membrane-bound systems is modulated by the physical state of its environment, despite the fact that the enzyme might be lipid- or nonlipid-dependent.

Absence of substrate inhibition and freezing-inactivation of the mosquito acetylcholinesterase are caused by alterations of hydrophobic interactions

Membrane-bound acetylcholinesterase (AChE) from mosquito showed the characteristic substrate inhibition of this enzyme, but 105000×g supernatants of freshly extracted enzyme did not. Addition of chaotropic anions, a freeze-thaw cycle and autolysis of the amphiphilic acetylcholinesterase to its non-amphiphilic derivatives resulted in return of the substrate inhibition feature along with an apparent increment in the enzyme activity. These results suggested that the lipidic environment of the mosquito AChE is temporarily perturbed when extracted. The enzyme is probably trapped in non-sedimenting mixtures composed of endogenous amphiphilic molecules. The occurrence of this phenomenon was not affected by the presence of Triton X-100 and other detergents, either alone or in combination with sodium chloride. Freezing in the presence of strong chaotropic anions (perchlorate, iodide and thiocyanate) caused the irreversible inactivation of the mosquito AChE. Crude and incomplete purified fractions of the enzyme were more sensitive than a more purified preparation. With both the purified AChE and the non-purified AChE, amphiphilic AChE was more freeze labile. Freezing at −10°C enhanced inactivation of non-purified fractions. At this temperature, even weak chaotropic anions (fluoride, chloride and nitrate), while in combination with non-ionic detergents that solubilized mosquito AChE efficiently, reduced the enzyme activity of these fractions. In this case, recovery of the enzyme activity by incubation at 25°C was inversely correlated with the effectiveness of the chaotropic anion. Gel filtration failed to show any change in the hydrodynamic radius of the freezing-inactivated AChE. Therefore, this phenomenon is explained as different degrees of denaturation of the enzyme in direct association with the chaotropic strength. Thus, antichaotropic anions, such as sulfate, should improve the stability of the mosquito acetylcholinesterase during extraction, purification and storage.

The effects of ethanol on the structural stability of acetylcholine receptor and the activity of various molecular forms of acetylcholinesterase

The actions of ethanol on the structural stability of acetylcholine receptor (AchR)-enriched membrane vesicles and the activity of various molecular forms of acetylcholinesterase (AchE) were investigated, using the receptor and the enzyme isolated from the electric organ of Torpedo californica. In the presence of ethanol up to 200 mM, the thermogram of AchR-enriched membranes exhibited no significant decrease in the temperature ( t d) of receptor transition at 57°C, but a decrease in the enthalpy change ( ΔH d) indicated a slight ethanol-induced structural perturbation. The presence of 12.5 nmol α-bungarotoxin also caused a decrease in ΔH d. A complete loss of the receptor transition was observed at a higher concentration 500 nmol of α-bungarotoxin and no recovery of the transition was found with the addition of 200 mM ethanol. The results suggested a noncompetitive interaction of ethanol with the receptor. In the presence of 200–1000 mM ethanol, the activity of two soluble forms of AchE, a higher (117 S) aggregate and a lower (10 S) aggregate was not significantly affected. Comparing the activity of these two aggregates over a wide concentration range of ethanol (200–2000 mM) revealed no obvious difference in the level of ethanol effect between them. However, after removal of ethanol, the higher aggregate form of AchE exhibited a greater recoverability of the activity, suggesting a possible slightly greater structure-functional stability for it. Studies of soluble AchE and membrane-bound AchE showed that the presence of 200 or 600 mM ethanol caused a greater level of inhibition in membrane-bound enzyme than in soluble enzyme, possible due to a disruption of protein-lipid interaction needed to maintain the conformation of membrane-bound AchE. Interestingly, at a much higher concentration of ethanol (2.0 M), membrane-bound AchE became more resistant to ethanol than did the soluble forms of AchE. In this case, the effective concentration of ethanol felt by the enzyme was expected to be less for membrane-bound AchE, owing to ethanol's solubility in lipids.

Proteolytic stimulation and solubilization of membrane-bound acetylcholinesterase from muscle sarcotubular system.

Incubation of membranes derived from sarcotubular system of rabbit skeletal muscle with increasing concentrations of Triton X-100 produced both stimulation of the AChE activity and solubilization of this enzyme. Mild proteolytic treatment of microsomal membranes produced a several fold activation of the still membrane-bound acetylcholinesterase (AChE) activity. Attempts were made to solubilize AChE from microsomal membranes by proteolytic treatment. About 30-40% of the total enzyme activity could be solubilized by means of trypsin or papain. Short trypsin treatment of the microsomal membranes produced first an activation of the membrane-bound enzyme followed by solubilization. Incubation of muscle microsomes for a short time with papain yielded a significant portion of soluble enzyme. Membrane-bound enzyme activation was measured after a prolonged incubation period. These results are compared with those of solubilization obtained by treatment of membranes with progressive concentrations of Triton X-100. The occurrence of molecular forms in protease-solubilized AChE was investigated by means of centrifugation analysis and slab gel electrophoresis. Centrifugation on sucrose gradients revealed two main components of 4.4S and 10-11S in either trypsin or papain-solubilized AChE. These components behaved as hydrophilic species whereas the Triton solubilized AChE showed an amphipatic character. Application of slab gel electrophoresis showed the occurrence of forms with molecular weights of 350,000; 175,000; 165,000; 85,000 and 76,000. The stimulation of membrane-bound AChE by detergents or proteases would indicate that most of the enzyme molecules or their active sites are sequestered into the lipid bilayer through lipid-protein or protein-protein interactions and these are broken by proteolytic digestion of the muscle microsomes.

Effect of spinal cord ischemia on axonal transport of cholinergic enzymes in rabbit sciatic nerve

The fast axonal transport of acetylcholinesterase (AChE) and the slow transport of choline acetyltransferase (ChAT) were measured by the stop-flow ligation technique in the sciatic nerve of rabbits 6 and 24 h after ischemia performed by the occlusion of the abdominal aorta which lasted 40 min. Activities of these enzymes were also measured in punched samples of the spinal cord (L 5–6). Results were correlated with those obtained from the sham-operated control group. Six h after ischemia, its only apparent effect was a different distribution of accumulated enzymes in the central nerve segments. Twenty-four h after ischemia, the transport of AChE was markedly depressed; proximodistal accumulation decreased by 68%, whereas enzyme activity in the intact contralateral nerve and in the ventral horns of the spinal cord was preserved. No effect of ischemia on the retrograde axonal transport of AChE was observed in this experimental model. Cytoplasmic ChAT is much more susceptible to necrotic degeneration than membrane-bound AChE; 24 h after ischemia its activity decreased significantly in all investigated parts of the sciatic motoneurones but the rate of slow axonal transport did not seem to be affected.

Interaction of lanthanum chloride with human erythrocyte membrane in relation to acetylcholinesterase activity

Lanthanum chloride (1 mM) inhibits the activity of acetylcholinesterasein vitro in the human erythrocyte membrane. Lineweaver-Burk analysis indicates that lanthanum chloride induced inhibition of acetylcholinesterase activity is competitive in nature. The Arrhenius plot shows that the transition temperature of erythrocyte membrane-bound acetylcholinesterase is significantly reduced in the presence of lanthanum chloride. These results suggest that lanthanum chloride increases the fluidity of the erythrocyte membrane and this may be a cause of inhibition of membrane-bound acetylcholinesterase activity.

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, where-upon 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.

Human and rat erythrocyte membranes as a model for studying the effects of organic solvents on membrane-bound acetylcholinesterase in vitro

The effects of toluene, trichloroethylene and ethanol on membrane acetylcholinesterase (AChE) activity in human and rat intact erythrocytes and erythrocyte ghosts were studied in vitro over a range of concentrations (300–3000 ppm) and at three different incubation temperatures (37, 15 and 5°C). Toluene and trichloroethylene decreased the AChE activity in intact erythrocytes and in erythrocyte ghosts in both species. Trichloroethylene had a more marked effect on erythrocyte ghosts than on intact cells. At low concentrations, toluene generally had greater AChE-inhibiting potency than trichloroethylene. At the concentrations studied, ethanol had no effect on AChE activity. The effects of decreases in incubation temperature on solvent-induced AChE inhibition were very variable. We consider both the human and rat erythrocyte membrane to be a good model for studies of the anaesthetic effects of organic solvents on cell membranes. The effects of organic solvents on AChE, an integral component of the cell membrane, may be one of the critical factors determining the membrane effects of organic solvents even in nerve cells.

Molecular form of human lymphocyte membrane-bound acetylcholinesterase

The membrane-bound acetylcholinesterase (AchE) from human peripheral blood lymphocyte gives only one symmetrical peak on sucrose density gradient centrifugation in the presence of Triton X-100 detergent, with the calculated sedimentation coefficient of 6.5 S. However, this dimeric form of AchE was converted to a monomeric 3.8 S form when treated with 2-mercaptoethanol and iodoacetic acid. The results are consistent with studies which have shown by sodium dodecyl sulfate gel electrophoresis that the enzyme is built up of two identical monomers interlinked by disulfide bond(s). Under reducing conditions, revealed a single species of 70000 molecular weight, whereas under non-reducting conditions, another species of 140000 molecular weight of the AchE was found. Polyacrylamide gel electrophoresis indicated a single band with AchE activity in the presence of Triton X-100. In contrast, in the absence of the same detergent multiple band pattern could be observed. These results suggest that membrane-bound AchE enzyme is present in homogenous dimeric form on human lymphocyte membrane.

Comparative study on the therapeutic ultrasound effects on erythrocyte membrane-bound and free acetylcholinesterase.

Kinetics of erythrocyte acetylcholinesterase activity alterations exposed to ultrasound of therapeutic intensities of 0.88 MHz and 0.05-1.5 W/cm2 was studied. The differences were studied between the mechanisms of the inactivation of membrane-bound and free enzyme: the diminution of active enzyme sites for membrane-bound acetylcholinesterase and the decrease of enzyme-substrate affinity for the free form during sonication. The combined mechanical stresses in the ultrasonic field did not produce inactivation of free enzyme, as compared to the membrane-bound enzyme. Exponential ultrasonic/acoustochemical inactivation curves were obtained for the soluted crystalline form of acetylcholinesterase.

Neurons segregate clusters of membrane-bound acetylcholinesterase along their neurites.

Immunocytochemical studies with a monoclonal antibody show that acetylcholinesterase (AcChoEase; EC 3.1.1.7) is distributed in clusters along the fibers of cultured sympathetic neurons but is essentially absent from cell bodies. Although tissue-cultured sympathetic neurons synthesize several oligomeric forms of AcChoEase, only the hydrophobic globular (G4) form of AcChoEase is present within these clusters. This G4 form is asymmetrically distributed within neurons and is transported preferentially into nerve fibers following its synthesis in the cell bodies. Thus G4 is found in clusters on neurons and is readily distinguishable from the hydrophilic forms on the surfaces of myotubes. The association of a specialized form of AcChoEase in densities on neurons in culture indicates that neurons and myotubes have distinct mechanisms for localizing AcChoEase molecules on their surfaces and suggests that these two types of electrically excitable cells have different requirements for organizing synaptic components on their plasma membranes.

Combined effects of succinylcholine and calcium on membrane-bound acetylcholinesterase activity.

The effects of succinylcholine and calcium (Ca2+), alone and together, on membrane-bound acetylcholinesterase ("true-type" cholinesterase) were examined using human erythrocyte ghosts to elucidate the combined pharmacological activity of succinylcholine and calcium in in vivo system. Succinylcholine inhibited the acetylcholinesterase by a mixed style. Calcium alone exhibited an inhibitory effect on the enzyme, but a biphasic effect together with succinylcholine: marked restoration of the enzyme activity at calcium concentrations lower than 6 mM and depression at its higher concentrations. It is suggested that calcium induces a conformational change of the enzyme protein leading to the altered binding capacity of succinylcholine. In anesthetic practice, therefore, the use of calcium may not be indicated for the treatment of SCh phase II block.