Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:3.1.1.7 (acetylcholinesterase)
28,390 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Acetylcholinesterase (AChE; EC 3.1.1.7) extracted in 1% Triton X-100 from rabbit brain was purified 2,000-fold by chromatography on agarose conjugated with a monoclonal antibody directed against human red blood cell cholinesterase. After elution from the immunoadsorbent with pH 11 buffer, the preparation was purified further by affinity chromatography on phenyltrimethylammonium-Sepharose 4B with decamethonium elution. Overall yield of purified enzyme was 37% of the AChE originally solubilized, with a specific activity of 2,950 units/mg protein. Electrophoresis under reducing conditions in 7.5% sodium dodecyl sulfate polyacrylamide gels revealed only one silver-staining polypeptide band. A streamlined purification procedure enabled the isolation of electrophoretically homogeneous AChE to be completed in fewer than 7 days, at yields exceeding 50%. Electrophoretic analysis of purified AChE indicated an apparent MW of 71,000 for the monomeric subunit. Gel filtration and sucrose density gradient centrifugation in the presence of Triton X-100 showed little difference between the properties of the native and the purified enzyme. The molecular mass of the main species was estimated from the gel filtration and sedimentation data to be 280,000 daltons. Kinetic parameters of the purified protein (Km = 0.16 +/- 0.01 mM) were close to those of the native enzyme (Km = 0.12 +/- 0.01 mM) when examined with acetylthiocholine iodide as substrate. The two-step immunopurification procedure presented in this communication offers a convenient route to homogeneous neural AChE in quantities useful for detailed biochemical and immunochemical study.
...
PMID:Two-step immunoaffinity purification of acetylcholinesterase from rabbit brain. 396 30

The detergent-soluble form of acetylcholinesterase was purified from the electric organ of the electric rays Narke japonica and Torpedo californica, and its properties were examined. The electric organ of N. japonica and T. californica contains three types of acetylcholinesterase: low-salt-soluble, asymmetric or tailed, and detergent-soluble forms. Results showed that in N. japonica, asymmetric forms were predominant, whereas in T. californica the detergent-soluble form was predominant. Low-salt-soluble acetylcholinesterase constituted 10% of the total acetylcholinesterase in both species. Detergent-soluble acetylcholinesterase was purified by immunoaffinity chromatography with a monoclonal antibody (Nj-601) to acetylcholinesterase. Triton X-100 extracts of these electric organs were applied to a column of Nj-601-Sepharose, and the bound acetylcholinesterase was eluted quantitatively by lowering the pH to 2.8. This simple procedure gave good yields. The purified enzymes gave single peaks at 6 S on sucrose gradients in the presence of detergent and polydisperse aggregates in the absence of detergent. Reduction of disulfide bonds gave peaks at 4.4 S. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the purified acetylcholinesterases gave bands with Mr of about 130 000 in the unreduced state and with Mr of 66 000 in addition to a very faint band of Mr 130 000 in the reduced state. The Mr-66 000 polypeptides were labeled with diisopropylfluorophosphate. Thus, the detergent-soluble acetylcholinesterases exist as dimers of the Mr-66 000 components. Two-dimensional electrophoresis of the purified enzymes indicated their homogeneity. The isoelectric points of both enzymes were 5.1 under the conditions employed. The two enzymes had very similar amino acid compositions, and contained more than 14% of neutral sugars and glucosamine. Monoclonal antibodies were raised to detergent-soluble acetylcholinesterase by the hybridoma technique; eight were obtained. All of them recognized the catalytic subunits of detergent-soluble and asymmetric acetylcholinesterase, and reacted only with detergent-soluble acetylcholinesterase in immunoblots. Four of the monoclonal antibodies inhibited the activities of both the detergent-soluble and asymmetric forms of acetylcholinesterase.
...
PMID:Detergent-soluble form of acetylcholinesterase in the electric organ of electric rays. Its isolation, characterization and monoclonal antibodies. 397 94

Acetylcholinesterase (EC 3.1.1.7) from fetal bovine serum (FBS) was purified to electrophoretic homogeneity. The procedure involved procainamide affinity chromatography with native FBS, followed by chromatography on Sepharose 6B and DEAE-Sephadex. The acetylcholinesterase was purified approximately 44,000-fold, and 13 mg was obtained corresponding to an overall yield of about 45%. The purified acetylcholinesterase was stable at 4 degrees C for at least 8 weeks but was labile to freezing; however, in 50% glycerol the enzyme was stable at -20 degrees C for at least 12 weeks. FBS acetylcholinesterase exhibited typical substrate inhibition, had a Km of 120 microM, and a turnover number of 5300 s-1 with the substrate acetylthiocholine. The enzyme was highly sensitive to the specific acetylcholinesterase inhibitor 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one. FBS acetylcholinesterase was characterized as a G4 form of acetylcholinesterase and was distinguished from bovine erythrocyte acetylcholinesterase on the basis of lectin gel binding, [3H] Triton X-100 binding, amino acid composition, number of catalytic subunits/molecule, and hydrodynamic properties. FBS acetylcholinesterase had a Stokes radius of 76 A as judged by gel filtration, and from this a molecular weight of 340,000 daltons was calculated. The enzyme had a subunit weight of approximately 83,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; paraoxon titration indicated a relative active site mass of 75,000 daltons. The amino acid composition of FBS acetylcholinesterase was similar to the human erythrocyte acetylcholinesterase (Rosenberry, T. L., and Scoggin, D. M. (1984) J. Biol. Chem. 259, 5643-5652). A monoclonal antibody directed against human erythrocyte acetylcholinesterase, AE-2, (Fambrough, D. M., Engel, A. G., and Rosenberry, T. L. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 1078-1082) cross-reacted with FBS acetylcholinesterase.
...
PMID:Acetylcholinesterase from fetal bovine serum. Purification and characterization of soluble G4 enzyme. 398 Apr 78

The Aviation Research Laboratory has developed a methodology for evaluating toxicant effects on pilot performance. Flight data are collected using a digital flight simulator, the ILLIMAC (ILLInois Micro Aviation Computer), during holding patterns and instrument landing system approaches. The flight data are recorded by a separate microcomputer, which also presents the Sternberg memory searching task. A preliminary study examined pilot performance in the simulator and cholinesterase inhibition by insecticides in agricultural pilots. The correlation between the physiological parameters and the pilot performance data was determined. Experiments are planned to determine the effects of a variety of drugs on pilot performance. Neurotoxicants to be studied include ethanol, three antiemetic drugs, and atropine sulfate.
...
PMID:Measuring the effects of neurotoxicants on flight simulator performance. 398 7

The effects of gossypol on membrane functions of the human erythrocyte were studied. Gossypol (10 microM) had no effect on spontaneous hemolysis, osmotic fragility, cell volume, cholinesterase activity, hexose transport, ouabain-sensitive inorganic cation transport, ouabain-insensitive inorganic cation transport and nucleoside transport. Conversely, 10 microM gossypol inhibited inorganic anion transport by approximately 90% for three different substrates, i.e., phosphate, sulfate and chloride. Inhibition of inorganic anion transport was specific as 10 microM gossypol had no effect on the eight aforementioned membrane-related functions of the human erythrocyte. Inhibition inorganic anion transport was characterized using sulfate as the substrate and had the following features: it was potent, with a Ki of approximately 3 microM; it was rapid, with onset occurring in less than 1 min; it was potently blocked by physiological concentrations of albumin and plasma with 50% blocking achieved at 0.03% (w/v) albumin; it occurred by a noncompetitive kinetic mechanism; it was independent of medium Ca++, Mg++ or pH. Gossypol was bound to human erythrocytes and cell membranes isolated from erythrocytes. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid is a potent inhibitor of anion transport and can be covalently bound to band 3. Covalently bound 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid blocked a fraction of gossypol binding to erythrocyte membranes.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Effect of gossypol on erythrocyte membrane function: specific inhibition of inorganic anion exchange and interaction with band 3. 403 82

A small hydrophobic domain in isolated human erythrocyte acetylcholinesterase is responsible for the interaction of this enzyme with detergent micelles and the aggregation of the enzyme on removal of detergent. Papain has been shown to cleave this hydrophobic domain and to generate a fully active hydrophilic enzyme that shows no tendency to interact with detergents or to aggregate [Dutta-Choudhury, T.A., & Rosenberry, T.L. (1984) J. Biol. Chem. 259, 5653-5660]. We report here that the intact enzyme could be reconstituted into phospholipid liposomes while the papain-disaggregated enzyme showed no capacity for reconstitution. More than 80% of the enzyme reconstituted into small liposomes could be released by papain digestion as the hydrophilic form. Papain was less effective in releasing the enzyme from large liposomes that were probably multilamellar. In a novel application of affinity chromatography on acridinium resin, enzyme reconstituted into small liposomes in the presence of excess phospholipid was purified to a level of 1 enzyme molecule per 4000 phospholipid molecules, a ratio expected if each enzyme molecule was associated with a small, unilamellar liposome. Subunits in the hydrophilic enzyme form released from reconstituted liposomes by papain digestion showed a mass decrease of about 2 kilodaltons relative to the intact subunits according to acrylamide gel electrophoresis in sodium dodecyl sulfate, a difference similar to that observed previously following papain digestion of the soluble enzyme aggregates. The data were consistent with the hypothesis that the same hydrophobic domain in the enzyme is responsible for the interaction of the enzyme with detergent micelles, the aggregation of the enzyme in the absence of detergent, and the incorporation of the enzyme into reconstituted phospholipid membranes.
...
PMID:A small hydrophobic domain that localizes human erythrocyte acetylcholinesterase in liposomal membranes is cleaved by papain digestion. 404 29

Groups of guinea pigs were injected with a range of dosages for sarin (0, 140, 279, 557 micrograms/kg) followed by pralidoxime (2-PAM) and atropine sulfate (16 mg/kg). Poisoning by sarin in these animals elevated plasma pralidoxime content in a dose-dependent manner within 10 min of intoxication. Plasma levels after administration of 3.12 mg/kg of 2-PAM were elevated from a control mean of 6.18 micrograms/ml to a maximum of 13.78 micrograms/ml in animals given 557 micrograms/kg of sarin at 2 min after the injection of the therapeutic compounds. This suggests that pathophysiological changes following intoxication by potent inhibitors of cholinesterase result in a decrease in the rate and extent of distribution of therapeutic compounds. This effect is most likely a consequence of changes in cardiovascular functions influencing blood flow to various organs.
...
PMID:Sarin intoxication elevates plasma pralidoxime. 406 Jan 91

Pretreatment of mice with 4-(o-benzylphenoxy)-N-methylbutylamine hydrochloride (bifemelane) protected against effects of anemic hypoxia. Befemelane delayed the loss of the righting reflex (from 17.8 +/- 1.3 to 21.9 +/- 1.2 min, p less than 0.05) and death (from 19.6 +/- 1.3 to 23.3 +/- 1.1, p less than 0.05) in mice with anemic hypoxia (induced with NaNO2). Pretreatment with bifemelane ameliorated the reduction in the synthesis of acetylcholine from labeled precursors in anemic hypoxia. Namely, it reduced the inhibition of acetylcholine synthesis from labeled choline (from 3.8 +/- 0.5 to 9.4 +/- 1.2 pmole/mg protein at 30 mg/kg, p less than 0.01), but not significant at 15 mg/kg. However it (15 mg/kg) caused a significant increase in the incorporation of [U-14C] glucose into acetylcholine compared to the value for hypoxic animals (from 5 +/- 0.5 to 9 +/- 1 dpm/mg protein, p less than 0.001). Under normal conditions, concentrations of acetylcholine and glucose in the brain were significantly increased by the 30 mg/kg of bifemelane, while the synthesis of acetylcholine from choline was significantly decreased. This reduction of synthesis might be caused by the increased acetylcholine concentrations in the brain. Fifteen mg/kg of bifemelane significantly increased the concentrations of glucose, 14C-acid soluble fraction and the synthesis of acetylcholine from [U-14C] glucose. In the in vitro experiments, cholinesterase activity was significantly inhibited by the bifemelane (1.47 microM). However, its inhibitory effects were about 1/9000 of physostigmine sulfate, which might be too weak to increase the acetylcholine concentration in the brain.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:[Effects of 4-(o-benzylphenoxy)-N-methylbutylamine hydrochloride (bifemelane) on the synthesis of acetylcholine in anemic hypoxia]. 409 87

Studies were carried out to assess the prospects of adapting an enzyme administration procedure developed with rat liver gulonolactone oxidase to other enzymes of therapeutic interest. The enzyme is administered intraperitoneally as the glutaraldehyde-reacted immunoprecipitate. A gulonolactone oxidase from a different source, chicken kidney, also shows catalytic capability following administration. This finding suggests that other enzymes modified by this procedure might also act in vivo. Four out of five enzymes tested (asparaginase, serum cholinesterase, rat and chicken gulonolactone oxidases) have significant catalytic activity and relatively minor changes in affinity for substrate after the modification, and only one (histidase) is inactivated by the modification. Analysis of immunoprecipitates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of these enzymes indicates that they consist largely of enzyme and immunoglobulin G. All five of these modified enzymes are not toxic even with repetitive administrations whereas unmodified asparaginase is allergenic to a majority of guinea pigs tested. The modification described is very simple and rapid and is, therefore, a practical means of preparing certain enzymes for therapeutic administration.
...
PMID:Adaptability of an enzyme replacement therapy to other enzymes with potential therapeutic applications. 409 51

Muscle membranes were partially purified from rat leg muscles. Externally oriented membrane functions were used to monitor and characterize the resulting membrane fractions. Na(+)K(+)-stimulated Mg(++)-adenosinetriphosphatase, acetylcholinesterase, and cholinergic receptor activities are present and enriched in the density-gradient subfractions of crude sarcolemma when compared with the first pellet. The physical separation of the cholinesterase and receptor activities on the gradient subfractions is demonstrated. Receptor activity, determined by specific (125)I-labeled alpha-bungarotoxin binding, appears in fractions with densities similar to other plasma membranes (D(4) (20) 1.1015-1.1520). Acetylcholinesterase, on the other hand, is preferentially distributed in lighter density fractions (D(4) (20) 1.0507-1.0780) and parallels the gradient distribution of the ATPase. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a high-molecular-weight glycoprotein sediments with the higher density fractions only. The data suggest a molecular dissection of the layers of the sarcolemma. The receptor is tentatively felt to be an integral component of the junctional plasma membrane. Acetylcholinesterase is felt to be superficially located on the ectolamina of the junctional sarcolemma, and may be woven within the matrix of the intersynaptic basement membrane.
...
PMID:In vitro analysis of the general properties and junctional receptor characteristics of skeletal muscle membranes. Isolation, purification, and partial characterization of sarcolemmal fragments. 427 96


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---