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

Molecular forms of acetylcholinesterase (AChE) in fresh electric organ tissue are elongated structures in which a multisubunit head containing the catalytic sites is attached to a fibrous tail. The principal form, 18S AChE, is of MW ca. 1,100,000 and aggregates reversibly at low ionic strength. Trypsin converts it to an 11S globular tetramer devoid of the tail and lacking the capacity to aggregate reversibly in low salt. Amino acid analysis, collagenase and pepsin digestion and immunological techniques were utilized to demonstrate that the fibrous tail of the elongated forms of AChE is a collagen triple helix. The distal portion of the tail contains a region responsible for the capacity for aggregation at low ionic strength. This latter property may be related to the postulated role of the tail in anchoring AChE to the fibrillar matrix of the basal lamina.
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PMID:Electric eel acetylcholinesterase: a multisubunit enzyme containing a collagen tail. 626 36

We have extracted acetylcholinesterase from young chick retinas by homogenization in different solutions combining high salt concentration, ionic and nonionic detergents, and EDTA, looking for an optimum procedure for the solubilization of collagen-tailed, asymmetric structural forms of the enzyme. High salt and EDTA seem to be the only necessary requirements for the solubilization of acetylcholinesterase as the A12 form (20S), and the presence of detergent in the homogenization medium does not significantly improve the yield of tailed enzyme. Extraction in the absence of detergent has the potential advantage of a threefold enrichment of tailed enzyme, because only about one-third of the total retinal acetylcholinesterase activity is solubilized. Divalent cations, especially Ca2+, seem to be involved in the attachment of the tailed enzyme to the retinal membranes, at the tail level. High salt-EDTA-extracted 20S acetylcholinesterase (without detergent) aggregates in the presence of exogenous Ca2+ and becomes "insoluble." However, the aggregated 20S acetylcholinesterase can be completely recovered and brought back into solution by further addition of EDTA. Besides, the aggregation can be prevented by the inclusion of Triton X-100 in the homogenization buffer or by adding the detergent concurrently with Ca2+. It is postulated that the acetylcholinesterase collagenous tail is coated by acidic lipid molecules hydrophobically bound to the tail protein so that Ca2+ ionic bridges would actually link these lipid molecules (and consequently the tail) to the membrane matrix. Removal of the lipid coat (e.g., by Triton X-100) produces tailed acetylcholinesterase molecules that no longer aggregate in the presence of Ca2+ and are fully accessible to collagenase digestion.
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PMID:Solubilization of collagen-tailed acetylcholinesterase from chick retina: effect of different extraction procedures. 627 24

1. Desensitization of acetylcholine (ACh) receptors was studied at the frog neuromuscular junction under voltage clamp.2. ACh was applied directly to junctional receptors by stimulating the motor nerve with trains of impulses. End-plate currents (e.p.c.s) were used to estimate the total number of channel openings by the junctional ACh receptors, and miniature end-plate currents (m.e.p.c.s) were used to measure changes in post-synaptic sensitivity. Under the conditions of these experiments the changes in m.e.p.c. amplitudes were shown to be post-synaptic in origin and thus provided a measure of desensitization.3. When the acetylcholinesterase was inhibited with diisopropylfluorophosphate, neostigmine, or collagenase treatment to prolong the duration of the nerve-released ACh in the synaptic cleft, desensitization developed during repetitive stimulation of 1000 impulses at 5-33 impulses/sec and then recovered after the conditioning trains, with a time constant of about 25 sec.4. When the acetylcholinesterase was active so that the duration of ACh in the synaptic cleft resulting from each nerve impulse was brief (< 300 musec), desensitization developed in response to 300-500 pairs of nerve stimuli if the interval between the impulses of each pair was 25 msec or less. When the interval was 30 msec or greater, however, measurable desensitization did not occur, even if the total number of channel openings was many times greater than in the experiments with shorter intervals or inhibited esterase where desensitization readily occurred.5. The desensitization observed to pairs of impulses was enhanced by chlorpromazine and decreased when the post-synaptic membrane was depolarized, properties similar to those described previously for desensitization to bath and ionophoretic application of ACh.6. These results indicate that desensitization to nerve-released transmitter is not a simple consequence of receptor activation, is not due to blockade of the open receptor channels by ACh, and does not result from ACh binding directly to desensitized receptors with a resulting shift in the receptor population towards the desensitized state.7. We suggest that the desensitization observed to nerve-released transmitter is a two-step process with both steps initiated by ACh. In the first step ACh converts some receptors into a desensitizable state which has an apparent lifetime of less than 30 msec; in the second step ACh desensitizes the desensitizable state.
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PMID:A study of desensitization of acetylcholine receptors using nerve-released transmitter in the frog. 627 65

The effects of beta-endorphin (lipotropin 61-91) and related naturally-occurring peptides upon acetylcholinesterase activity in rat hind-limb muscles was investigated. beta-endorphin weakly inhibited the activity in a plasma membrane-enriched fraction. The inhibition by beta-endorphin of the membrane-associated acetylcholinesterase was less marked when the fractions were prepared from muscles which had been denervated 4-6 days previously. The membrane-associated acetylcholinesterase was solubilised from normal muscle preparations and separated by sucrose density gradient centrifugation into three major peaks (16S, 10S and 4S). beta-Endorphin inhibited the activity in the 16S peak but not that in the 10S and 4S peaks, whilst tensilon, a competitive inhibitor of acetylcholinesterase, inhibited the activity of all three peaks. beta-Endorphin inhibited the activity in the 16S peak but not that in the 10S and 4S peaks, whilst tensilon, a competitive inhibitor of acetylcholinesterase, inhibited the activity of all three peaks. beta-Endorphin inhibited the 16S activity in a concentration-dependent manner and its action was partly prevented if naloxone was added simultaneously. Purified natural porcine and bovine beta-endorphin were equipotent in terms of effective concentration range but the maximum inhibition was greater with the bovine peptide. beta-Lipotropin was approximately 4 times less potent than beta-endorphin, whilst C-fragment (lipotropin 61-87) was 100 times less potent. Prolonged treatment with collagenase did not reduce the catalytic activity of 16S acetylcholinesterase, but it was no longer susceptible to the inhibitory action of beta-endorphin. Kinetic studies indicated a complex type of inhibition by beta-endorphin (hyperbolic Lineweaver-Burke plot). Methionine enkephalin inhibited acetylcholinesterase in a weakly non-competitive manner and its action was not abolished if the enzyme was predigested with collagenase. beta-Endorphin produces a novel form of inhibition of acetylcholinesterase, acting only on the 16S (A12 or 'motor endplate-specific') form of the enzyme. The findings are discussed in the light of evidence that beta-endorphin-related immunoreactivity is expressed in motor nerve axons in the immature rat.
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PMID:Selective inhibition of 'motor endplate-specific' acetylcholinesterase by beta-endorphin and related peptides. 628 23

Myotubes of a mouse muscle-cell line (C2) synthesize in culture a 16S form of acetylcholinesterase that is normally found only in regions of adult mouse muscle that contain endplates. The 16S enzyme in C2 cell extracts has the properties expected of acetylcholinesterase forms that have a collagen-like tail. In intact cells, the active site of the 16S acetylcholinesterase is protected by a membrane-impermeable inhibitor, and this form of the enzyme can be removed by treatment of the cells with collagenase. Thus the enzyme is extracellular. Its extraction by high ionic strength solutions lacking detergent suggests that the 16S form is associated with the extracellular matrix by ionic interactions. Histochemical staining shows focal patches of acetylcholinesterase activity on the cell surface. Collagenase treatment, which removes only the 16S form, abolishes this staining pattern, indicating that the patches consist of the 16S enzyme. We conclude that the 16S enzyme in C2 myotubes occurs in focal patches on the cell surface, where it is associated with the extracellular matrix.
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PMID:Association of the synaptic form of acetylcholinesterase with extracellular matrix in cultured mouse muscle cells. 628 45

The receptor for alpha-latrotoxin, the major protein component of the black widow spider venom, was investigated by the use of the purified toxin and of polyclonal, monospecific anti-alpha-latrotoxin antibodies. Experiments on rat brain synaptosomes (where the existence of alpha-latrotoxin receptors was known from previous studies) demonstrated that the toxin-receptor complex is made stable by glutaraldehyde fixation. At saturation, each such complex was found to bind on the average five antitoxin antibody molecules. In frog cutaneous pectoris muscles, the existence of a finite number of high-affinity receptors was revealed by binding experiments with 125I-alpha-latrotoxin (Kd = 5 X 10(-10) M; bmax = 1.36 +/- 0.16 [SE] X 10(9) sites/mg tissue, dry weight). Nonpermeabilized muscles were first treated with alpha-latrotoxin, and then washed, fixed, dissociated into individual fibers, and treated with anti-alpha-latrotoxin antibodies and finally with rhodamine-conjugated sheep anti-rabbit antibodies. In these preparations, muscle fibers and unmyelinated preterminal nerve branches were consistently negative, whereas bright specific fluorescent images, indicative of concentrated alpha-latrotoxin binding sites, appeared in the junctional region. These images closely correspond in size, shape, and localization to endplates decorated by the acetylcholinesterase reaction. The presynaptic localization of the specific fluorescence found at frog neuromuscular junctions is supported by two sets of findings: (a) fluorescent endplate images were not seen in muscles that had been denervated; and (b) the distribution of fluorescence in many fibers treated with alpha-latrotoxin at room temperature was the one expected from swollen terminal branches. Swelling of terminals is a known morphological change induced by alpha-latrotoxin in this preparation. When muscles were treated with either proteolytic enzymes (trypsin, collagenase) or detergents (Triton X-100) before exposure to alpha-latrotoxin, the specific fluorescent endplate images failed to appear. Taken together these findings indicate that the alpha-latrotoxin receptor is an externally exposed protein highly concentrated in the nerve terminal plasma membrane. Its density (number per unit area) at the frog neuromuscular junction can be calculated to be approximately 2,400/micron2.
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PMID:Specific localization of the alpha-latrotoxin receptor in the nerve terminal plasma membrane. 633 Jan 24

Electrophorus electricus acetylcholinesterase is a large polymorphic enzyme. Its native forms 18 S, 14 S and 8.5 S possess a tail having a collagen-like structure. It was suggested that this tail is involved in the anchorage of the enzyme at the terminal of the synapse. Watkins et al. [1] showed that all forms of the enzyme having a collagen segment also bind to sphingomyelin liposomes with almost no binding to phosphatidylcholine (PC) liposomes. In agreement with the above results, the binding of acetylcholinesterase reported here was independent of the following liposomal parameters (a) curvature, (b) the physical state of the bilayer, (c) the gel to liquid crystalline phase transition of sphingomyelin, (d) stereospecificity of the sphingomyelin, (e) acyl chain of the sphingomyelin. The binding was reduced with increasing PC content in sphingomyelin vesicles. The binding has no effect on the bilayer integrity. The enzymatic activity can be released from the vesicles by incubation with collagenase. The association of the enzyme with the liposomes had minimal effect on its kinetic parameters (Km, Vmax). The only detectable effect was increasing enzyme stability at low enzyme concentration. This suggested that the binding of the enzyme to sphingomyelin liposomes reduced its surface denaturation. Such association was not unique to acetylcholinesterase since collagen showed similar behavior. Collagen binding to sphingomyelin liposomes was 5-10-times larger than to PC liposomes. The exact details of the interaction of collagen and collagen-like peptides with sphingomyelin bilayers are yet unknown although it differs from the well documented hydrophobic or electrostatic interactions [7]. This work proposes hydrogen bonding as a third mechanism which involves the interface region of sphingolipids molecules and the collagen or collagen-like tail of acetylcholinesterase. This binding is also of interest due to its correlation to the accumulation of sphingomyelin and collagen during aging and the development of atherosclerosis in blood vessels of mammals.
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PMID:Characterization of the association of Electrophorus electricus acetylcholinesterase with sphingomyelin liposomes. Relevance to collagen-sphingomyelin interactions. 649 89

The characterization of individual acetylcholinesterase (AChE) molecular form subcellular pools in adult mammalian skeletal muscle is a critical point when considering such questions as the origin, assembly, and neurotrophic regulation of these molecules. By correlating the results of differential extraction, in vitro collagenase digestion, and in situ pharmacologic probes of AChE molecular forms in endplate regions of adult rat anterior gracilis muscle, we have shown that: 1) 4.0S (G1) and 6.0S (G2) AChE are predominantly membrane-bound and intracellular; if an extracellular and/or soluble fraction of these forms exists, it cannot be adequately resolved by our methods; 2) 9-11S (globular) AChE activity is distributed between internal and external pools, as well as membrane-associated and soluble fractions; 3) 16.0S (A12) AChE is not an integral membrane protein and exists both intracellularly (25-30%) and extracellularly (70-75%).
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PMID:Subcellular localization of acetylcholinesterase molecular forms in endplate regions of adult mammalian skeletal muscle. 650 36

Enzymatic hydrolysis of acetylcholine (ACh) was determined in intact frog sartorius muscles or their homogenates. The Vmax was 29 nmol min-1 in intact muscles and 46 nmol min-1 per muscle in homogenates, and the Km was 6 and 0.2 mM, respectively. The muscle was divided into small segments, which were homogenized; the junctional cholinesterase (ChE) accounted for 60% of total enzyme activity. At low substrate concentrations the rate of hydrolysis was up to 30 times higher in homogenates than in intact muscles. This difference was greatly reduced at very high substrate concentrations. It appears that most of the ChE in intact muscle is 'occluded' to external ACh, mainly because the ChE at the edges of the synaptic cleft prevents the ACh from reaching the enzyme situated further inwards, which consequently does not contribute to its hydrolysis; homogenization makes all synaptic ChE accessible to added ACh. Incubation of sartorius muscles with collagenase caused an 80% decrease in ChE activity (determined in homogenates) of end-plate-containing parts which became similar to that in end-plate-free parts on which collagenase had little effect. Histochemistry showed that the tendon-muscle junction contained folds which were stained intensively for ChE. Diethyldimethylpyrophosphonate , neostigmine, eserine, and di-isopropyl fluorophosphonate inhibited ChE activity in this order of potency. The I50 values (i.e. the concentrations of the drugs which caused a 50% inhibition) were about 5 times higher in intact than in homogenized tissue. Neostigmine, 0.15 and 0.4 microM, increased the time constant of miniature end-plate currents 1.3- and 1.8-fold, and slowed down ChE activity of muscle homogenates by 1.4 and 2.1 times, respectively, without significantly affecting ACh hydrolysis by intact muscles. This indicates that synaptic ChE is not present in large excess. It is concluded that ChE activity measured in homogenates presents a better picture of in situ ChE activity than that measured in whole muscles especially for evaluating the effect of ChE inhibitors. A mathematical model for ChE-hindered diffusion of ACh is presented in an Appendix.
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PMID:Acetylcholinesterase activity in intact and homogenized skeletal muscle of the frog. 661 Jul 44

The cholinesterase activity of Xenopus laevis oocytes was assessed using [3H]acetylcholine in a simple radiometric procedure. The cholinesterase activity of mature (stage V-Vl) oocytes was very sensitive to inhibition by the specific acetylcholinesterase inhibitor, BW284-C5l, and relatively insensitive to an inhibitor of non-specific, or butyrylcholinesterase. The Km and Vmax of the acetylcholinesterase measured in homogenates of oocytes were 312 microM and 4.6 nmol-oocyte 1-h 1, respectively. Triton X-100 increased the enzyme activity of homogenates four- to five-fold while collagenase treatment displaced into the medium none of the acetylcholinesterase activity from either homogenates or intact oocytes. Cations were found generally to diminish the acetylcholinesterase activity of oocyte homogenates, and lanthanum ions inhibited acetylcholine hydrolysis with an IC50 of 0.63 mM. Subcellular fractionation of oocytes revealed that the bulk of enzyme activity was associated with particulate fractions. Acetylcholinesterase activity was also detected on the surface, and in homogenates, of immature oocytes. Peak enzyme activity resided in stage IV oocytes. Eggs obtained from females induced to spawn were found to have acetylcholinesterase activity in homogenates but little or no hydrolytic activity was detected on the egg surface. These results provide a point of departure for further investigations of the functional significance of this enzyme in Xenopus oocytes.
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PMID:Acetylcholinesterase activity of Xenopus laevis oocytes. 666 98


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