Globular Forms Of Acetylcholinesterase Research Articles

Anti-acetylcholinesterase activity of Corallocarpus epigaeus tuber: In vitro kinetics, in silico docking and molecular dynamics analysis

Biological activities of Corallocarpus epigaeus tuber (CET) were determined and its active constituents were identified. CET was sequentially extracted with various solvents. The methanol extract was further fractionated using silica coloumn chromatography to obtain active constituents-rich fraction called CET-MAF. The phytochemicals in CET-MAF was identified using GC-MS analysis. The in vitro antioxidant activity and inhibition against various globular forms of acetylcholinesterase (AChE) by CET-MAF were determined. In silico analysis of CET-MAF active constituents interaction with human AChE was carried out. CET-MAF showed in vitro antioxidant activity. CET-MAF inhibited various globular forms of AChE, G1 (IC50 = 147 µg/ml; Ki=13.6 µg/ml), G2 (IC50=146 µg/ml; Ki=8.6 µg/ml) and G4 (IC50 = 158 µg/ml; Ki = 9.3 µg/ml) in mixed manner. Molecular docking analysis revealed the interaction of active constituents such as 5-acetoxypentadecane (5AP) and alpha-linolenic acid (ALA) of CET-MAF with active site of AChE. Molecular dynamics simulation for 500 ns revealed the stable binding of these compounds with AChE. MMBGSA calculation revealed the binding energy of -52.95 and -55.49 kcal/mol for interaction of ALA and 5AP with AChE, respectively. Altogether, CET-MAF showed biological activities in vitro which can be used against cognitive dysfunction.

Acetylcholinesterase from the invertebrate Ciona intestinalis is capable of assembling into asymmetric forms when co-expressed with vertebrate collagenic tail peptide

To learn more about the evolution of the cholinesterases (ChEs), acetylcholinesterase (AChE) and butyrylcholinesterase in the vertebrates, we investigated the AChE activity of a deuterostome invertebrate, the urochordate Ciona intestinalis, by expressing in vitro a synthetic recombinant cDNA for the enzyme in COS-7 cells. Evidence from kinetics, pharmacology, molecular biology, and molecular modeling confirms that the enzyme is AChE. Sequence analysis and molecular modeling also indicate that the cDNA codes for the AChE(T) subunit, which should be able to produce all three globular forms of AChE: monomers (G(1)), dimers (G(2)), and tetramers (G(4)), and assemble into asymmetric forms in association with the collagenic subunit collagen Q. Using velocity sedimentation on sucrose gradients, we found that all three of the globular forms are either expressed in cells or secreted into the medium. In cell extracts, amphiphilic monomers (G(1)(a)) and non-amphiphilic tetramers (G(4)(na)) are found. Amphiphilic dimers (G(2)(a)) and non-amphiphilic tetramers (G(4)(na)) are secreted into the medium. Co-expression of the catalytic subunit with Rattus norvegicus collagen Q produces the asymmetric A(12) form of the enzyme. Collagenase digestion of the A(12) AChE produces a lytic G(4) form. Notably, only globular forms are present in vivo. This is the first demonstration that an invertebrate AChE is capable of assembling into asymmetric forms. We also performed a phylogenetic analysis of the sequence. We discuss the relevance of our results with respect to the evolution of the ChEs in general, in deuterostome invertebrates, and in chordates including vertebrates.

Choline acetyltransferase, acetylcholinesterase, and nicotinic acetylcholine receptors of human gingival and esophageal epithelia.

A non-neuronal cholinergic system that includes neuronal-like nicotinic acetylcholine receptors (nAChRs) has recently been described in epithelial cells that line the skin and the upper respiratory tract. Since the use of nicotine-containing products is associated with morbidity in the upper digestive tract, and since nicotine may alter cellular functions directly via nAChRs, we sought to identify and characterize a non-neuronal cholinergic system in the gingival and esophageal epithelia. mRNA transcripts for alpha3, alpha5, alpha7, and beta2 nAChR subunits, choline acetyltransferase, and the asymmetric and globular forms of acetylcholinesterase were amplified from gingival keratinocytes (KC) by means of polymerase chain-reactions. These proteins were visualized in the gingival and esophageal epithelia by means of specific antibodies. Variations in distribution and intensity of immunostaining were found, indicating that the repertoire of cholinergic enzymes and receptors expressed by the cells changes during epithelial maturation, and that an upward concentration gradient of free acetylcholine exists. Blocking of the nAChRs with mecamylamine resulted in reversible loss of cell-to-cell adhesion, and shrinking and rounding of cultured gingival KC. Activation of the receptors with acetylcholine or carbachol caused stretching and peripheral ruffling of the cytoplasmic aprons, and formation of new intercellular contacts. These results demonstrate that both the keratinizing epithelium of attached gingiva and the non-keratinizing epithelium lining the upper two-thirds of the esophageal mucosa possess a non-neuronal cholinergic system. The nAChRs expressed by these epithelia are coupled to regulation of cell adhesion and motility, and may provide a target for the deleterious effects of nicotine.

Acetylcholinesterase in tentacles of octopus vulgaris (cephalopoda. histochemical localization and characterization of a specific high salt-soluble and heparin-soluble fraction of globular forms

Transverse sections of Octopus tentacles were stained for acetylcholinesterase (AChE) activity. An intense staining, that was suppressed by preincubation in 10 −5 M eserine, was detected in a number of neuronal cells, nerve fibres and neuromuscular junctions of intrinsic muscles of the arm. Octopus acetylcholinesterase was found as two molecular forms: an amphiphilic dimeric form (G2) sensitive to phosphatidylinositol phospholipase C and a hydrophilic tetrameric (G4) form. Sequential solubilization revealed that a significant portion of both G2 and G4 forms was recovered only in a high salt-soluble fraction (1 M NaCl, no detergent). Heparin (2 mg/ml) was able to solubilize G2 and G4 forms with the same efficiency than 1 M NaCl. The solubilizing effect of heparin was concentration-dependent and was reduced by protamine (2 mg/ml). This suggests that heparin operates through the dissociation of ionic interactions existing in situ between globular forms of AChE and cellular or extracellular polyanionic components. Interaction of AChE molecular forms with heparin has been reported so far in only a few instances and its physiological meaning is uncertain. G2 and G4 forms, interacting or not with heparin, all belong to a single pharmacological class of AChE. This suggests the existence of a single AChE gene. Amphiphilic and hydrophilic subunits thus likely result either from the processing of a single AChE transcript by alternative splicing (as in vertebrate AChE) or from a post-translation modification of a single catalytic peptide.

Agrin-induced specializations contain cytoplasmic, membrane, and extracellular matrix-associated components of the postsynaptic apparatus

The aims of the studies reported here were to determine the extent to which the specializations induced by agrin on cultured chick myotubes resemble the postsynaptic apparatus and examine how these specializations form. We found that agrin induces the formation of specializations at which at least 6 components of the postsynaptic apparatus are concentrated: one cytoplasmic component [a 43 kDa acetylcholine receptor (AChR)-associated protein], 3 membrane components [AChRs and globular forms of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE)], and 2 extracellular matrix-associated proteins (A12 asymmetric AChE and a heparan sulfate proteoglycan). The accumulation of AChE and BuChE into agrin-induced aggregates occurred in the absence of any change in the amount, rate of synthesis, accumulation, and release, or molecular forms of either enzyme. Thus, agrin affects primarily the distribution of these components of the postsynaptic apparatus and not their metabolism. Agrin-induced formation of AChR aggregates was not prevented by inhibition of protein synthesis, consistent with our previous results that agrin-induced accumulation of AChRs occurs by lateral migration. The accumulation of components of the extracellular matrix would seem less likely to occur by lateral migration and so might require release of newly synthesized proteins; indeed, formation of aggregates of heparan sulfate proteoglycan was prevented by inhibitors of protein synthesis. Thus, different components of the postsynaptic apparatus accumulate in agrin-induced specializations by different mechanisms.

Differentiation and distribution of acetylcholinesterase molecular forms in the mouse cochlea.

The activities of the globular and asymmetric forms of acetylcholinesterase (AChE) were measured in the whole cochlea and cochlear turns of the developing postnatal mouse. The globular AChE forms (G4, G2 and G1) were present in each cochlear turn at birth. An asymmetric AChE form (A12) was detected in the midturn on the 4th postnatal day, and in the base and apex on the 7th postnatal day. The activities of all AChE molecular forms increased rapidly during the second postnatal week and reached a plateau by the 19th postnatal day. In the 26-day old mouse, G4 constitutes the largest proportion of total cochlear AChE (57%), G2/G1 being 37% and A12 being 6%. The distribution of the AChE forms among the different turns is as follows: the combined value of the activities of G2 and G1 AChE was the same in each turn; G4 was the major form in the base and midturn; and A12, the least abundant AChE form of all, was localized mainly in the base. Our results indicate that in the cochlea (1) the content of molecular forms is similar to that of other neuronal systems, (2) the expression of AChE molecular forms is developmentally regulated, and (3) the AChE isoenzymes develop and are distributed differentially along the cochlear length; resulting near maturity in the greater proportional expression of G4 and A12 in the base and midturn and G2/G1 in the apex.

Purification and properties of the membrane-bound form of acetylcholinesterase from chicken brain. Evidence for two distinct polypeptide chains.

The major molecular form of acetylcholinesterase (AChE) from chicken brain is a membrane-bound glycoprotein with an apparent sedimentation coefficient of 11.4 S. Analysis of the purified protein by gel filtration, velocity sedimentation, and sodium dodecyl sulfate-gel electrophoresis shows that the solubilized enzyme is a globular tetramer with an apparent Mr = 420,000. This membrane-bound form of AChE is hydrophobic and readily aggregates in the absence of detergent. These aggregates are concentration-dependent, relatively stable in the presence of high salt concentrations, yet readily dissociate upon addition of detergent to the 11.4 S form, indicating that the interactions are hydrophobic. Polyclonal and monoclonal antibodies raised against chicken brain AChE purified by ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis precipitate AChE enzyme activity. However, these antibodies do not cross-react with the enzyme from chicken muscle which preferentially hydrolyses butyrylcholine. Immunoprecipitation of isotopically labeled enzyme molecules from tissue cultured brain cells and analysis by sodium dodecyl sulfate-gel electrophoresis shows that AChE consists of two polypeptide chains with apparent Mr = 105,000 (alpha) and 100,000 (beta) in a 1:1 ratio. Immunoblotting of brain AChE with either the polyclonal or monoclonal antibodies indicates that the alpha and beta chains share antigenic determinants. Furthermore, both polypeptide chains can be labeled with [3H]diisopropyl fluorophosphate, indicating that they each contain a catalytic site. This is the first indication that globular forms of AChE may consist of multiple polypeptide chains.

Collagen-tailed and globular forms of acetylcholinesterase in the developing chick visual system

We have carried out a comparative study of the developmental profiles of the enzyme acetylcholinesterase, and of its collagen-tailed and globular structural forms, solubilized in the presence of 1 M NaCl, 1% (w/v) sodium cholate and 2 mM EDTA, in the chick retina and optic lobes. The overall acetylcholinesterase activities, both per mg protein and per embryo or chick, are substantially higher in tectum than in retina, from embryonic day 16. The A(12) collagen-tailed form of the enzyme is present in similar amounts in the embryonic retina and optic tectum; however, while the A(12) activity increases significantly in retina after birth, both by percentage and in absolute terms, the tectal tailed enzyme follows a declining developmental profile, reaching a minimum after 6 months of life. On the other hand, the globular G(4) species shows developmental profiles, both in retina and tectum, rather similar to those obtained for the overall enzyme activity, while the G(2) and G(1) forms are present in comparable concentrations in both tissues. Besides, G(4) is the predominant globular form in the chick optic lobe after hatching, G(2) and G(1) being enriched in the embryonic tectum. In the case of retina, however, all the globular forms contribute more evenly to the total acetylcholinesterase activity, along the developmental period considered. The potential significance of some of the postnatal developmental profiles is discussed in terms of the progressive adjustment of retina and tectum to the requirements of visual function.

Molecular forms of acetylcholinesterase in developing Torpedo embryos

We have studied the evolution of acetylcholinesterase molecular forms during the embryonic development of Torpedo marmorata, in the electric organs and in the electric lobes of the central nervous system. In the early stages of development (35 mm embryos, ‘myogenic phase’ of electric organ development), globular forms of acetylcholinesterase (G 4 and G 2) are abundant in both tissues and the collagen-tailed form A 12 is already present. In the electric organs, this form accumulates rapidly after the 55–60 mm stage (‘electrogenic phase’), when synapse formation first commences. Although the molecular characteristics of the collagen-tailed forms, and particularly their aggregation properties, do not appear to change during development, their solubilization requires higher concentrations of MgCl 2, as the electrocytes mature, suggesting that they become more tightly integrated in a better organized basal lamina. The smaller collagen-tailed form A 8 shows a transient increase which coincides approximately with the maximal accumulation of A 12, suggesting that it is an intermediate in its synthesis. The accumulation of the hydrophobic G 2, which eventually becomes predominent in the adult electric organs, lags behind that of A 12. The functional significance of this important fraction of acetylcholinesterase is therefore not that of a pool of precursor for the synthesis of A 12. In the electric lobes, the tetrameric form (G 4) is abundant during development, as well as G 2 and G 1 at certain stages, but the A 12 form is predominant in the adult.

Asymmetric and globular forms of acetylcholinesterase in mammals and birds.

We have identified six molecular forms of acetylcholinesterase (AcChoE: acetylcholine hydrolase, EC 3.1.1.7) in extracts from bovine superior cervical ganglia. We show that three of them resemble the collagen-tailed forms of Electrophorus AcChoE in their hydrodynamic parameters, low-salt aggregation properties, and collagenase sensitivity. The six molecular forms of bovine AcChoE appear structurally homologous to the six forms of electric fish AcChoE that have previously been characterized. They include globular molecules (monomers, dimers, and tetramers) and asymmetric aggregating molecules that possess a collagen-like tail associated with one, two, and three tetramers. We propose to call the globular forms G1, G2, and G4 and the asymmetric forms A4, A8, and A12, the subscripts indicating the number of catalytic subunits. In spite of quantitative differences in their molecular parameters, the AcChoE forms from rat and chicken are clearly homologous to those of bovine AcChoE. Thus the nomenclature we introduce is very probably valid for the main AcChoE molecular forms, at least in vertebrates, and should help to clarify structural relationships and homologies among them. This model, however, does not claim to represent entirely the complex polymorphism of AcChoE, because more or less hydrophobic variants of the G forms have been observed, and because other molecular associations cannot be excluded. We discuss the significance of the globular and collagen-tailed structure for the molecular localization of AcChoE.