A membrane-associated dimer of acetylcholinesterase from Xenopus skeletal muscle is solubilized by phosphatidylinositol-specific phospholipase C

Inestrosa, Nibaldo C.; Fuentes, María-Elena; Anglister, Lili; Futerman, Anthony H.; Silman, Israel

doi:10.1016/0304-3940(88)90809-9

Cited by 30 publications

(10 citation statements)

References 16 publications

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“…Similar results on the release of the dimeric G2 form of acetylcholinesterase by PtdIns-specific phospholipase C have been shown before in several tissues, including erythrocytes [8], Torpedo electric organ [9] and Xenopus skeletal muscle [29]. The present report shows for the first time that the liver G2 acetylcholinesterase can be solubilized from an intact eucaryotic cell in culture by treatment with PtdIns-specific phospholipase C. This result indicates that the dimeric form of acetylcholinesterase is located at the plasma membrane, facing the extracellular medium.…”

Section: Discussionsupporting

confidence: 64%

Different membrane‐bound forms of acetylcholinesterase are present at the cell surface of hepatocytes

Perelman

Brandan

1989

European Journal of Biochemistry

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In the present study we have determinated the acetylcholinesterase molecular forms present in rat liver hepatocytes; we have also studied the association of acetylcholinesterase with the cell surface of the hepatocytes. Subcellular fractionation indicated that rough endoplasmic reticulum and plasma-membrane-enriched fractions contains G4 and G1 acetylcholinesterase forms bound to membranes. Hepatocytes incubated with phosphatidylinositol-specific phospholipase C released about 70% of the surface acetylcholinesterase. Sedimentation analysis showed that all the solubilized acetylcholinesterase activity comes exclusively from a G2 dimer. The G4 hydrophobic form of acetylcholinesterase accounts for the additional cell-surface activity. The existence of these two forms of acetylcholinesterase on the surface of hepatocytes was further established by analyzing the phosphatidylinositol-specific phospholipase C sensitivity of the acetylcholinesterase molecular forms present in isolated rat liver plasma membranes.Acetylcholinesterase is the enzyme responsible of the hydrolysis of the neurotransmitter acetylcholine [l, 21. The enzyme exists in multiple molecular forms which can be separated by velocity sedimentation. The acetylcholinesterase molecular forms can be classified as globular (monomers GI, dimers G1 and tetramers G4) and asymmetric forms (containing one, two or three tetramers plus a collagen-like tail: A4, As and AI2) [I -41. Asymmetric forms of acetylcholinesterase are associated with the extracellular matrix by heparan sulfate proteoglycans [5,6]. Globular acetylcholinesterases have been identified as both soluble and membrane-bound forms and some of them appear to have hydrophobic domains that attach the enzyme to the plasma membrane [3, 71. The G2 forms of acetylcholinesterase are apparently anchored to the membrane through a glycolipid moiety which contains phosphatidylinositol (PtdIns) covalently linked to the C-terminal [8 -1 I]; the G4 acetylcholinesterase form contains a non-catalytic hydrophobic 20-kDa polypeptide subunit [12]. Futerman et al. [I31 have communicated that a G2 dimer of acetylcholinesterase was present in rat liver homogenates. Here we show that isolated hepatocytes and plasma membrane fractions contain both GZ and G4 forms of acetylcholinesterase. In particular, the dimer was mainly localized in the cell surface from where it can be entirely solubilized by treatment with PtdIns-specific phospholipase C.Correspondence to E. Brandan, Molecular Neurobiology Unit,

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Section: Discussionsupporting

confidence: 64%

Different membrane‐bound forms of acetylcholinesterase are present at the cell surface of hepatocytes

Perelman

Brandan

1989

European Journal of Biochemistry

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“…While AChE T is found in all classes of vertebrates, AChE H exists in cartilaginous fish ( Torpedo spp.) [33], perhaps amphibians ( Xenopus laevis ) [34], and mammals [35], but has not been reported in jawless or bony fish, reptiles, or birds, raising questions about the evolution of this splice variant [26].…”

Section: Introductionmentioning

confidence: 99%

“…R and H variants of BChE have not been reported. However, according to the Xenopus tropicalis genome project [38] and other evidence [34], [39]–[42], an H variant of BChE appears to be present in amphibian Xenopus species. The atypical BChEs of T. marmorata and A. dussumieri are T variants (BChE T ), assembling a collection of globular and asymmetric forms [17], [20].…”

Section: Introductionmentioning

confidence: 99%

Evolution of Acetylcholinesterase and Butyrylcholinesterase in the Vertebrates: An Atypical Butyrylcholinesterase from the Medaka Oryzias latipes

Pezzementi

Nachon²,

Chatonnet

2011

PLoS ONE

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Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are thought to be the result of a gene duplication event early in vertebrate evolution. To learn more about the evolution of these enzymes, we expressed in vitro, characterized, and modeled a recombinant cholinesterase (ChE) from a teleost, the medaka Oryzias latipes. In addition to AChE, O. latipes has a ChE that is different from either vertebrate AChE or BChE, which we are classifying as an atypical BChE, and which may resemble a transitional form between the two. Of the fourteen aromatic amino acids in the catalytic gorge of vertebrate AChE, ten are conserved in the atypical BChE of O. latipes; by contrast, only eight are conserved in vertebrate BChE. Notably, the atypical BChE has one phenylalanine in its acyl pocket, while AChE has two and BChE none. These substitutions could account for the intermediate nature of this atypical BChE. Molecular modeling supports this proposal. The atypical BChE hydrolyzes acetylthiocholine (ATCh) and propionylthiocholine (PTCh) preferentially but butyrylthiocholine (BTCh) to a considerable extent, which is different from the substrate specificity of AChE or BChE. The enzyme shows substrate inhibition with the two smaller substrates but not with the larger substrate BTCh. In comparison, AChE exhibits substrate inhibition, while BChE does not, but may instead show substrate activation. The atypical BChE from O. latipes also shows a mixed pattern of inhibition. It is effectively inhibited by physostigmine, typical of all ChEs. However, although the atypical BChE is efficiently inhibited by the BChE-specific inhibitor ethopropazine, it is not by another BChE inhibitor, iso-OMPA, nor by the AChE-specific inhibitor BW284c51. The atypical BChE is found as a glycophosphatidylinositol-anchored (GPI-anchored) amphiphilic dimer (G2 a), which is unusual for any BChE. We classify the enzyme as an atypical BChE and discuss its implications for the evolution of AChE and BChE and for ecotoxicology.

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“…The end plate strips were maintained in ice-cold saline solution (0.9% NaCl). The tissues were then homogenized for 5 min using a glass-to-glass homogenizer at full speed, in 1:10 (w/v) detergent buffer: 10 mM Tris-HCl, pH 7.4, 1% Triton X-100, and protease inhibitors (1 mM N-ethylmaleimide, 1 mM benzamidine, 0.1 mg/ml bacitracin, 100 mM caproic acid, 5 mM EDTA) (22). The homogenates were centrifuged at 15,000 rpm for 25 min in a fixed rotor using a Kubota KR-20000T centrifuge.…”

Section: Methodsmentioning

confidence: 99%

A Major Portion of Synaptic Basal Lamina Acetylcholinesterase Is Detached by High Salt- and Heparin-containing Buffers from Rat Diaphragm Muscle and Torpedo Electric Organ

Casanueva

García‐Huidobro

Campos

et al. 1998

Journal of Biological Chemistry

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Collagen-tailed asymmetric acetylcholinesterase (AChE) forms are believed to be anchored to the synaptic basal lamina via electrostatic interactions involving proteoglycans. However, it was recently found that in avian and rat muscles, high ionic strength or polyanionic buffers could not detach AChE from cell-surface clusters and that these buffers solubilized intracellular non-junctional asymmetric AChE rather than synaptic forms of the enzyme. In the present study, asymmetric AChE forms were specifically solubilized by ionic buffers from synaptic basal lamina-enriched fractions, largely devoid of intracellular material, obtained from the electric organ of Torpedo californica and the end plate regions of rat diaphragm muscle. Furthermore, foci of AChE activity were seen to diminish in size, number, and staining intensity when the rat synaptic basal lamina-enriched preparations were treated with the extraction buffers. In the case of Torpedo, almost all the AChE activity was removed from the pure basal lamina sheets. We therefore conclude that a major portion of extracellular collagen-tailed AChE is extractable from rat and Torpedo synaptic basal lamina by high ionic strength and heparin buffers, although some non-extractable AChE activity remains associated with the junctional regions.The enzyme acetylcholinesterase (AChE) 1 plays a key role in cholinergic neurotransmission (1). Its predominant form at the neuromuscular junction is the collagen-tailed asymmetric form, A 12 , which is located on the extracellular surface positioned for the hydrolysis of acetylcholine. Most of this junctional AChE is associated with the basal lamina (BL), located between the nerve ending and the muscle plasma membrane (2, 3), and can be removed from the cell surface of muscle tissue (4, 5) and mouse myotubes (6) by treatment with collagenase, indicating that the collagenic tail of the enzyme is involved in its anchorage to the BL (7,8). Although the precise mechanisms by which asymmetric AChE forms are anchored to the BL remain elusive (9), there is compelling evidence to suggest that heparan sulfate proteoglycans (HSPGs) or related proteoglycans are involved (7,10). This evidence includes the recent finding that A 12 has two heparin-binding consensus sequences in its collagenic tail (11). Asymmetric AChE forms have a high binding affinity for BL components, particularly HSPGs (12) which are themselves major constituents of basement membranes (13,14). Heparin and heparan sulfate have also been shown to release asymmetric AChE activity from rat muscle end plate regions (15) and BL sheets purified from the electric organ of Discopyge (16). The demonstration that A 12 could bind and be selectively eluted from heparin-agarose columns, whereas non-collagenous forms and A 12 after collagenase treatment could not, proved the direct interaction of A 12 with heparin in vitro (11,17). Direct interactions with heparin/heparan sulfate moieties in vivo have also been demonstrated. Asymmetric AChE forms were shown to bind the surface of HSPGri...

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A membrane-associated dimer of acetylcholinesterase from Xenopus skeletal muscle is solubilized by phosphatidylinositol-specific phospholipase C

Cited by 30 publications

References 16 publications

Different membrane‐bound forms of acetylcholinesterase are present at the cell surface of hepatocytes

Different membrane‐bound forms of acetylcholinesterase are present at the cell surface of hepatocytes

Evolution of Acetylcholinesterase and Butyrylcholinesterase in the Vertebrates: An Atypical Butyrylcholinesterase from the Medaka Oryzias latipes

A Major Portion of Synaptic Basal Lamina Acetylcholinesterase Is Detached by High Salt- and Heparin-containing Buffers from Rat Diaphragm Muscle and Torpedo Electric Organ

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