Solubilization of 20S acetylcholinesterase from chick retina

Barat, Ana; Escudero, Elena; Gómez-Barriocanal, Javier; Ramı́rez, Galo

doi:10.1016/0006-291x(80)90109-6

Cited by 14 publications

(5 citation statements)

References 12 publications

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“…It should be noted that these changes in the amount of tailed enzyme solubilized under the different conditions considered were not accompanied by changes in the total amount of enzyme solubilized. This fact and our previous results (Barat et al, 1980b) suggest that when EDTA is not used, or its effect is overcome by concomitant addition of suitable amounts of divalent cations, the 20s tailed form of the enzyme is destroyed during the preparative process [this is especially clear when using detergent, since in this case over 96% of the enzyme in the homogenate is solubilized, whether EDTA is present or not in the homogenization medium (Table 1)]. Furthermore, we have experimentally confirmed that the insoluble pellets remaining after extraction of retinal tissue with buffer-salt-Triton X-100, with or without EDTA (about 2% of the total tissue AChE activity), yielded the same amount (about 0.5%) of tailed AChE upon re-extraction in EDTA-containing buffer-saltdetergent solutions.…”

Section: Role Of Divalent Cationssupporting

confidence: 82%

“…In a first approach, using collagenase digestion and two-step salt-detergent extractions, we were able to show the presence of small amounts (1-2%) of tailed AChE in several regions of the chick central nervous system (Barat et al, 1 9 8 0~; Villafruela et al, 1980). More recently (Barat et al, 1980b), we have reported that the addition of EDTA to the initial homogenization solution results in the solubilization of a sizable amount of A,, AChE from chick retina. In the present paper we examine in detail the requirements for the solubilization of intact collagen-tailed AChE species from chick retina and discuss the potential role of divalent cations (and more specifically of Ca2+) in the attachment of the tailed AI2 AChE to membranes.…”

Section: Gomez-barriocanal Et Almentioning

confidence: 98%

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Solubilization of Collagen‐Tailed Acetylcholinesterase from Chick Retina: Effect of Different Extraction Procedures

Gómez-Barriocanal

Barat

Escudero

et al. 1981

Journal of Neurochemistry

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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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Section: Role Of Divalent Cationssupporting

confidence: 82%

Section: Gomez-barriocanal Et Almentioning

confidence: 98%

Solubilization of Collagen‐Tailed Acetylcholinesterase from Chick Retina: Effect of Different Extraction Procedures

Gómez-Barriocanal

Barat

Escudero

et al. 1981

Journal of Neurochemistry

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“…In chicken brain, Ramirez et al have demonstrated the existence of a small proportion of the A,, form, which could be released by collagenase (Villafruella et al 1980), and shown that this form could be solubilized from the retina with EDTA, but not in the presence of divalent cations (Barat et al, 1981). In the case of bovine SCG and caudate nucleus, EDTA did increase the apparent proportion of A forms, because of a better solubilization or stabilization of these molecules, but its presence was not a necessary condition.…”

Section: Discussionmentioning

confidence: 99%

Molecular Forms of Acetylcholinesterase in Bovine Caudate Nucleus and Superior Cervical Ganglion: Solubility Properties and Hydrophobic Character

Grassi

Vigny

Massoulié

1982

Journal of Neurochemistry

104

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In the present paper, we report an analysis of acetylcholinesterase molecular forms in the bovine caudate nucleus and superior cervical ganglion. We show that: (1) The superior cervical ganglion contains a significant proportion (∼ 15%) of collagen‐tailed forms (mostly A12 and A8), but these molecules are found only as traces (ca. 0.002%) in the caudate nucleus, even in favorable extraction conditions (i.e., in the presence of 1 m‐NaCl, 5 mm‐EDTA, 1% Triton X‐100). (2) The bulk of acetylcholinesterase corresponds to globular forms, mostly the tetrameric G4 and the monomeric G1 forms, with a smaller proportion of the dimeric G2 form. (3) The tetrameric enzyme exists as a minor soluble component (GS4) that does not interact with Triton X‐100, and a major hydrophobic component (GH4) that is partially solubilized in the absence of detergent in the caudate nucleus, but not in the superior cervical ganglion. (4) The monomeric G1 form presents a marked hydrophobic character, as indicated by its interaction with Triton X‐100, although it may be solubilized in large part in the absence of detergent in both tissues. (5) The detergentsolubilized forms aggregate upon removal of detergent. This property disappears after partial purification of G4) that does not interact with Triton X‐100, and a major hydrophobic component (GH4, but is restored upon addition of an inactivated crude extract, indicating that it is attributable to interactions with other hydrophobic components. (6) The proportions of molecular forms solubilized in detergent‐free buffers vary with the ionic composition of the medium. Repeated extractions of caudate nucleus in Tris‐HCl buffer produce a larger overall yield of G1 form (e.g., 40%) than appears in a single quantitative detergent solubilization (<15%). This G1 form apparently derives in part from a pool of GH4 form. (7) However, detergents that allow a quantitative solubilization of acetylcholinesterase yield the same proportions of forms (about 85% G4) independently of the ionic conditions. (8) Modifications of the molecular forms occur spontaneously during purification, or storage of the crude aqueous ex‐tracts, in a manner that depends on the ionic conditions. In Tris‐HCl buffer, G1 is converted into a well‐defined 7.5S form. In Ringer, polydisperse components are formed. The effects observed in Ringer cannot be reproduced by addition of 5 mm‐Ca2‐ to the Tris buffer either during or after extraction. (9) Proteases, such as pronase, convert the hydrophobic forms into molecules that do not appear to interact with Triton X‐100, and do not aggregate in its absence. These results raise fundamental questions regarding the status of acetylcholinesterase in situ, the structure and interactions of its molecular forms. They are discussed with reference to previous publications.

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“…Previous studies have established that both AChE and BChE are found in the tissues as a set of asymmetric (A 12 , A 8 , and A 4 ) and globular forms (G 4 , G 2 , and G 1 ), which are characterized by sedimentation properties and cellular localization (9). The globular AChE forms predominate in the retina, whereas asymmetric and globular AChE molecules coexist in the RPE (10)(11)(12)(13)(14)(15). In addition to their role in cholinergic transmission, cholinesterases may also play a role during morphogenesis and neurodegenerative diseases (12,16,17 ).…”

Section: Introductionmentioning

confidence: 99%

Effect of Streptozotocin‐Induced Diabetes on Activities of Cholinesterases in the Rat Retina

Sánchez‐Chávez

Salceda

2000

IUBMB Life

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The effect of streptozotocin-induced diabetes on cholinesterases activities was studied in the retina and, for comparison, in other nervous and nonnervous tissues. Streptozotocin diabetes did not affect acetylcholinesterase activity in the retina but increased its activity in the cerebral cortex (100%) and in serum (55%), and decreased it by 30-40% in erythrocytes. The butyrylcholinesterase activity was decreased by 30-50% in retina and hippocampus and to a lesser extent in retinal pigment epithelium from rats treated with streptozotocin for one week. Changes observed in cholinesterase activities were not correlated with the fasting blood glucose concentration. The results suggest that diabetes might influence a specific subset of cells and isoforms of cholinesterases. This, in turn, could lead to alterations associated with diabetes complications.

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Solubilization of 20S acetylcholinesterase from chick retina

Cited by 14 publications

References 12 publications

Solubilization of Collagen‐Tailed Acetylcholinesterase from Chick Retina: Effect of Different Extraction Procedures

Solubilization of Collagen‐Tailed Acetylcholinesterase from Chick Retina: Effect of Different Extraction Procedures

Molecular Forms of Acetylcholinesterase in Bovine Caudate Nucleus and Superior Cervical Ganglion: Solubility Properties and Hydrophobic Character

Effect of Streptozotocin‐Induced Diabetes on Activities of Cholinesterases in the Rat Retina

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