Solubilization of 20S acetylcholinesterase from the chick central nervous system

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

doi:10.1016/0304-3940(80)90147-0

Cited by 14 publications

(3 citation statements)

References 7 publications

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“…The improved ex traction of the quail forms with the MgC me dium and earlier reports of improved extrac tion by cholate containing medium by Ramir ez and colleagues [1,33] illustrate the impor tance of the techniques used to solubilize and separate AChE forms.…”

Section: Discussionmentioning

confidence: 99%

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Regulation of Acetylcholinesterase Forms in Quail and Chicken Muscle Cultures

Bulger

Randall²,

Nieberg³

et al. 1982

Dev Neurosci

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High molecular forms of acetylcholinesterase (AChE) have been difficult to obtain in primary cultures of muscle from mammals and birds. This research used pectoral muscle cultures from Japanese quail and chick embryos grown in medium containing embryo extract and horse serum to study the growth and extraction conditions necessary for study of high molecular weight 20S AChE forms found in these birds. The results confirmed the presence of a 20S AChE form in quail muscle cultures and showed that its extraction from the cells was considerably improved by using a Mg-cholate extraction buffer rather than the more commonly used NaCl-Triton X buffer. A striking finding was that removal of serum from the medium for 1–2 days caused the preferential increase in a 20S form in the quail and the resolution of one from background activity in chick muscle cultures. Removal of embryo extract had no effect on levels of the AChE forms. The results suggest that serum factors inhibit the formation of the high molecular weight, motor end plate associated form of AChE in aneural cultures of avian muscle, and that effects of factors such as neural extracts and nerves on AChE forms in cultured muscle should be examined using a defined basal media.

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Section: Discussionmentioning

confidence: 99%

“…It solubilized virtually 100% of the AChE ac tivity from homogenates of chicken muscle cultures and more than 85% of the AChE ac tivity of quail muscle culture homogenates. NaT buffer, commonly used for this purpose [1,26,27,33], was less effective in solubiliz ing AChE. …”

Section: Extraction O F Ac He From Culturesmentioning

confidence: 99%

Regulation of Acetylcholinesterase Forms in Quail and Chicken Muscle Cultures

Bulger

Randall²,

Nieberg³

et al. 1982

Dev Neurosci

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“…These lipidic membranes may be either extrajunctional plasma membranes or, more probably, sarcoplasmic reticulum membranes, as recently suggested by electron microscopy. This lipid membrane-associated 16S AcChoEase is probably physicochemically homologous to the chick retina (20S) asymmetric AcChoEase, which has been proposed to be anchored into the cell plasma membrane (25). In contrast, BtChoEase is nearly absent from ECM material.…”

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confidence: 95%

Acetylcholinesterase of mammalian neuromuscular junctions: presence of tailed asymmetric acetylcholinesterase in synaptic basal lamina and sarcolemma.

Dreyfus¹,

Rieger²,

Pinçon‐Raymond³

1983

Proc. Natl. Acad. Sci. U.S.A.

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A sarcolemma-rich fraction can be isolated after subeellular fractionation of mouse intercostal muscles by sedimentation on a discontinuous sucrose gradient. The quantitative recovery of the acetylcholine receptor in this fraction is about 50%, which indicates the presence of a high proportion of postsynaptic membranes. Acetylcholinesterase (AcChoEase; EC 3.1.1.7) is found mainly in three different layers: the top layer, which contains soluble AcChoEase, the intermediate layer (fraction A), and the last, AcChoR-rich, layer (fraction C). The relative proportions of the molecular forms of AcChoEase are different in the three layers.The "16S" AcChoEase is in a higher proportion in both types of membrane fractions (A and C) compared to soluble AcChoEase. Both total AcChoEase and 16S AcChoEase are enriched in the A and C fractions. In the C fraction, the sequential use of homogenizations in the presence of detergent and high ionic strength allows the "solubilization" of two distinct AcChoEase pools. One is detergent-soluble and mainly composed of slow-sedimenting forms; the other one is detergent-insoluble, high-ionic strengthsoluble, and composed mainly of collagen-like, tailed, asymmetric (16S) AcChoEase. Thus, most of the asymmetric AcChoEase is specifically localized in the synaptic extracellular matrix of the mammalian muscle fiber. However, in the A fraction, most of the 16S AcChoEase found is solubilized by detergent alone, suggesting an association with microsomal membranes. It may mean that at least some of the basal lamina-embedded 16S AcChoEase is preassembled intracellularly in the sarcoplasmic reticulum.The 16S form of acetylcholinesterase (AcChoEase; EC 3.1.1.7) is highly concentrated in the regions of muscle containing endplates (1) and is absent or barely detectable after denervation (1, 2). Membrane fractionation experiments have been widely used to determine the cellular loci of the AcChoEase accumulation and localization. For example, muscle microsomal or sarcoplasmic reticulum, and also sarcolemmal fractions, have been prepared and analyzed for their enrichment in AcChoEase activity or content of various molecular forms. In particular, the sarcolemmal fraction isolated by McLaughlin et al.(3) was enriched for both 16S AcChoEase and extracellular matrix components (basal lamina). There is indirect evidence, essentially from studies on electric fish AcChoEase, that the asymmetric forms of AcChoEase, which are composed of tetrameric protomers and a collagen-like multistranded tail (4-8), can interact with basal lamina components such as glycosaminoglycans of unknown nature (9), fibrous material (10), or fibronectin (11). In mammalian skeletal muscle, 16S AcChoEase has been found to be collagenase sensitive (12) and thus is probably homologous to the electric fish tailed enzyme. In view of its privileged localization in the region rich in motor endplates and its possible crucial role in cholinergic neurotransmission, it is of prime importance to determine the cellular structures with which it...

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