Monomerization of tetrameric bovine caudate nucleus acetylcholinesterase. Implications for hydrophobic assembly and membrane anchor attachment site

Heider, Harald; Brodbeck, Urs

doi:10.1042/bj2810279

Cited by 26 publications

(21 citation statements)

References 30 publications

(28 reference statements)

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“…This situation is at variance to that of G4 forms of vertebrate cholinesterases where such dissociation of tetramers is usually not achieved by reducing agents [31, 321 or removal of S-S bonds by limited proteolyis [33,341. The forces holding the subunits together in the G4 form of AChE were recently shown to result from a hydrophobic sequence located near the Cterminus of the catalytic peptide [35]. It is clear that this type of non-covalent hydrophobic interactions is not involved in the stabilization of the quaternary structure in the 14s, 14f and 12s forms.…”

Section: Molecular Forms In Each Classmentioning

confidence: 99%

Acetylcholinesterases of the nematode Steinernema carpocapsae

Arpagaus¹,

Richier²,

Bergé³

et al. 1992

European Journal of Biochemistry

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We analyzed the molecular forms of acetylcholinesterase (AChE) in the nematode Steinernema carpocapsae. Two major AChEs are involved in acetylcholine hydrolysis. The first class of AChE is highly sensitive to eserine = 0.05 pM). The corresponding molecular forms are: an amphiphilic 14s form converted into a hydrophilic 14.5s form by mild proteolysis and two hydrophilic 12s and 7s forms. Reduction of the amphiphilic 14s form with 10 mM dithiothreitol produces hydrophilic 7s and 4s forms, indicating that it is an oligomer of hydrophilic catalytic subunits linked by disulfide bond(s) to a hydrophobic structural element that confers the amphiphilicity to the complex. Sedimentation coefficients suggest that 4S, 7S, 12s forms correspond to hydrophilic monomer, dimer, tetramer and that the 14s form is also a tetramer linked to one structural element. The second class of AChE is less sensitive to eserine (IC50 =0.1 mM). Corresponding molecular forms are hydrophilic and amphiphilic 4s forms (monomers) and a major amphiphilic 7s form converted into a hydrophilic dimer by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C. This amphiphilic 7s form thus possesses a glycolipid anchor. It appears that Steinernemu (a very primitive invertebrate) presents AChEs with two types of membrane association that closely resemble those described for amphiphilic G2 and G4 forms of AChE in more evolved animals.Acetylcholinesterase (AChE) and butyrylcholinesterase are highly polymorphic enzymes in vertebrates. Six different molecular forms can be expressed in a tissue-specific and agedependent manner. Three globular forms (Gl, G2 and G4 forms) correspond to monomers, dimers and tetramers of catalytic subunits, and three asymmetric forms (A forms) are associations of one, two or three tetramers to a collagen 'tail' [I].In invertebrates, only AChE has been described so far and its molecular diversity is more restricted : for example, there is no convincing evidence for the existence of collagen-tailed, asymmetric forms in these organisms. In insects, only G1 and G 2 forms have been characterized. The major molecular form is an amphiphilic dimer (2, 31 linked to the plasma membrane by a glycolipid anchor [4 -61. A glycolipid-anchored AChE has also been characterized in the trematode Schistosoma mansoni [7].

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Section: Molecular Forms In Each Classmentioning

confidence: 99%

Acetylcholinesterases of the nematode Steinernema carpocapsae

Arpagaus¹,

Richier²,

Bergé³

et al. 1992

European Journal of Biochemistry

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“…As shown previously, monomerization and release of the hydrophobic anchor from tetrameric DS-AChE from human proteolysis (Gennari et al, 1987;Heider and Brodbeck, 1992). Also, proteinase K was shown to release mammalian brain tetrameric AChE from a crude membrane fraction of bovine caudate nucleus, presumably by splitting the hydrophobic anchor (Fuentes and Inestrosa, 1992).…”

Section: Discussionmentioning

confidence: 86%

“…As shown previously (Liao et al, 1991 and, human and bovine brain DS-AChE contain N-linked carbohydrates which could be removed by treatment with N-glycosidase F. Solid-phase immunoassays, as well as Western blotting, showed that deglycosylation of these two enzymes did not iffect the reaction of mAb 132-5 with the treated antigens (assayed with the same amount of antigen before and after the treatment). As shown by Heider and Brodbeck (1992), limited digestion with trypsin monomerizes DS-AChE but leaves the catalytic activity predominantly intact. When DS-132-4, 132-5 and 132-6.…”

Section: Cholinesterasesmentioning

confidence: 99%

“…Limited tryptic digestion and proteinase K treatment of DS-AChE of human and bovine brain were performed as described by Heider and Brodbeck (1992) and by Gennari et al (1987), respectively.…”

Section: Enzymic Treatmentmentioning

confidence: 99%

“…Hydrophobic labelling of bovine brain DS-AChE was performed as described by Heider and Brodbeck (1992) using [12sI]TID (Amersham). ['251]TID-labelled DS-AChE contained about 3000 cpdunit, and almost no AChE activity was lost during the radiolabelling procedure.…”

Section: -Wiuoromethyl-3-(rn-[1~i]iodophenyl) Diazirine ([Lzsi]tid) mentioning

confidence: 99%

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Monoclonal antibodies against brain acetycholinesterases which recognize the subunits bearing the hydrophobic anchor

Liao

Mortensen

Nørgaard‐Pedersen

et al. 1993

European Journal of Biochemistry

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Monoclonal antibodies were raised against amphiphilic detergent-soluble (DS) acetylcholinesterase (AChE) from human brain caudate nucleus. Three mAb, 132-4 (IgG,), 132-5 (IgG,) and 132-6 (IgG,), specific for brain DS-AChE were selected and subcloned. These mAb reacted with native as well as heat-denatured and SDS-denatured DS-AChE, indicating that the epitopes to which mAb bound are continuous determinants. The mAb cross-reacted with DS-AChE from bovine and mouse brain and with brain DS-AChE from river trout (Sufmo rrurra f o m f u r i o ) and lake trout (SuZmo rrurru f o m u Zacusrris). No cross-reaction was detected with the following antigens : salt-soluble (SS) AChE from bovine brain, glycophospholipid-anchored AChE from human and bovine erythrocytes, DS-butyrylcholinesterase and SS-butyrylcholinesterase (BtChE) from the brains of human and bovine, DS-BtChE from chicken and BtChE from human serum. Deglycosylation of brain DSAChE with N-glycosidase F did not abolish the binding of mAb to DS-AChE. After reduction of brain DS-AChE by dithiothreitol, the mAb no longer reacted with the antigen, indicating that a disulfide bridge is important for the epitope. Monomerization of brain DS-AChE by trypsin and limited proteinase K treatment also abolished the binding of mAb to DS-ACE. Sucrose-densitygradient centrifugation showed that mAb reacted only with native tetrameric forms, but not with dimeric and monomeric forms. Western blot, after SDSPAGE under non-reducing conditions, showed that mAb reacted with those subunits carrying the hydrophobic anchor (i.e. tetramers, himers and heavy dimers) but not with those devoid of it (light dimers or monomers). Since mAb 132-4,132-5 and 132-6 recognized DS-ACE from fish up to mammalian brain in the evolutionary tree, it is concluded that the epitope to which these mAb bind, is conserved in nature.

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Molecular forms of acetyl- and butyrylcholinesterase in normal and dystrophic mouse brain

Moral‐Naranjo¹,

Cabezas‐Herrera²,

Vidal³

1996

J. Neurosci. Res.

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In searching for possible differences in the composition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) forms in dystrophic brain, the distribution of various enzyme molecules in normal (NB) and dystrophic (DB) 129B6F1/J mouse brain has been investigated. The tissue was sequentially extracted with saline (S1) and with saline‐Triton X‐100 buffers (S2) to release soluble and membrane‐bound cholinesterases. About 15% of the AChE and 35% of the BuChE activities in NB were recovered in S1, and the rest in S2, G4, G2, and G1 AChE and BuChE forms were identified in the soluble fractions obtained from NB and DB. The shift in sedimentation values of the separated AChE and BuChE species in sucrose gradients made with and without detergents revealed the occurrence of hydrophilic (H) and amphiphilic (A) variants of cholinesterases in the extracts. The amphiphilic properties of the several AChE and BuChE molecules were analyzed by Triton X‐114 phase‐partitioning and by phenyl‐agarose chromatography. A12 (1%), G4A (72%), G4H (8%), and G2A + G1A (19%) AChE molecules, and G4A (34%), G4H (19%), and G2A + G1A (47%) BuChE forms, were identified in NB. The G4A AChE and BuChE isoforms differed in their interaction with Triton X‐114 and with a hydrophobic matrix. Neither the extent of cholinesterase solubilization, nor the distribution of individual enzyme forms, was significantly altered in DB. The lack of specific differences in the distribution of AChE and BuChE forms between NB and DB suggests that the biosynthetic pathway leading to the various enzyme forms is altered in muscle but not in dystrophic mouse brain. © 1996 Wiley‐Liss, Inc.

show abstract

Monomerization of tetrameric bovine caudate nucleus acetylcholinesterase. Implications for hydrophobic assembly and membrane anchor attachment site

Cited by 26 publications

References 30 publications

Acetylcholinesterases of the nematode Steinernema carpocapsae

Acetylcholinesterases of the nematode Steinernema carpocapsae

Monoclonal antibodies against brain acetycholinesterases which recognize the subunits bearing the hydrophobic anchor

Molecular forms of acetyl- and butyrylcholinesterase in normal and dystrophic mouse brain

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