Amphiphilic and hydrophilic forms of acetylcholinesterase from sheep platelets

Citation
Mr. Marcos et al., Amphiphilic and hydrophilic forms of acetylcholinesterase from sheep platelets, BBA-BIOMEMB, 1415(1), 1998, pp. 163-173
Citations number
42
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES
ISSN journal
00052736 → ACNP
Volume
1415
Issue
1
Year of publication
1998
Pages
163 - 173
Database
ISI
SICI code
0005-2736(199812)1415:1<163:AAHFOA>2.0.ZU;2-J
Abstract
Acetylcholinesterase (AChE, EC 3.1.1.7) was extracted from sheep platelets by successive homogenizations, yielding low-salt soluble (LSS), high-salt s oluble (HSS) and detergent-soluble (DS) fractions. These accounted, respect ively, for about 30%, 7% and 60% of total AChE activity. Applications of hy drophobic chromatography on phenyl-agarose to three solubilized fractions r evealed that hydrophilic forms were almost exclusively located in the LSS f raction (approximate to 27% of total AChE), whereas most amphiphilic forms were present in DS extracts (approximate to 59% of total AChE), the remaini ng forms being distributed among aqueous soluble fractions. Enzyme molecula r forms in the solubilized extracts were identified by centrifugation in 5- 20% sucrose gradients containing Triton X-100 or Brij 97 to differentiate b etween hydrophilic or amphiphilic species, A predominance of hydrophilic di meric forms (approximate to 22%), with small amounts of hydrophilic monomer s (5%) and amphiphilic dimers and monomers (3%), was found in soluble AChE (LSS fraction). Amphiphilic AChE forms extracted in the HSS and DS fraction s had a single peak in the sedimentation profiles with sedimentation coeffi cients of about 6S in gradients with Triton X-100; these were slightly shif ted in the presence of Brij 97. After treatment with dithiothreitol, this m olecular form solubilized in DS was converted to another molecular form wit h a lower sedimentation coefficient. Our results show that amphiphilic glob ular dimers are the dominant molecular form in sheep Platelet AChE, suggest ing a partial conversion of this membrane-bound form into soluble dimers an d monomers, mainly with a hydrophilic character, through the action of eith er endogenous proteases and phospholipases or residual endogenous reducing agents. (C) 1998 Elsevier Science B.V. All rights reserved.