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.