Amphiphilic and hydrophilic forms of acetylcholinesterase from sheep platelets.
Acetylcholinesterase (AChE, EC 3.1.1.7) was extracted from sheep platelets by successive homogenizations, yielding low-salt soluble (LSS), high-salt soluble (HSS) and detergent-soluble (DS) fractions. These accounted, respectively, for about 30%, 7% and 60% of total AChE activity. Applications of hydrophobic chromatography on phenyl-agarose to three solubilized fractions revealed that hydrophilic forms were almost exclusively located in the LSS fraction ( approximately 27% of total AChE), whereas most amphiphilic forms were present in DS extracts ( approximately 59% of total AChE), the remaining forms being distributed among aqueous soluble fractions. Enzyme molecular forms in the solubilized extracts were identified by centrifugation in 5-20% sucrose gradients containing Triton X-100 or Brij 97 to differentiate between hydrophilic or amphiphilic species. A predominance of hydrophilic dimeric forms ( approximately 22%), with small amounts of hydrophilic monomers (5%) and amphiphilic dimers and monomers (3%), was found in soluble AChE (LSS fraction). Amphiphilic AChE forms extracted in the HSS and DS fractions had a single peak in the sedimentation profiles with sedimentation coefficients of about 6S in gradients with Triton X-100; these were slightly shifted in the presence of Brij 97. After treatment with dithiothreitol, this molecular form solubilized in DS was converted to another molecular form with a lower sedimentation coefficient. Our results show that amphiphilic globular dimers are the dominant molecular form in sheep platelet AChE, suggesting a partial conversion of this membrane-bound form into soluble dimers and monomers, mainly with a hydrophilic character, through the action of either endogenous proteases and phospholipases or residual endogenous reducing agents.
Rocío Marcos
M
M
Department of Biochemistry and Molecular Biology, University of Salamanca, Edificio Departamental, Lab. 106, Plaza Drs. de la Reina s/n, 37007 Salamanca, Spain.
Sánchez-Yagüe
J
J
Hernández-Hernández
A
A
Llanillo
M
M
D000110:Acetylcholinesterase / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D000818:Animals; D001792:Blood Platelets / Q000201:enzymology*; D002853:Chromatography, Liquid; D012756:Sheep; D012995:Solubility
D000110:Acetylcholinesterase