Properties of bovine erythrocyte acetylcholinesterase solubilized by phosphatidylinositol-specific phospholipase C1.
The properties of acetylcholinesterase solubilized from bovine erythrocyte membrane by phosphatidylinositol (PI)-specific phospholipase C of Bacillus thuringiensis or with a detergent, Lubrol-PX, were studied. The activity of Lubrol-PX-solubilized acetylcholinesterase was broadly distributed in the fractions having Ve/Vo = 1.0-2.0 in gel filtration on a Sepharose 6B column. The intermediary fractions (Ve/Vo = 1.3-1.7) were collected as "the middle active Sepharose 6B eluate" and characterized on the basis of enzymology and protein chemistry. When this eluate was treated with PI-specific phospholipase C, the major activity peak was obtained in the later fractions with Ve/Vo = 1.75-2.0 on the same column chromatography. Lubrol-solubilized and phospholipase C-treated acetylcholinesterase preparations were different in the thermostability, the elution profiles of chromatography on Mono Q, butyl-Toyopearl and phenyl-Sepharose columns, and the affinity to phospholipid micelles. On treatment with PI-specific phospholipase C, Lubrol-solubilized acetylcholinesterase became more thermostable. The phospholipase C-treated enzyme was eluted at lower NaCl concentration from the Mono Q column than the Lubrol-solubilized enzyme. The most important difference was observed in the hydrophobicity of these two enzyme preparations. The Lubrol-solubilized enzyme shows high affinity to phospholipid micelles and hydrophobic adsorbents such as butyl-Toyopearl and phenyl-Sepharose. However, this hydrophobicity was lost when acetylcholinesterase was solubilized from bovine erythrocyte membrane by PI-specific phospholipase C. The presence of myo-inositol was confirmed in the purified preparation of acetylcholinesterase by gas chromatography (GC)-mass spectrometry (MS).(ABSTRACT TRUNCATED AT 250 WORDS)
Taguchi
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R
Faculty of Pharmaceutical Sciences, Nagoya City University, Aichi.
Ikezawa
H
H
D000110:Acetylcholinesterase / Q000032:analysis*; D000478:Alkylation; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D004795:Enzyme Stability; D004912:Erythrocytes / Q000201:enzymology*; D008401:Gas Chromatography-Mass Spectrometry; D006358:Hot Temperature; D007525:Isoelectric Focusing; D010084:Oxidation-Reduction; D010716:Phosphatidylinositols; D012995:Solubility; D010738:Type C Phospholipases
D010716:Phosphatidylinositols; D000110:Acetylcholinesterase; D010738:Type C Phospholipases