Abstract
Acetylcholinesterase extracted with Triton X-100 from bovine brain caudate nuclei was purified by affinity chromatography to apparent homogeneity. The purified enzyme was labeled with [3H]diisopropyl fluorophosphate at the active sites and with the photoactivated reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine, a compound which has been shown to be selective for the hydrophobic membrane-binding domains of several other proteins. The subunit structure was analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate before and after disulfide reduction. After reduction, a single 3H-labeled band at 70 kDa was stained by silver, but most of the 125I label corresponded to a 20-kDa species. Prior to reduction, five 3H-labeled and silver-stained bands were apparent at 70, 140, 160, 260, and greater than 360 kDa. These species were presumed to represent monomer and disulfide-linked oligomers of 70-kDa catalytic subunits. 125I label was selectively associated with the 160-, 260-, greater than 360-, and a 90-kDa species. Quantitative gel slicing of 3H- and 125I-labeled nonreduced enzyme supported a structural model in which the tetrameric enzyme is a dimer of nonidentical catalytic subunit dimers, one of which involves a direct intersubunit disulfide linkage between two 70-kDa catalytic subunit monomers and the second of which contains two disulfide linkages through an intervening 125I-labeled 20-kDa noncatalytic subunit. This 20-kDa subunit is proposed to contain the membrane attachment site. The brain enzyme did not contain components characteristic of the glycolipid anchors of erythrocyte acetylcholinesterases. However, part of the 125I label was associated with fatty acids, indicating that at least a portion of the brain enzyme membrane anchor is composed of nonamino acid components.
Highlights
$Department of Cell Eiobgy, Catholic University of Chile, globular AChEs have been identified
Acetylcholinesterase extracted with Triton X-100 Gz AChE forms have been purified from human and bovine from bovine brain caudate nuclei was purified by af- erythrocyte membranes [6, 7], torpedo electric organ [8], and finity chromatography to apparent homogeneity
The Drosophila heads? and theirhydrophobic domains have been purified enzymewas labeled with [3H]diisopropylfluo- shown to consist of novel glycolipid anchors which appear to rophosphate at the active sites and with the photoac- contain phosphatidylinositoland are covalently linked to the tivatedreagent 3-(trifluoromethyl)-3-(~-[12~Ili~op-o l ~ p t i d eC termini [10,11,12,13]: G, AChE forms are predomphenyl } ~ ~ i r i nae,compound which has been shown inant in mammalian brain [2] and have been purified from to be selective for thehydrophobic membrane-binding caudate nuclei of bovine and human brain [14,15,16], but no domains of several other proteins
Summary
$Department of Cell Eiobgy, Catholic University of Chile, globular AChEs have been identified
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