Biochemical characterization of sheep platelet acetylcholinesterase after detergent solubilization.
The biochemical characterization of detergent-solubilized acetylcholinesterase (AChE) from subcellular particles of sheep platelets and the effects of different effectors on AChE activity from solubilized platelet crude membranes have been undertaken and studied. Solubilization of AChE with detergent increased the thermal stability of the enzyme from all particulate fractions. Solubilized AChE from the mitochondria-granule fraction was the most thermostable at 55 degrees C. The Km values against acetylthiocholine chloride and the Arrhenius plot obtained were very similar for the AChE from all the solubilized fractions. There were no differences in the ability of solubilized AChE from different subcellular fractions to bind concanavalin A (Con A). In solubilized platelet crude membranes, benzyl alcohol was a potent AChE inhibitor at a concentration of 10(-2) M, whereas ethanol was not. Mg2+ cations and, to a lesser extent, Ca2+ and Mn2+ cations, activated AChE at concentrations higher than 1 mM. Serine hydrolase inhibitors and cholinesterase-specific inhibitors were very effective in the inactivation of AChE, whereas EDTA and EGTA had no effect. Of all the monosaccharides tested, only N-acetylneuraminic acid exerted an inhibitory effect on AChE activity. Immobilized-lectin binding studies demonstrated the interaction of solubilized crude membrane-bound AChE with Con A, lentil lectin and wheat germ agglutinin. Taken together, these data suggest the presence of a unique form of the membrane-bound AChE which has at least alpha-mannose and N-acetylglucosamine residues in the glycan chain.
Departamento de Bioquímica y Biología Molecular, Facultad de Biología, Universidad de Salamanca, Spain.
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EM
Martín-Valmaseda
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Sánchez-Yagüe
J A
JA
Cabezas
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Llanillo
D000110:Acetylcholinesterase / Q000097:blood*; D000117:Acetylglucosamine / Q000032:analysis; D000122:Acetylthiocholine / Q000378:metabolism; D000818:Animals; D019905:Benzyl Alcohol; D001592:Benzyl Alcohols / Q000494:pharmacology; D001792:Blood Platelets / Q000201:enzymology* / Q000648:ultrastructure; D002241:Carbohydrates / Q000032:analysis; D002462:Cell Membrane / Q000201:enzymology; D002800:Cholinesterase Inhibitors; D003902:Detergents; D004795:Enzyme Stability; D006358:Hot Temperature; D007700:Kinetics; D008358:Mannose / Q000032:analysis; D019158:N-Acetylneuraminic Acid; D012756:Sheep; D012794:Sialic Acids / Q000494:pharmacology; D012995:Solubility; D013347:Subcellular Fractions / Q000201:enzymology; D013816:Thermodynamics
D001592:Benzyl Alcohols; D002241:Carbohydrates; D002800:Cholinesterase Inhibitors; D003902:Detergents; D012794:Sialic Acids; D000122:Acetylthiocholine; D000110:Acetylcholinesterase; D019158:N-Acetylneuraminic Acid; D019905:Benzyl Alcohol; D008358:Mannose; D000117:Acetylglucosamine