BIOCHEMICAL-CHARACTERIZATION OF SHEEP PLATELET ACETYLCHOLINESTERASE AFTER DETERGENT SOLUBILIZATION

The biochemical characterization of detergent-solubilized acetylcholin esterase (AChE) from subcellular particles of sheep platelets and the effects of different effectors on AChE activity from solubilized plate let crude membranes have been undertaken and studied. Solubilization o f AChE with detergent increased the thermal stability of the enzyme fr om all particulate fractions. Solubilized AChE from the mitochondria-g ranule fraction was the most thermostable at 55 degrees C. The K-m val ues against acetylthiocholine chloride and the Arrhenius plot obtained were very similar for the AChE from all the solubilized fractions. Th ere were no differences in the ability of solubilized AChE from differ ent subcellular fractions to bind concanavalin A (Con A). In solubiliz ed platelet crude membranes, benzyl alcohol was a potent AChE inhibito r at a concentration of 10(-2) M, whereas ethanol was not. Mg2+ cation s and, to a lesser extent, Ca2+ and Mn2+ cations, activated AChE at co ncentrations higher than 1 mM. Serine hydrolase inhibitors and choline sterase-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 int eraction of solubilized crude membrane-bound AChE with Con A, lentil l ectin and wheat germ agglutinin. Taken together, these data suggest th e presence of a unique form of the membrane-bound AChE which has at le ast alpha-mannose and N-acetylglucosamine residues in the glycan chain .