Flounder (Platichthys flesus) muscle contains two types of cholinesterases, that differ in molecular form and in substrate specificity. Both enzymes were purified by affinity chromatography. About 8% of cholinesterase activity could be attributed to collagen-tailed asymmetric acetylcholinesterase sedimenting at 17S, 13s and 9S, which showed catalytic properties of a true acetylcholinesterase. 92% of cholinesterase activity corresponded to an amphiphilic dimeric enzyme sedimenting at 6 s in the presence of Triton X-100. Treatment with phospholipase C yielded a hydrophilic form and uncovered an epitope called the cross-reacting determinant, which is found in the hydrophilic form of a number of glycosyl-phosphatidylinositol-anchored proteins. This enzyme showed catalytic properties intermediate to those of acetylcholinesterase and butyrylcholinesterase. It hydrolyzed acetylthiocholine, propionylthiocholine, butyrylthiocholine and benzoylthiocholine. The K , and the maximal velocity decreased with the length and hydrophobicity of the acyl chain. At high substrate concentrations the enzyme was inhibited. The P(IC,~) values for BW284C51 and ethopropazine were between those found for acetylcholinesterase and butylcholinesterase. For purified detergent-soluble cholinesterase a specific activity of 8000 IU/mg protein, a turnover number of 2.8 x lo7 h-', and 1 active site/subunit were determined.
Different forms of acetylcholinesterase (AChE), EC 3.1.1.7, were demonstrated in human brain caudate nucleus. One form was solubilized at high ionic strength, the other with Triton X-100. The detergent-extractable form was purified to homogeneity by affinity chromatography. This form of AChE is amphiphile-dependent; i.e., it was active only in the presence of amphiphiles (detergents or lipids). Further, the enzyme was shown to bind detergents and to interact hydrophobically with Phenyl-Sepharose. In the presence of detergents the enzyme is a tetramer (subunit molecular weight, 78,000) which aggregates on the removal of detergents. Human brain AChE showed a reaction of identity with human erythrocyte AChE in crossed-line immunoelectrophoresis. The high-salt-soluble brain enzyme did not cross-react with the erythrocyte enzyme. The two classes of AChE seem not to be related, as they show no common antigenic determinant.
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