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SUMMARYThe properties of the cholinesterase activity in homogenates of whole rat diaphragm and of innervated ( +EP) and non-innervated ( -EP) regions of the muscle have been investigated. Under standard assay conditions, over 90% of the cholinesterase activity of whole muscle homogenates was due to specific acetylcholinesterases. The specific activity of acetylcholinesterase was higher in +EP regions of muscle than in -EP regions. About 40% of the total activity was calculated to be specifically associated with the endplates. When a high speed supernatant fraction of muscles homogenized in 1 M NaC1, 0.5% Triton X-100 was subjected t o velocity sedimentation in a sucrose gradient, and three species of acetylcholinesterase activity with sedimentation constants of 4 S, 10 S and 16 S were observed. All three forms were stable under the conditions of sedimentation and had buoyant densities of approximately 1.28. All three hydrolyzed P-methylacetylcholine at approximately 30 % the rate that acetylcholine was hydrolyzed. The 10 S and 16 S forms were inhibited by concentrations of acetylcholine over 1.25 mM, but no substrate inhibition was observed with the 4 S enzyme. Velocity sedimentation of extracts from +EP and -EP regions of muscle demonstrated that the 4 S and 10 S forms of the enzyme were distributed throughout the muscle while the 16 S form was found only in +EP regions. Extracts of the phrenic nerve contained only 4 S and 10 S forms. Thus, the 16 S form of acetylcholinesterase is specifically associated with endplate regions of muscle and may correspond t o the endplate enzyme. Seven days after denervation of the diaphragm, both endplate-specific cholinesterase activity and the cholinesterase activity in -EP regions of muscle were decreased. Although the activity of all three forms of acetylcholinesterase were decreased in denervated muscle, the largest proportional decrease occurred in the activity of the 16 S form.Abbreviations: EDTA, ethylenediaminetetraacetate; Tris, tris(hydroxymethy1) aminomethane; 284~51, 1 : 5-bis-(4-allyl-dimethyl-ammoniumphenyl) -pentane-3-one bromide; +EP, containing endplates; -EP, without endplates 343 344

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