Multiple forms of acetylcholinesterase and their distribution in endplate and non‐endplate regions of rat diaphragm muscle

Abstract

The 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 NaCl, 0.5% Triton X‐100 was subjected to 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 β‐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 to 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.