Neural regulation of cholinesterase in newt skeletal muscle. I. Distribution and characterization of the enzyme species
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AbstractThe molecular forms of acetylcholinesterase (AChE) in triceps muscles of newts (Notophthalmus viridescensiwere identified and their distribution in the muscle determined. Six forms of AChE were separated from crude muscle homogenates using velocity sedimentation on sucrose gradients. Their sedimentation coefficients were 16.5S, 1226, 9.8S, 7S, 5.5S, and 3.7S. They were identified as acetylcholinesterases by their ability to hydrolyze acetyl‐β‐methylcholine, but not butyrylthiocholine. The 7S form, however, has not been as thoroughly characterized as it can only be consistently identified in sucrose gradients of Tris‐Triton X‐100‐NaC1 extracts of muscle previously extracted with Tris‐Triton X‐100. The Tris‐Triton X‐100 extraction removes the 5.55 and 9.8S AChE forms which mask the 7S form in sucrose gradients of unfractionated material. The region of muscle containing the motor endplates was enriched in the 16.5S form, whereas the musculotendinous junction contained a high proportion of the 12.8S form. Contamination by AChE present in blood and nerve, did not account for the distribution of the 12.8S species within the muscle. The 3.7S and 5.5S forms were soluble in Tris buffer and are probably located in the cytoplasm. The 9.8S forms required 1MNaCl for solubilization, indicating that it is associated with an osmotic shock‐resistant intracellular compartment, possibly the sarcotubular system. Triton X‐100 was required to solubilize the 16.5S, 12.8S and 7S forms indicating that they may be membrane bound. Crude muscle homogenates and each of the forms of AChE isolated on sucrose gradients yielded nine bands of AChE activity when subjected to polyacrylamide gel electrophoresis (PAGE). The bands of AChE were apparently monomers and multimers from which the larger forms of AChE were assembled.
