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J. Biol. Chem., Vol. 268, Issue 25, 19152-19159, 09, 1993

Localization of "non-extractable" acetylcholinesterase to the vertebrate neuromuscular junction

SG Rossi and RL Rotundo
Department of Cell Biology and Anatomy, University of Miami School of Medicine, Florida 33101.

Asymmetric forms of acetylcholinesterase (AChE) are thought to be the predominant forms of this enzyme at vertebrate neuromuscular junctions where they attach to the synaptic basal lamina via a collagen-like tail. High salt and heparin-containing buffers are capable of solubilizing asymmetric AChE molecules from skeletal muscle; however, detachment of AChE specifically from synaptic basal lamina using these procedures has not been demonstrated. To determine whether AChE can be solubilized from mature neuromuscular junctions, adult quail muscle fibers were extracted with buffered detergent solutions containing either 0.05 M NaCl, 1 m NaCl, 0.5-2 mg/ml heparin, 8 M urea, or 4 m guanidine HCl, and the remaining AChE molecules were localized by indirect immunofluorescence. Analysis of extracted AChE oligomeric forms showed that low salt buffers containing heparin and high salt buffers were capable of solubilizing substantial amounts of catalytically active collagen-tailed AChE, whereas none of these buffers were capable of detaching AChE from synaptic basal lamina. In contrast, digestion with purified collagenase detached asymmetric forms from the non-extractable fraction and removed the AChE from the neuromuscular junctions. Parallel experiments using rat gastrocnemius muscle and enzyme histochemistry to detect AChE gave similar results. These studies indicate that the junctional AChE molecules are firmly attached to the extracellular matrix and that all the conventional extraction buffers used to solubilize the asymmetric collagen-tailed forms of AChE are incapable of detaching this enzyme from the synaptic basal lamina.
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