LOCALIZATION OF NON-EXTRACTABLE ACETYLCHOLINESTERASE TO THE VERTEBRATE NEUROMUSCULAR-JUNCTION

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 ta il. High salt and heparin-containing buffers are capable of solubilizi ng asymmetric AChE molecules from skeletal muscle; however, detachment of AChE specifically from synaptic basal lamina using these procedure s has not been demonstrated. To determine whether AChE can be solubili zed from mature neuromuscular junctions, adult quail muscle fibers wer e 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 immunofluo rescence. 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-tail ed AChE, whereas none of these buffers were capable of detaching AChE from synaptic basal lamina. In contrast, digestion with purified colla genase detached asymmetric forms from the non-extractable fraction and removed the AChE from the neuromuscular junctions. Parallel experimen ts 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 tha t all the conventional extraction buffers used to solubilize the asymm etric collagen-tailed forms of AChE are incapable of detaching this en zyme from the synaptic basal lamina.