Transplantation of quail collagen-tailed acetylcholinesterase molecules onto the frog neuromuscular synapse.
The highly organized pattern of acetylcholinesterase (AChE) molecules attached to the basal lamina of the neuromuscular junction (NMJ) suggests the existence of specific binding sites for their precise localization. To test this hypothesis we immunoaffinity purified quail globular and collagen-tailed AChE forms and determined their ability to attach to frog NMJs which had been pretreated with high-salt detergent buffers. The NMJs were visualized by labeling acetylcholine receptors (AChRs) with TRITC-alpha-bungarotoxin and AChE by indirect immunofluorescence; there was excellent correspondence (>97%) between the distribution of frog AChRs and AChE. Binding of the exogenous quail AChE was determined using a species-specific monoclonal antibody. When frog neuromuscular junctions were incubated with the globular G4/G2 quail AChE forms, there was no detectable binding above background levels, whereas when similar preparations were incubated with the collagen-tailed A12 AChE form >80% of the frog synaptic sites were also immunolabeled for quail AChE attached. Binding of the A12 quail AChE was blocked by heparin, yet could not be removed with high salt buffer containing detergent once attached. Similar results were obtained using empty myofiber basal lamina sheaths produced by mechanical or freeze-thaw damage. These experiments show that specific binding sites exist for collagen-tailed AChE molecules on the synaptic basal lamina of the vertebrate NMJ and suggest that these binding sites comprise a "molecular parking lot" in which the AChE molecules can be released, retained, and turned over.
Rotundo
R L
RL
Department of Cell Biology and Anatomy, University of Miami School of Medicine, Florida 33136, USA. rrotundo@molbio.med.miami.edu
Rossi
S G
SG
Anglister
L
L
D000110:Acetylcholinesterase / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001485:Basement Membrane / Q000201:enzymology; D001665:Binding Sites; D003094:Collagen; D005455:Fluorescent Antibody Technique; D066298:In Vitro Techniques; D009469:Neuromuscular Junction / Q000201:enzymology* / Q000378:metabolism; D011784:Quail; D011894:Rana pipiens; D011950:Receptors, Cholinergic / Q000378:metabolism
D011950:Receptors, Cholinergic; D003094:Collagen; D000110:Acetylcholinesterase