Endplate Acetyicholinesterase in Muscle

When I wrote this paper, I was a junior faculty member in the Department of Neurobiology at Harvard Medical School, where I had been recently joined by Regis Kelly, a colleague whom I had met when we were postdoctoralfellows in biochemistry at Stanford. At Harvard, we were trying to develop a biochemical approach to the neuromtscularjunclion, which at that time was the synapse whose electrophysiological function was best understood. The important role of acetylcholinesterase at the junction was well known. HistOdseIniCaI expenments by Couteaux and others had shown it to be highly concentrated there, and electrophysiological experiments demonstrated that itacted toterminate the action of the transmitter, acetykholine, on the postsynaptic membrane. Our etpesiments began with an accidental finding. We were trying to purify the surface membranes of muscle fibers and were using acetykholinesterase, which we assumed to be an integral membrane protein, as a marker for the postaynaptic membrane. To remove contaminating connective thas.ie, we treated a partiallypurified surface membranepreparation with collagenase. To oursurprise we found that this treatment solubilized acetylchollnesterase from the membranes. We repeated the experiment on intact muscle and found that incubation with collagenase released active enzyme from the muscle into the medium without affecting the nirsilological properties ofthenisde fiber membrane. Phyilóiogicat and histochemical experiments showed thatthe released acetyIchofln1~~ plate. Moreover, it came only from the endplate acetylchollnesterase in parts of themuscle tacking endplateswas unaffected bycollagenase treatment. These results, which we publIshed In Nature,’ suggested to us not only that the esidplate enzyme was associated with extraceffular matrix material, but also that it must be different from the rest of the acetykholinesterase in the muscle. I then attempted to extract and purify acetylcbolinesterase from muscle to see If I could find a disonctive en~iatefoorn.Conventional biochendcal methods did not seeni to work however. The enzyme aggregated easily, as4 using a variety of column procedures, I was unable to obtaindiscrete, reproducible peaks of activity.Two key ideas broke the logjam. First, Kelly as.gr.dasl analysisby sucrose gradient sedimentation. This method gave two distinct fractions, but showed no differ~ between regions of muscle with endplates and those without. Then, I found a paper in French by j. Massoulid and F. Rieger on acetylchollnesterase from the electric organ of eel. 2 With the aid of a dictionary, I laboriously translated this paper word.by.word into an English version, which Istill have.Theeffort was worth it. This paper, along with several other, from Massoulié’s laboratory, reported that eel electric organ contained a species of acetylcs.ollnesterase that was highly asymmetric and that aggregated at low salt concentrations, but was soluble in 1M NaCI. Application of the high salt method to muscle immediately revealed a new sucrose gradient peak thatcorresponded to the asymmetricform and that was found only in endplate regionsofmuscle. These were the results reported in the Journal of Neurobiology article. Taken together, the results of our two papers suggested thata special fonts of acetylcholinesterase is presentatendplates and thatit is nota menthrane protein, hut is associated with the extracellular matrix,perhaps by a mechanlon hwoleing collagen. Subsequent work by I. Simian 3 and by P. Taylor’ on acetylcholinesterase from electric fish showed that the asymmetric form indeed hasa special structure in which three tetramers of catalytic subunit are aftached to a longcollagen-like tail. 114. McMalsan and coworkers 5 then demonstrated directly that acetylcholinesterase atthe neuromuscularjunction is part of the basal lamina, presimsably attached by the tail. The Journal of Neurvjikkgy paper thus formed an initial rmk in the conthgdsg Investigation of the relation between the Pl.~oio*4heictionand molecular çkolsynaptic acera