The isolation of pure cholinergic synaptic vesicles from the electric organs of elasmobranch fish of the family Torpedinidae

Abstract

1. Zonal centrifuging permitted the separation, on the milligram scale and in a form largely free from contamination by soluble cytoplasmic protein or membrane fragments derived from other structures, of synaptic vesicles from the purely cholinergic terminals of the electric organ of Torpedo. Up to lOOg of tissue could be processed in a single run. 2. As much as 46 % of the bound acetylcholine from the original tissue preparation was recovered as a single peak of density equivalent to 0.38M-sucrose-0.21 M-NaCl and with a concentration of up to 680nmol of acetylcholine/mg of protein. 3. The limiting concentration of acetylcholine in isolated vesicles when allowance had been made for non-vesicular protein appeared to be about 600nmol/mg of protein. 4. Vesicle counts by a 'bead-tagging' procedure indicated an acetylcholine content of about 360,umol/ml of vesicles; thus the vesicle protein content would be about 60% (w/v). 5. Calculations showed that the core of the vesicle, accounting for about 55 % of the vesicle volume, could be largely filled with acetylcholine and protein.

Functional evolution of the microfibril-associated glycoproteins

“…The long branch also indicates that Torpedo MAGP is highly divergent. We originally attributed those results to a distinctive evolutionary pathway driven by its expression in the electric organ, a unique organ composed of modified muscle fibers (Whittaker et al, 1972). However, the Torpedo sequence presents a number of residue substitutions similar to those seen in bony vertebrate MAGP2 (e.g.…”

ATP-Dependent calcium uptake by cholinergic synaptic vesicles isolated fromtorpedo electric organ

“…In the past, this cholinergic tissue led to the isolation pure synaptic vesicles [19,20], but this procedure gave a rather diluted fraction in spite of its high ACh content. This difficulty was not overcome by the scaled-up method using zonal rotors developed by Whittaker et al [41]. We present here a technique leading to a concentrated fraction of synaptic vesicles purified to near morphological homogeneity.…”

Coordinated release of ATP and ACh from cholinergic synaptosomes and its inhibition by calmodulin antagonists

“…The preparation of choice for such experiments has been the electric organ of the marine ray, which has a dense, purely cholinergic innervation. While the purification of synaptic vesicles from electric organ demonstrated that they contain high concentrations of ACh (Whittaker et al, 1972;Wagner et al, 1978) the direct release of this vesicular ACh has been difficult to observe. In fact, Israel and colleagues carried out a series of experiments demonstrating that, under certain conditions, ACh can be released directly from cytoplasmic pools (Israel et al,198 1) and that ACh release can be observed from proteoliposomes reconstituted from synaptosomal membranes (Israel et al, 1984).…”