Purification of small dense vesicles from stimulated Torpedo electric tissue by glass bead column chromatography

Abstract

Synaptic vesicles were isolated from perfused tissue blocks of Torpedo electric organ using sucrose density gradient centrifugation in swing-out rotors. After application of [ 3H]acetate and low frequency stimulation (0.1 Hz) a denser peak of [ 3H]acetylcholine could be separated from the main and coincident peak of the vesicle constitutents acetylcholine and adenosine 5'-triphosphate in accordance with previous findings using zonal centrifugation ( Zimmermann & Denston, Neuroscience 2, 715–730, 1977). Further separation of subcellular particles sedimenting in the range of synaptic vesicles, by chromatography through columns of porous glass beads, yielded three main fractions which were eluted in the order, large (esterase-containing) membrane particles in the void volume, larger synaptic vesicles containing acetylcholine of low specific radioactivity (peak I) and smaller vesicles containing acetylcholine of higher specific radioactivity (peak II). After stimulating the electric tissue (which causes the appearance of a large proportion of synaptic vesicles about 25% smaller in diameter; Zimmermann & Denston, Neuroscience 2, 695–714, 1977), the peak of larger vesicles (peak I) also contains vesicles of smaller diameter. The glass bead column thus separates membrane fragments from synaptic vesicles, but only partially resolves larger and smaller vesicles. This is also reflected by the decrease in the ratio of the specific radioactivity of acetylcholine of peak I to that of peak II, from 8.2 for unstimulated control to 4.0 for stimulated tissue. The results demonstrate that using glass bead chromatography the smaller vesicles, which appear on stimulation-induced transmitter release, contain acetylcholine of high specific radioactivity and can be completely separated from any membrane contaminants which could possibly contain a pool of nonvesicle-bound acetylcholine.