Chemical Composition of Cholinergic Synaptic Vesicles from Torpedo marmorata Based on Improved Purification

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Abstract
Cholinergic synaptic vesicles from the electric organ of T. marmorara were purified to a constant composition and a higher transmitter content than previously reported. By optimizing the extraction conditions and using a 2-step purification on discontinuous and continuous sucrose density gradients, 10-fold higher acetylcholine and ATP values per weight of protein were obtained. The purity of the vesicle preparation was confirmed by EM, absence of marker enzymes, behaviour in density gradient centrifugation, and by a specific and reproducible protein composition. Vesicles contain 6.9 .mu.mol acetylcholine and 1.0 .mu.mol ATP per mg protein. The lipid/protein ratio of 3.5 (w/w) indicates a lipid-rich membrane. The value suggests the absence of a proteinaceous core. On dodecylsulfate gel electrophoresis a distinct protein pattern is obtained with components ranging from 20,000-160,000 in MW. Vesiculin, reported earlier to be a low-MW vesicle protein, is not detected. One of the major bands comigrates with muscle actin from the same animal. Further characterization of this protein by 2-dimensional gel electrophoresis suggested that it is an actin-like polypeptide. Evidence for a specific association of this actin-like protein with vesicles and its possible involvement in the neurosecretory process is discussed.