Chemical composition of cholinergic synaptic vesicles from Torpedo marmorata based on improved purification

Abstract

Cholinergic synaptic vesicles from the electric organ of Torpedo marmorata have been purified to a constant composition and a higher transmitter content than previously reported. By optimising the extraction conditions and using a two-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 electronmicroscopy, absence of marker enzymes, behaviour in density gradient centrifugation, as well as by a specific and reproducible protein composition. Vesicles contain 6.9 mumol acetylcholine and 1.0 mumol 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. Upon dodecylsulphate gel electrophoresis a distinct protein pattern is obtained with components ranging from 20000 to 160000 in molecular weight. Vesiculin, reported earlier to be a low-molecular-weight vesicle protein, is not detected. One of the major bands comigrates with muscle actin from the same animal. Further characterisation of this protein by two-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.