Purification of synaptic vesicles from elasmobranch electric organ and the use of biophysical criteria to demonstrate purity

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Abstract
Cholinergic synaptic vesicles were purified from the electric organs of 2 related marine elasmobranchs, Torpedo californica and Narcine brasiliensis, to a specific activity higher than previously obtained. The homogeneity of the vesicles was demonstrated by biophysical criteria. The purification scheme consisted of differential centrifugation, flotation equilibrium in sucrose density gradients and permeation chromatography on glass bead columns of average pore size 3000 .ANG.. The criteria for purity were that bound acetylcholine, bound nucleotide triphosphate, protein and lipid P behave identically when vesicles were analyzed by procedures which depend on vesicle size, density and charge. Contaminants were not detected when vesicles were fractionated by preparative and analytical velocity sedimentation, by preparative equilibrium sedimentation using glycerol density gradients or by electrophoresis in Ficoll density gradients. Pure synaptic vesicles, purified 290-fold from the initial homogenate, contained per milligram of protein: 8 .mu.mol of acetylcholine, 3 .mu.mol of ATP and 7 .mu.mol of lipid P. These procedures may be of general value in the purification of membrane vesicles.

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