Mg‐ATPase and Torpedo Cholinergic Synaptic Vesicles

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Abstract
The Mg-ATPase activity in cholinergic synaptic vesicles from the electric organ of T. marmorata was investigated in view of possible contamination of vesicles by other subcellular fractions. After dilution in concentrated sucrose, the vesicular fraction isolated on a sedimentation sucrose gradient was purified further on a flotation density gradient. This treatment allows separation of the vesicles according to their content. The 2 vesicular content makers, acetylcholine [ACh] and ATP, are recovered as sharp coincident peaks at a density close to 0.48 M sucrose. Empty vesicles were identified in denser regions by the protein pattern on gel electrophoresis which is identical to the pattern obtained for filled vesicles. Refractionation of vesicles depleted of their ACh content by valinomycin leads to an extreme picture, with a massive shift of the vesicles toward dense regions. A ouabain-insensitive Mg-ATPase is indeed associated with the vesicle membrane, but the activity is fully apparent only when vesicles are permeabilized either as the result of the fractionation procedure or after detergent treatment. The relative insensitivity of the Mg-ATPase assoociated with the synaptic vesicles to oligomycin, N,N''-dicyclohexylcarbodiimide and azide indicates that this enzyme differs from the classic F1F0 mitochondrial enzyme. The most striking finding is the sensitivity to vanadate of the vesicular Mg-ATPase, which suggests the involvement of a phosphorylated intermediate. The possibility that the enzyme has an inward orientation is discussed.