Mg-ATPase and Torpedo cholinergic synaptic vesicles.
The reported presence of Mg-ATPase activity in cholinergic synaptic vesicles from the electric organ of Torpedo marmorata was reinvestigated 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. It appears that this treatment allows separation of the vesicles according to their content. The two vesicular content markers, acetylcholine and ATP, are recovered as sharp coincident peaks at a density close to 0.48 M sucrose. Empty vesicles are 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 acetylcholine content by valinomycin leads to an extreme picture, with a massive shift of the vesicles toward denser regions. We have then shown that 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 associated 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. On the basis of both the difference in inhibitor sensitivity between untreated and detergent-treated vesicles and of the pronase experiments, the possibility that the enzyme has an inward orientation is discussed.
Diebler
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MF
Lazereg
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D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase; D002458:Cell Fractionation; D003902:Detergents; D004557:Electric Organ / Q000201:enzymology*; D007425:Intracellular Membranes / Q000201:enzymology; D013572:Synaptic Vesicles / Q000201:enzymology*; D014101:Torpedo
D003902:Detergents; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase