Characterization of a Mg2+-ATPase and a proton pump in cholinergic synaptic vesicles from the electric organ of Torpedo marmorata

Abstract

Cholinergic synaptic vesicles from the electric organ of Torpedo marmorata are associated with a Mg2+-ATPase insensitive to ouabain and oligomycin. Treatment of vesicle membranes with dichloromethane releases a Mg2+-ATPase with apparent molecular mass of around 250 kDa as determined by gel filtration. The vesicular ATPase resembles the mitochondrial F1-ATPase in these properties. Gel electrophoresis of the solubilized ATPase shows however that only a single 50-kDa band is present as compared to the a-subunit (52 kDa) and P-subunit (50 kDa) of electric orgah mitochondrial F1-ATPase present in this range of molecular mass range. In agreement, covalent photoaffinity labelling of isolated vesicles with azido-ATP shows a 50-kDa band.Vesicle ghosts were found to accumulate [14C]methylamine in an ATP-dependent manner indicating the presence of an inwardly directed proton pump.We conclude that cholinergic vesicles contain a proton pump probably driven by the MgZ+-ATPase here described, which generates an electrochemical gradient across the vesicle membrane and is necessary for uptake and storage of acetylcholine within the vesicles.The cholinergic synaptic vesicles from the electric organs of Torpedinidae have proven to be a valuable model system for the study of the structure and function of synaptic vesicles and the mechanisms of acetylcholine storage and release in cholinergic nerve terminals [l, 21.Studies on isolated vesicles have shown that uptake of acetylcholine in vitro is an electrogenic process [3,4] supported by external ATP [5] suggesting the presence of an ATP-driven proton pump in the vesicle membrqne. These observations are in agreement with the finding using a chemical method [6] or 31P-NMR [7] that the pH within vesicles is acidic relative to the neutral external medium and has a value around 5.2-5.7. It was also shown [8, 9, 101 that treatment of isolated vesicles with ionophores causes release of acetylcholine providing additional evidence that an electrochemical gradient across the membrane is necessary for transmitter storage. Direct evidence for the presence of a proton pump has however not been provided yet.The association of vesicles with an ATPase has been described by Breer et al. [l 11 and confirmed by Rothlein and Parsons [12] and Michaelson et al. [13]; the latter showed in addition that the ATPase is externally oriented. These studies described at Ca2 +-activated and/or Mg2 +-activated ATPase but do not distinguish between the possibility that one or more different ATPases (e.g. a Mg2+-ATPase and a Ca2+-ATPase) are present, since isolation and further characterization was not carried out. We confirm here that vesicles contain a Mg2+-ATPase but in addition isolate it and partially characterize it. Furthermore we provide evidence that an ATP-dependent proton pump is part of the vesicle. MATERIALS AND METHODS Subcellailur.fractionationVesicles were extracted from frozen electric organs of Torpedo marmorata, the extract centrifuged for 30 min at 10 500 rev. min-' in a Sorvall SS 34 rotor an...

Ion channels in synaptic vesicles from Torpedo electric organ.

“…The biochemical effects of fusion were assessed by measuring the Mg-ATPase activity in the presence of ouabain and oligomycin (12). The values obtained, 505 ± 33 mol of Pi per mg of protein per min at 30'C (n = 3) before and 430 ± 61 (n = 2) after fusion, suggest that this particular vesicular enzyme, and supposedly other vesicular proteins, may undergo the fusion reaction without gross alterations in their properties.…”

Uptake of GABA by rat brain synaptic vesicles isolated by a new procedure.

“…In the mammalian nervous system, uptake of serotonin (Maron (©IRL Press Limited, Oxford, England et al, 1979) and catecholamines (Angelides, 1980) have been reported using vesicle preparations of variable purity. The presence of such an enzyme in purified synaptic vesicles has recently been demonstrated (Harlos et al, 1984;Jahn and Maycox, 1988). In addition, the uptake of glutamate by synaptic vesicles has been described recently (Disbrow et al, 1982;Ueda, 1983, 1985;Jahn and Maycox, 1988).…”

Isolation and Characterization of Secretory Granules Storing a Vasoactive Intestinal Polypeptide‐Like Peptide in Torpedo Cholinergic Electromotor Neurones

“…Mg*+-dependent ATPase (EC 3.6.1.3) was determined in the presence of oligomycin and ouabain as described by Harlos et al (1984). Thiamine pyrophosphatase (EC 3.6.1.…”