We have employed azido derivatives of ATP and AMP to identify the ATP translocase of synaptic vesicles. Azido-AMP inhibits transport of both ATP and AMP in vitro. The affinity of the translocase for the azido derivatives is similar to that of the native ligands. Upon UV irradiation of vesicles incubated with radiolabeled azido-AMP or -ATP, a molecular weight (Mr) 34000 polypeptide is selectively modified. On two-dimensional gel electrophoresis, the single radiolabeled polypeptide has a pI of approximately 7.7. Analysis of the fractions obtained when vesicles were purified on linear sucrose density gradients reveals that the Mr 34000 polypeptide is highly enriched in the vesicle-containing fractions. The findings support the notion that this polypeptide is identical with a previously described vesicle-specific component of the same molecular size [Stadler, H., & Tashiro, T. (1979) Eur. J. Biochem. 101, 171-178], and we conclude on the basis of uptake inhibition and photoaffinity labeling results that this protein is directly involved in ATP translocation of synaptic vesicles.

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...

We have employed sulfhydryl group reagents in an attempt to determine the mechanism by which the transport of nucleotides into synaptic vesicles is controlled. Transport proved to be sensitive to N-ethylmaleimide; radiolabelled N-ethylmaleimide was used to locate the sulfhydryl group to the translocase-associated molecule previously identified as a polypeptide of MI 34000 [Lee and Witzemann (1983) Biochemistry 22,6123-61301. The nucleotide uptake was 75% inhibited by the mercurials p-hydroxymercuribenzoate and p-chloromercuriphenylsulfonate. Uptake was also sensitive to the reagents phenylarsine oxide and iodosobenzoic acid, which are specific for dithiols. These results indicate that a readily accessible dithiol is critical for nucleotide transport. Using the lipophilic oxidants iodosobenzoic acid and plumbagin, we demonstrated that nucleotide uptake was inhibited upon oxidation of the dithiol but that this did not involve an alteration in the affinity of the translocase for its substrate.Reactive sulfhydryl groups often play an important role in the transport of molecules across biological membranes. The ADP/ATP carrier from beef heart mitochondria [l], the glucose transporter of the intestinal brush border membrane [2, 31 and Ca2+ transport by the Ca2+, Mg2+-adenosine triphosphate of the sarcoplasmic reticulum [4], for example, all show high sensitivity to sulfhydryl-blocking reagents. In many cases, the sensitive sulfhydryl groups are involved in a dithiol-disulfide interconversion which in turn affects the affinity of the carrier for the substrate. This has been best demonstrated in the case of hexose and lactose transport in bacteria [5]. The importance of such redox-sensitive steps in the diverse transport systems for Ca2+ (61, Pi [7], y-aminobutyric acid in brain synaptosomes [8], and glucose [9, 101 as well as for hexose and lactose mentioned above, has led Robillard and Konings [ll] to propose that these dithioldisulfide interconversions play a general role in regulating membrane transport.Cholinergic synaptic vesicles from the electromotor tissue of Torpedo marmorata contain an adenine nucleotide translocase with general specificity for transporting ATP, AMP, and ADP [12]. This translocase has been identified with a membrane polypeptide of MI 34000 [13,14].For both synaptic vesicles from electric organ as well as chromaffin granules, loading with ATP occurs prior to the import of neurotransmitter [15, 161. This fact has led to the hypothesis that the presence of ATP is essential for the uptake of the neurotransmitter [12, 151. Since acetylcholine uptake and vesicle recycling may be regulated by vesicular ATP concentrations it is important to understand the molecular mechanism of ATP uptake into vesicles. We have initiated experiments to determine whether sensitive thiol or disulfide groups, located on the translocase, would affect ATP transport into synaptic vesicles.From the experiments reported here, free sulfhydryl groups can be shown to be involved in ATP translocation and to play a critical rol...

No abstract