Nucleotide uptake by isolated cholinergic synaptic vesicles: Evidence for a carrier of adenosine 5'-triphosphate

The in vitro uptake of [ 3H]nucleotides was studied using cholinergic syaptic vesicles isolated from Torpedo electric organ, with a resting membrane potential of 50–60 mV. The osmotically sensitive uptake of [ 3H]adenosine 5'-triphosphate (ATP) was markedly influenced by temperature and external pH, and was maximal after 40–50 min; longer incubation resulted in loss of accumulated radiolabel. Similar characteristics were also observed for adenosine 5'-mono- and diphosphate and guanosine and uridine triphosphates, all of which acted as competitive substrates for the saturable system which transported ATP (K T 1.15 mM). Breakdown of [ 3H]nucleotides in the medium was not a significant factor, and adenosine, guanosine and adenine were very poorly incorporated. Under conditions of V max, vesicle to medium ratios of [ 3H]ATP of 20–25 were observed; the amount of radiolabel was equivalent to 20–50% of the initial endogenous amount of ATP in the vesicles. Atractyloside specifically inhibited nucleotide transport with no modification of hemicholinium-3 sensitive acetylcholine uptake. Antisera raised (a), to whole Torpedo vesicle extract, and (b), to a single purified vesicle polypeptide, greatly stimulated ATP uptake without effect on simultaneous influx of either acetylcholine or glucose. It is concluded that isolated vesicles contain a nucleotide carrier of wide pharmacological specificity (possibly the 34,000 molecular weight protein of Stadler & tashiro [1979]), which is likely to be of physiological relevance. Implications for vesicular refilling mechanisms are discussed.