Characterization of nucleotide transport into rat brain synaptic vesicles

ATP transport to synaptic vesicles from rat brain has been studied using the fluorescent substrate analogue 1,N-6-ethenoadenosine 5'-triphosphate (epsilon-ATP). The increase in intravesicular concentration was lime dependent for the first 30 min, epsilon-ATP being the most abundant nucleotide. The complexity of the saturation curve indicates the existence of kinetic and allosteric cooperativity in the nucleotide transport, which exhibits various affinity states with K-0.5, values of 0.39 +/- 0.06 and 3.8 +/- 0.1 mM with epsilon-ATP as substrate. The V-max values obtained were 13.5 +/- 1.4 pmol min(-1) . mg of protein(-1) for the first curve and 28.3 +/- 1.6 pmol . min(-1) . mg of protein(-1) considering both components. This kinetic behavior can be explained on the basis of a mnemonic model. The nonhydrolyzable adenine nucleotide analogues adenosine 5'-O-3-(thiotriphosphate), adenosine 5'-O-2-(thiodiphosphate), and adenosine 5'-(beta,gamma-imino)triphosphate and the diadenosine polyphosphates P-1,P-3-di(adenosine)triphosphate, P-1,P-4-di(adenosine)tetraphosphate, and P-1,P-5-di(adenosine)pentaphosphate inhibited the nucleotide transport. The mitochondrial ATP/ADP exchange inhibitor atractyloside, N-ethylmaleimide, and polysulfonic aromatic compounds such as Evans blue and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid also inhibit epsilon-ATP vesicular transport.