Nucleotide vesicular transporter of bovine chromaffin granules. Evidence for a mnemonic regulation

The nucleotide vesicular transport has been studied with the fluorescent substrate analogues, the (1,N6-ethenoadenosine) nucleotides. The transport experiments were carried out with granular preparations from bovine adrenal medulla, and epsilon-ATP, epsilon-ADP, and epsilon-AMP were quantified after separation by high performance liquid chromatography. The granular concentration increase of all three nucleotides was time-dependent. The concentration dependence of epsilon-nucleotide transport to chromaffin granules did not follow the Michaelis-Menten kinetics and presented a similar three-step curve with cooperativity. This shape can be considered to be the result of the addition of three sigmoidal curves with their corresponding kinetic parameters. epsilon-ATP exhibited K values of 0.25, 1, and 3 mM and Vmax values of 0.02, 0.04 and 0.19 nmol.min-1.mg of protein-1, for the first, second, and third curves for each step, respectively. epsilon-ADP exhibited K values of 0.15, 0.9, and 3.6 mM and Vmax values of 0.025, 0.035, and 0.3 nmol.min-1.mg of protein-1, respectively for the first, second, and third curves. epsilon-AMP exhibited K values of 0.2, 1.2, and 3.2 mM, and Vmax values of 0.01, 0.04, and 0.055 nmol.min-1.mg of protein-1, also for the first to third steps. The Hill numbers for epsilon-ATP, epsilon-ADP, and epsilon-AMP were not constant but a function of the transport saturation. The nonhydrolyzable ATP analogues AMPPNP, ATP gamma S, and ADP beta S were activators of epsilon-nucleotide transport at concentrations under 1 mM and inhibitors at higher concentrations. Atractyloside and N-ethylmaleimide partially inhibited the nucleotide granular transport. High extragranular ATP concentrations specifically induced the exit of the previously transporter granular epsilon-ATP.