NUCLEOTIDE VESICULAR TRANSPORTER OF BOVINE CHROMAFFIN GRANULES - EVIDENCE FOR A MNEMONIC REGULATION

The nucleotide vesicular transport has been studied with the fluoresce nt substrate analogues, the (1,N-6-ethenoadenosine) nucleotides. The t ransport 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 chromatogr aphy. The granular concentration increase of all three nucleotides was time-dependent. The concentration dependence of epsilon-nucleotide tr ansport to chromaffin granules did not follow the Michaelis-Menten kin etics and presented a similar three-step curve with cooperativity. Thi s shape can be considered to be the result of the addition of three si gmoidal curves with their corresponding kinetic parameters. epsilon-AT P exhibited K values of 0.25, 1, and 3 mM and V-max 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 val ues of 0.15, 0.9, and 3.6 mM and V-max values of 0.025, 0.035, and 0.3 nmol.min(-1) mg of protein(-1), respectively for the first, second, a nd third curves. epsilon-AMP exhibited K values of 0.2, 1.2, and 3.2 m M, and V-max values of 0.01, 0.04, and 0.055 nmol.min(-1) mg of protei n(-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, A TP gamma S, and ADP beta S were activators of epsilon-nucleotide trans port at concentrations under 1 mM and inhibitors at higher concentrati ons. Atractyloside and N-ethylmaleimide partially inhibited the nucleo tide granular transport. High extragranular ATP concentrations specifi cally induced the exit of the previously transported granular epsilon- ATP.