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.
Gualix
J
J
Departamento de Bioquimica, Facultad de Veterinaria, Universidad Complutense, Madrid, Spain.
Abal
M
M
Pintor
J
J
Garcia-Carmona
F
F
Miras-Portugal
M T
MT
D000227:Adenine Nucleotides / Q000378:metabolism*; D000313:Adrenal Medulla / Q000378:metabolism; D000818:Animals; D001278:Atractyloside / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002417:Cattle; D002451:Cell Compartmentation; D002474:Cell-Free System; D002837:Chromaffin Granules / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D000226:Mitochondrial ADP, ATP Translocases / Q000037:antagonists & inhibitors; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D000227:Adenine Nucleotides; D004791:Enzyme Inhibitors; D013439:Sulfhydryl Reagents; D001278:Atractyloside; D000226:Mitochondrial ADP, ATP Translocases; D005033:Ethylmaleimide