8-AZIDO-ADENINE NUCLEOTIDES AS SUBSTRATES OF ECTO-NUCLEOTIDASES IN CHROMAFFIN CELLS - INHIBITORY EFFECT OF PHOTOACTIVATION

The components of the ecto-nucleotidase pathway at the extracellular surface of adrenal chromaffin cells are the enzymatic activities responsible for the hydrolysis of granular nucleotide compounds released during the secretory response. The azido-nucleotides have been largely employed to characterize nucleotide binding sites. The 8-azido-adenine nucleotides were studied as substrates of ecto-nucleotidases in cultured chromaffin cells by HPLC procedures. 8-Azido-ATP (8-N3-ATP) was a good substrate for ecto-ATPase activity, the Km value was 256.30 ± 36.41 μM, and the Vmax value was 14.33 ± 0.84 nmol/min × 106 cells. 8-Azido-ADP (8-N3-ADP) was dephosphorylatedby the ecto-ADPase activity with a Km value of 595.29 ± 67.44 μM and Vmax value cf 6.86 ± 0.45 nmol/min × 106 cells. These kinetic parameters were similar to those obtained with ATP and ADP in the same culture and incubation conditions. 8-Azido-AMP (8-N3-AMP) was not hydrolyzed by the ecto-5′-nucleotidase activity. The 8-azido-nucleotides competitively inhibited the hydrolysis of adenine nucleotides, with Ki values in the same range as the Km. After uv photoactivation, the three 8-azido-nucleotides (100 MM) irreversibly inhibited and to a similar extent, between 40 and 55%, each of ecto-nucleotidase activities. UV photoactivation in the presence of nucleotides in the same concentration range was an effective protection from the inhibition. © 1993 Academic Press, Inc.