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 microM, and the Vmax value was 14.33 +/- 0.84 nmol/min x 10(6) cells. 8-Azido-ADP (8-N3-ADP) was dephosphorylated by the ecto-ADPase activity with a Km value of 595.29 +/- 67.44 microM and Vmax value of 6.86 +/- 0.45 nmol/min x 10(6) 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 microM) 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.