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.
Rodríguez-Pascual
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Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense de Madrid, Spain.
Torres
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Miras-Portugal
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MT
D015720:5'-Nucleotidase / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000528:radiation effects; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000528:radiation effects; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000313:Adrenal Medulla / Q000201:enzymology*; D000818:Animals; D001386:Azides / Q000378:metabolism* / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D007700:Kinetics; D013379:Substrate Specificity; D014466:Ultraviolet Rays
D001386:Azides; D000249:Adenosine Monophosphate; C017040:8-azidoadenosine 5'-triphosphate; C021291:8-azidoadenosine 5'-monophosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D015720:5'-Nucleotidase; D000251:Adenosine Triphosphatases; C027201:ectoATPase; C021622:8-azidoadenosine diphosphate