Determination of ATP impurity in adenine dinucleotides.
Adenine dinucleotides (ApnA) are extracellular signal molecules that are released from blood platelets, following stress, into the vascular system. The most abundant and best-characterized ApnA (Ap4A) interacts with a unique receptor on bovine aortic endothelial cells (BAEC) where it induces nitric oxide. Ap4A also interacts with P2 purinoceptors on BAEC to modulate Ca2+ mobilization and prostacyclin release; this behavior can be equally well explained by Ap4A being either a partial agonist to these receptors, or an antagonist in the presence of ATP contamination. To discern between these two possibilities, we have investigated the presence of such contaminants in ApnA preparations. The studies herein indicate that ApnAs (n = 3-6) contain ATP impurities; thus, when characterizing the ApnA interaction with ATP-binding sites, investigators must assure that the response elicited is not partly due to an ATP impurity. We here provide a means for detecting and estimating ATP impurities within Ap4A preparations while also eliminating them; the level of this contamination is estimated to be as low as 0.2%. We applied our method to distinguish the true effect of Ap4A at P2 purinoceptors; our findings are consistent with Ap4A acting as a partial agonist to these receptors. We also applied our method to characterizing the ApnA interaction with luciferase, and found that decontaminated ApnA (n = 4-6) are weak substrates for luciferase.
Pojoga
Luminita H
LH
Department of Genetics and Biochemistry, Clemson University, Clemson, South Carolina 29634-0324, USA.
Haghiac
Maricela L
ML
Moose
Jana E
JE
Hilderman
Richard H
RH
D000227:Adenine Nucleotides / Q000302:isolation & purification / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D011464:Epoprostenol / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D008163:Luminescent Measurements; D018048:Receptors, Purinergic P2 / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology
D000227:Adenine Nucleotides; D018048:Receptors, Purinergic P2; D000255:Adenosine Triphosphate; D011464:Epoprostenol; D008156:Luciferases; D000469:Alkaline Phosphatase