Determination of ATP impurity in adenine dinucleotides.
Determination of ATP impurity in adenine dinucleotides.
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腺嘌呤二核苷酸中 ATP 杂质的测定。
DOI：
10.1081/ncn-120030716
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发表时间：
2004
期刊：
影响因子：
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通讯作者：
Hilderman,RichardH
中科院分区：
文献类型：
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作者：
Pojoga,LuminitaH;Haghiac,MaricelaL;Moose,JanaE;Hilderman,RichardH
腺嘌呤二核苷酸（ApnA）是细胞外信号分子，在应激后从血小板释放到血管系统中。最丰富和最佳表征的ApnA（Ap 4A）与牛主动脉内皮细胞（BAEC）上的独特受体相互作用，诱导一氧化氮。Ap 4A还与BAEC上的P2嘌呤受体相互作用，以调节Ca 2动员和前列环素释放;这种行为同样可以很好地解释为Ap 4A是这些受体的部分激动剂，或在ATP污染存在下的拮抗剂。为了区分这两种可能性，我们研究了ApnA制剂中此类污染物的存在。本文中的研究表明，ApnA（n = 3-6）含有ATP杂质;因此，当表征ApnA与ATP结合位点的相互作用时，研究者必须确保引起的反应不是部分由ATP杂质引起的。我们在这里提供了一种检测和估计Ap 4A制剂中ATP杂质的方法，同时也消除了它们;这种污染的水平估计低至0.2%。我们应用我们的方法来区分Ap 4A在P2嘌呤受体的真实作用;我们的研究结果与Ap 4A作为这些受体的部分激动剂是一致的。我们还应用我们的方法来表征ApnA与荧光素酶的相互作用，并发现净化的ApnA（n = 4-6）是荧光素酶的弱底物。
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 Ca2mobilization 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.