Determination of cellular nicotinic acid-adenine dinucleotide phosphate (NAADP) levels
Determination of cellular nicotinic acid-adenine dinucleotide phosphate (NAADP) levels
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DOI：
10.1042/bj20031754
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发表时间：
2004-06-01
影响因子：
4.1
通讯作者：
Patel, S
中科院分区：
文献类型：
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作者：
Churamani, D;Carrey, EA;Patel, S
烟酸-腺嘌呤二核苷酸磷酸（NAADP）是一种新的细胞内钙动员信使。在海胆卵匀浆中，NAADP与其受体的结合不容易逆转;因此，与两种配体同时孵育制剂相比，预先与低浓度的NAADP孵育在抑制放射性标记的NAADP的后续结合方面更有效[Patel，丘吉尔and Galione（2000）Biochem.J.352，725-729]。我们扩展这一发现表明，NAADP是更有效的抑制随后的放射性配体结合在较低的匀浆浓度，效果再次很可能是由于受体-配体相互作用的不可逆性。通过简单的操作实验条件所提供的制备NAADP的增强的灵敏度已被应用于测定低水平的NAADP从人红细胞，大鼠肝细胞和大肠杆菌的酸性提取物中，而不干扰NADP分解。我们改进的方法定量NAADP应证明是有用的，在细胞内的信号作用的进一步评估。
Nicotinic acid-adenine dinucleotide phosphate (NAADP) is fast emerging as a new intracellular Ca2+-mobilizing messenger. In sea urchin egg homogenates, binding of NAADP to its receptor is not readily reversible; hence, prior incubation with low concentrations of NAADP is more effective in inhibiting subsequent binding of radiolabelled NAADP than incubating the preparation with the two ligands simultaneously [Patel, Churchill and Galione (2000) Biochem. J. 352, 725-729]. We extend this finding to show that NAADP is more effective still in inhibiting the subsequent radioligand binding at lower homogenate concentrations, an effect again quite probably due to the non-reversible nature of the receptor-ligand interaction. Enhanced sensitivity of the preparation to NAADP afforded by simple manipulation of the experimental conditions has been applied to determine low levels of NAADP in acid extracts from human red blood cells, rat hepatocytes and Escherichia coli without interference from NADP breakdown. Our improved method for the quantification of NAADP should prove useful in the further assessment of its signalling role within cells.