Lee H C, Aarhus R
Department of Physiology, University of Minnesota, Minneapolis 55455, USA.
Biochim Biophys Acta. 1998 Sep 16;1425(1):263-71. doi: 10.1016/s0304-4165(98)00079-8.
Nicotinic acid adenine dinucleotide phosphate (NAADP) mobilizes Ca2+ through a mechanism totally independent of cyclic ADP-ribose or inositol trisphosphate. Fluorescent analogs of NAADP were synthesized in this study to facilitate further characterization of this novel Ca2+ release mechanism. The base-exchange reaction catalyzed by ADP-ribosyl cyclase was utilized to convert nicotinamide 1,N6-ethenoadenine dinucleotide phosphate to a fluorescent product, nicotinic acid 1,N6-ethenoadenine dinucleotide phosphate (etheno-NAADP). The excitation spectrum of the product showed two maxima at 275 nm and 300 nm and an emission maximum at 410 nm. An aza derivative of etheno-NAADP was also synthesized by sequential treatments with NaOH and nitrite. The product, nicotinic acid 1,N6-etheno-2-aza-adenine dinucleotide phosphate (etheno-aza-NAADP) had excitation maxima at 280 nm and 360 nm and an emission maximum at 470 nm. The fluorescence of both analogs was sensitive to polarity and exhibited a 3-4-fold enhancement going from an aqueous buffer to an organic solvent. Proton-NMR measurements confirmed the presence of the etheno ring in both analogs. In the aza derivative the proton at the 2-position of the adenine ring was absent, consistent with the conversion of the 2-carbon to a nitrogen. Both analogs could activate Ca2+ release from sea urchin egg homogenates and the half-maximal concentrations for etheno-aza-NAADP and etheno-NAADP were at about 2.5 microM and 5 microM, respectively. At sub-threshold concentrations, both analogs could also function as antagonists, inactivating the NAADP-sensitive Ca2+ release with a half-maximal concentration of 60-80 nM. Microinjection of etheno-aza-NAADP into live eggs activated Ca2+ increase and triggered a cortical exocytotic reaction confirming its effectiveness in vivo. These fluorescent analogs are potentially useful for visualizing the novel Ca2+ stores that are sensitive to NAADP in live cells.
烟酰胺腺嘌呤二核苷酸磷酸（NAADP）通过一种完全独立于环ADP - 核糖或肌醇三磷酸的机制来动员钙离子。本研究合成了NAADP的荧光类似物，以促进对这种新型钙离子释放机制的进一步表征。利用ADP - 核糖基环化酶催化的碱基交换反应，将烟酰胺1，N6 - 乙烯腺嘌呤二核苷酸磷酸转化为荧光产物，即烟酸1，N6 - 乙烯腺嘌呤二核苷酸磷酸（乙烯基 - NAADP）。该产物的激发光谱在275nm和300nm处有两个最大值，发射最大值在410nm处。通过依次用氢氧化钠和亚硝酸盐处理，还合成了乙烯基 - NAADP的氮杂衍生物。产物，即烟酸1，N6 - 乙烯基 - 2 - 氮杂腺嘌呤二核苷酸磷酸（乙烯基 - 氮杂 - NAADP）在280nm和360nm处有激发最大值，发射最大值在470nm处。两种类似物的荧光对极性敏感，从水性缓冲液到有机溶剂时荧光增强3 - 4倍。质子核磁共振测量证实了两种类似物中乙烯基环的存在。在氮杂衍生物中，腺嘌呤环2位的质子不存在，这与2 - 碳转化为氮一致。两种类似物都能激活海胆卵匀浆中的钙离子释放，乙烯基 - 氮杂 - NAADP和乙烯基 - NAADP的半最大浓度分别约为2.5微摩尔和5微摩尔。在亚阈值浓度下，两种类似物也可作为拮抗剂，以60 - 80纳摩尔的半最大浓度使对NAADP敏感的钙离子释放失活。将乙烯基 - 氮杂 - NAADP显微注射到活卵中可激活钙离子增加并引发皮质胞吐反应，证实了其在体内的有效性。这些荧光类似物对于可视化活细胞中对NAADP敏感的新型钙离子储存可能是有用的。
