Vu C Q, Lu P J, Chen C S, Jacobson M K
Division of Medicinal Chemistry and Pharmaceutics, College of Pharmacy and the Lucille P. Markey Cancer Center, University of Kentucky, Lexington, 40536-0082, USA.
J Biol Chem. 1996 Mar 1;271(9):4747-54.
Cyclic adenosine diphosphoribose (cADPR), a metabolite of NAD, appears to modulate changes in intracellular free Ca2+ levels by activation of ryanodine-sensitive Ca2+ channels. We report here that an ADPR cyclase purified from Aplysia californica readily catalyzes the conversion of NADP to 2'-phospho-cyclic adenosine diphosphoribose (2'-P-cADPR), cyclized at N-1 of the adenine moiety. An enzyme from canine spleen previously shown to contain NAD glycohydrolase, ADPR cyclase, and cADPR hydrolase activities also utilized NADP and 2'-P-cADPR as substrates. The apparent Km value for NADP was 1.6 microM compared with 9.9 microM for NAD, and the Vmax with NADP was twice that with NAD, indicating that 2'-P-cADPR is a likely metabolite in mammalian cells. 2'-P-cADPR was as active as cADPR in eliciting Ca2+ release from rat brain microsomes, but was unable to elicit Ca2+ release following conversion to 2'-P-ADPR by the action of canine spleen NAD glycohydrolase. 2'-P-cADPR and 1-D-myo-inositol 1,4,5-trisphosphate (IP3) appear to act by distinct mechanisms as microsomes desensitized to IP3 still released Ca2+ in response to 2'-P-cADPR and vice versa. Also, inhibition of IP3-induced Ca2+ release by heparin had no effect on release by 2'-P-cADPR. Both 2'-P-cADPR and cADPR appear to act by a similar mechanism based on similar kinetics of Ca2+ release, similar dose-response curves, cross-desensitization, and partial inhibition of release by procaine. The results of this study suggest that 2'-P-cADPR may function as a new component of Ca2+ signaling and a possible link between NADP metabolism and Ca2+ homeostasis.
环腺苷二磷酸核糖（cADPR）是烟酰胺腺嘌呤二核苷酸（NAD）的一种代谢产物，它似乎通过激活对ryanodine敏感的钙离子通道来调节细胞内游离钙离子水平的变化。我们在此报告，从加州海兔中纯化出的一种ADPR环化酶能够轻易地催化烟酰胺腺嘌呤二核苷酸磷酸（NADP）转化为2'-磷酸环腺苷二磷酸核糖（2'-P-cADPR），其在腺嘌呤部分的N-1位环化。先前已证明犬脾脏中的一种酶含有NAD糖水解酶、ADPR环化酶和cADPR水解酶活性，该酶也将NADP和2'-P-cADPR用作底物。NADP的表观米氏常数（Km）值为1.6微摩尔，而NAD的为9.9微摩尔，且NADP的最大反应速度（Vmax）是NAD的两倍，这表明2'-P-cADPR可能是哺乳动物细胞中的一种代谢产物。2'-P-cADPR在引发大鼠脑微粒体释放钙离子方面与cADPR具有相同的活性，但在经犬脾脏NAD糖水解酶作用转化为2'-P-ADPR后无法引发钙离子释放。2'-P-cADPR和1-D-肌醇1,4,5-三磷酸（IP3）似乎通过不同机制起作用，因为对IP3脱敏的微粒体仍能响应2'-P-cADPR释放钙离子，反之亦然。此外，肝素对IP3诱导的钙离子释放的抑制作用对2'-P-cADPR引起的释放没有影响。基于钙离子释放的相似动力学、相似的剂量反应曲线、交叉脱敏以及普鲁卡因对释放的部分抑制作用，2'-P-cADPR和cADPR似乎通过相似机制起作用。本研究结果表明，2'-P-cADPR可能作为钙离子信号传导的一个新成分发挥作用，并且可能是NADP代谢与钙离子稳态之间的一个潜在联系。
