Kirchberger Tanja, Guse Andreas H
The Calcium Signalling Group, Department of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany.
Cold Spring Harb Protoc. 2013 Jun 1;2013(6):569-73. doi: 10.1101/pdb.prot073007.
Cyclic ADP-ribose (cADPR) is a Ca(2+)-mobilizing second messenger active in many cell types, tissues, and organisms. The mammalian NAD-glycohydrolase CD38 catalyzes formation of cADPR by removing nicotinamide and forming a new intramolecular bond between N1 of adenine and C1 of the "northern" ribose. In contrast to the ADP-ribosyl cyclase (ADPRC) from Aplysia californica, which almost exclusively catalyzes the formation of cADPR, CD38 mainly produces adenosine diphosphoribose (ADPR), while cADPR is found as a side product. Interestingly, CD38 also catalyzes the breakdown of cADPR to ADPR. These enzyme activities can be determined by incubating the substrates NAD or cADPR with either crude membranes, purified proteins, or intact cells expressing CD38; the latter is possible because the catalytic site of CD38 is on the cell surface. Analysis of substrate and products is performed by reverse-phase (RP) HPLC. Before HPLC analysis, cells and proteins must be removed from samples by centrifugation and/or ultrafiltration to stop further metabolism and to prevent HPLC columns from clogging.
环磷酸腺苷核糖（cADPR）是一种在许多细胞类型、组织和生物体中发挥作用的钙离子动员第二信使。哺乳动物的烟酰胺腺嘌呤二核苷酸糖水解酶CD38通过去除烟酰胺并在腺嘌呤的N1和“北方”核糖的C1之间形成新的分子内键来催化cADPR的形成。与加州海兔的ADP - 核糖基环化酶（ADPRC）几乎只催化cADPR的形成不同，CD38主要产生腺苷二磷酸核糖（ADPR），而cADPR只是副产物。有趣的是，CD38还催化cADPR分解为ADPR。这些酶活性可以通过将底物NAD或cADPR与粗制膜、纯化蛋白或表达CD38的完整细胞一起孵育来测定；后者是可行的，因为CD38的催化位点在细胞表面。底物和产物的分析通过反相（RP）高效液相色谱法进行。在进行高效液相色谱分析之前，必须通过离心和/或超滤从样品中去除细胞和蛋白质，以停止进一步的代谢并防止高效液相色谱柱堵塞。
