Measuring CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) activity by reverse-phase HPLC.

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.