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

TY - JOUR

T1 - Measuring CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) activity by reverse-phase HPLC

AU - Kirchberger, Tanja

AU - Guse, Andreas H

PY - 2013/6/1

Y1 - 2013/6/1

N2 - 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.

AB - 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.

KW - Animals

KW - Antigens, CD38

KW - Chromatography, High Pressure Liquid

KW - Cyclic ADP-Ribose

KW - Cytological Techniques

KW - Humans

KW - NAD

U2 - 10.1101/pdb.prot073007

DO - 10.1101/pdb.prot073007

M3 - SCORING: Journal article

C2 - 23734017

VL - 2013

SP - 569

EP - 573

JO - Cold Spring Harbor protocols

JF - Cold Spring Harbor protocols

SN - 1559-6095

IS - 6

ER -