Assay for ADP-ribosyl cyclase by reverse-phase high-performance liquid chromatography

Cyclic ADP-ribose (cADPR), a natural metabolite of beta-NAD(+), is a second messenger for Ca2+ signaling in T cells. As a tool for purification and identification of ADP-ribosyl cyclase(s) in T cells, a sensitive and specific enzymatic assay using 1,N-6-etheno-NAD(+) as substrate was developed. A major problem-the sensitivity of 1,N-6-etheno-cADPR toward the extraction medium perchloric acid-was solved by replacing the perchloric acid extraction procedure of nucleotides by a filtration step. Standard compounds for the HPLC analysis of ADP-ribosyl cyclases and NAD(+)-glycohydrolases, e.g., 1,N-6-etheno-cADPR, 1,N-6-etheno-ADPR, and 1,N-6-etheno-AMP, were produced by ADP-ribosyl cyclase from Aplysia californica and dinucleotide pyrophosphatase. The assay was applied to subcellular fractions prepared from human Jurkat T cells. As a result ADP-ribosyl cyclase and NAD(+)-glycohydrolase activity could be detected and precisely quantified in different subcellular fractions indicating the presence of different isoenzymes in T cells. (C) 2001 Elsevier Science.