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 Ca(2+) 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.
Schweitzer
K
K
University Hospital Hamburg-Eppendorf, Institute for Medical Biochemistry and Molecular Biology, Division of Cellular Signal Transduction, University of Hamburg, Martinistrasse 52, D-20246 Hamburg, Germany.
Mayr
G W
GW
Guse
A H
AH
D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D000246:Adenosine Diphosphate Ribose / Q000031:analogs & derivatives* / Q000032:analysis; D000818:Animals; D015703:Antigens, CD; D000943:Antigens, Differentiation / Q000032:analysis*; D001048:Aplysia / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D019169:Jurkat Cells; D007938:Leukemia; D008562:Membrane Glycoproteins; D009243:NAD / Q000031:analogs & derivatives* / Q000378:metabolism; D009244:NAD+ Nucleosidase / Q000032:analysis*; D013347:Subcellular Fractions / Q000201:enzymology; D013601:T-Lymphocytes
C019374:1,N(6)-ethenoadenosine diphosphoribose; D015703:Antigens, CD; D000943:Antigens, Differentiation; D008562:Membrane Glycoproteins; D009243:NAD; D000246:Adenosine Diphosphate Ribose; C011038:nicotinamide 1,N(6)-ethenoadenine dinucleotide; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; D009244:NAD+ Nucleosidase; D051997:ADP-ribosyl Cyclase 1