Determination of intracellular concentrations of the TRPM2 agonist ADP-ribose by reversed-phase HPLC.
Since the NAD metabolite ADP-ribose (ADPR) has recently gained attention as a putative messenger, a method was established for the quantification of intracellular ADPR by reversed-phase HPLC. Cellular nucleotides were extracted with trichloroacetic acid, and crude cell extracts purified by solid phase extraction using a strong anion exchange matrix. After optimization of the extraction procedure, cellular ADPR levels were determined using two different reversed-phase columns (C18 versus C12), operated in ion pair mode. Intracellular ADPR concentrations in human Jurkat T-lymphocytes and murine BW5147 thymocytes were determined to be 44+/-11 microM and 73+/-11 microM, respectively.
Gasser
Andreas
A
University Medical Center Hamburg-Eppendorf, Center of Experimental Medicine, Institute of Biochemistry and Molecular Biology I: Cellular Signal Transduction, Martinistr. 52, 20246 Hamburg, Germany.
Guse
Andreas H
AH
D000246:Adenosine Diphosphate Ribose / Q000032:analysis* / Q000302:isolation & purification; D000818:Animals; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007473:Ion Channels / Q000819:agonists*; D019169:Jurkat Cells; D008565:Membrane Proteins / Q000819:agonists*; D051379:Mice; D050053:TRPM Cation Channels; D013945:Thymoma
D007473:Ion Channels; D008565:Membrane Proteins; D050053:TRPM Cation Channels; C121934:TRPM2 protein, human; D000246:Adenosine Diphosphate Ribose