GDP-ribosyl cyclase activity as a measure of CD38 induction by retinoic acid in HL-60 cells.
Retinoic acid (RA) treatment of HL-60 cells induces surface expression of CD38. This lymphocytic antigen is also a novel bifunctional enzyme catalyzing the synthesis and hydrolysis of cyclic ADP-ribose (cADPR), a Ca2+ mobilizing metabolite of NAD+. The synthetic activity of CD38 is very difficult to detect because of the concurrent hydrolytic activity. In this study, a Ca2+ release assay capable of detecting submicromolar concentrations of cADPR was used to demonstrate the induction of ADP-ribosyl cyclase activity in HL-60 cells by RA. Concomitantly, cADPR hydrolase activity was also increased. The results were further substantiated by using a newly developed assay for GDP-ribosyl cyclase activity. This assay uses NGD+ as substrate instead of NAD+. The resulting fluorescent product, cyclic GDP-ribose, is resistant to hydrolysis and accumulates, making it a highly sensitive and convenient assay for CD38-like enzymes.
Graeff
R M
RM
Department of Physiology, University of Minnesota, Minneapolis 55455.
Mehta
K
K
Lee
H C
HC
D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D015703:Antigens, CD / Q000096:biosynthesis*; D000943:Antigens, Differentiation / Q000096:biosynthesis*; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D006868:Hydrolysis; D008562:Membrane Glycoproteins; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014212:Tretinoin / Q000494:pharmacology*
D015703:Antigens, CD; D000943:Antigens, Differentiation; D008562:Membrane Glycoproteins; D014212:Tretinoin; C090939:GDP-ribosyl cyclase; D009699:N-Glycosyl Hydrolases; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; D051997:ADP-ribosyl Cyclase 1