title: Enzymatic synthesis and characterizations of cyclic GDP-ribose. A procedure for distinguishing enzymes with ADP-ribosyl cyclase activity.
abstract: Cyclic nucleotides such as cAMP and cGMP are second messengers subserving various signaling pathways. Cyclic ADP-ribose (cADPR), a recently discovered member of the family, is derived from NAD+ and is a mediator of Ca2+ mobilization in various cellular systems. The synthesis and degradation of cADPR are, respectively, catalyzed by ADP-ribosyl cyclase and cADPR hydrolase. CD38, a differentiation antigen of B lymphocytes, has recently been shown to be a bifunctional enzyme catalyzing both the formation and hydrolysis of cADPR. The overall reaction catalyzed by CD38 is the formation of ADP-ribose and nicotinamide from NAD+, identical to that catalyzed by NADase. The difficulties in detecting the formation of cADPR have led to frequent identification of CD38 as a classical NADase. In this study, we show that both ADP-ribosyl cyclase and CD38, but not NADase, can cyclize nicotinamide guanine dinucleotide (NGD+) producing a new nucleotide. Analyses by high performance liquid chromatography and mass spectroscopy indicate the product is cyclic GDP-ribose (cGDPR) with a structure similar to cADPR except with guanine replacing adenine. Compared to cADPR, cGDPR is a more stable compound showing 2.8 times more resistance to heat-induced hydrolysis. These results are consistent with a catalytic scheme for CD38 where the cyclization of the substrate precedes the hydrolytic reaction. Spectroscopic analyses show that cGDPR is fluorescent and has an absorption spectrum different from both NGD+ and GDPR, providing a very convenient way for monitoring its enzymatic formation. The use of NGD+ as substrate for assaying the cyclization reaction was found to be applicable to pure enzymes as well as crude tissue extracts making it a useful diagnostic tool for distinguishing CD38-like enzymes from degradative NADases.
authors.affiliation: Department of Physiology, University of Minnesota, Minneapolis 55455.
authors.forename: R M
authors.initials: RM
authors.lastname: Graeff
authors.forename: T F
authors.initials: TF
authors.lastname: Walseth
authors.forename: K
authors.initials: K
authors.lastname: Fryxell
authors.forename: W D
authors.initials: WD
authors.lastname: Branton
authors.forename: H C
authors.initials: HC
authors.lastname: Lee
mesh_terms: D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D000943:Antigens, Differentiation / Q000378:metabolism; D001048:Aplysia / Q000201:enzymology; D001402:B-Lymphocytes / Q000201:enzymology; D001921:Brain / Q000201:enzymology; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004285:Dogs; D006156:Guanosine Diphosphate Sugars / Q000096:biosynthesis*; D006801:Humans; D008562:Membrane Glycoproteins; D009206:Myocardium / Q000201:enzymology; D009699:N-Glycosyl Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D009244:NAD+ Nucleosidase / Q000302:isolation & purification / Q000378:metabolism; D009492:Neurospora crassa / Q000201:enzymology; D010063:Ovum / Q000378:metabolism; D011755:Pyrophosphatases / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012617:Sea Urchins
chemical_list: D015703:Antigens, CD; D000943:Antigens, Differentiation; D006156:Guanosine Diphosphate Sugars; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; C090657:cyclic guanosine diphosphate-ribose; D009699:N-Glycosyl Hydrolases; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; D009244:NAD+ Nucleosidase; D051997:ADP-ribosyl Cyclase 1; D011755:Pyrophosphatases; C021899:nucleotide pyrophosphatase; D002118:Calcium