Enzymatic synthesis and characterizations of cyclic GDP-ribose. A procedure for distinguishing enzymes with ADP-ribosyl cyclase activity

• Published in: The Journal of biological chemistry Vol. 269; no. 48; pp. 30260 - 30267
• Main Authors: Graeff, R M, Walseth, T F, Fryxell, K, Branton, W D, Lee, H C
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 02-12-1994; American Society for Biochemistry and Molecular Biology
• Subjects: ADP-ribosyl Cyclase; ADP-ribosyl Cyclase 1; Animals; Antigens, CD - metabolism; Antigens, Differentiation - metabolism; Aplysia - enzymology; B-Lymphocytes - enzymology; Brain - enzymology; Calcium - metabolism; Cell Membrane - enzymology; Chromatography, High Pressure Liquid; Cloning, Molecular; Dogs; Guanosine Diphosphate Sugars - biosynthesis; Humans; Membrane Glycoproteins; Myocardium - enzymology; N-Glycosyl Hydrolases - isolation & purification; N-Glycosyl Hydrolases - metabolism; NAD+ Nucleosidase - isolation & purification; NAD+ Nucleosidase - metabolism; Neurospora crassa - enzymology; Ovum - metabolism; Pyrophosphatases - isolation & purification; Pyrophosphatases - metabolism; Recombinant Proteins - metabolism; Sea Urchins
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)43806-9

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.