ENZYMATIC-SYNTHESIS AND CHARACTERIZATIONS OF CYCLIC GDP-RIBOSE - A PROCEDURE FOR DISTINGUISHING ENZYMES WITH ADP-RIBOSYL CYCLASE ACTIVITY

Cyclic nucleotides such as cAMP and cGMP are second messengers subserv ing various signaling pathways. Cyclic ADP-ribose (cADPR), a recently discovered member of the family, is derived from NAD(+) and is a media tor of Ca2+ mobilization in various cellular systems. The synthesis an d degradation of cADPR are, respectively, catalyzed by ADP-ribosyl cyc lase and cADPR hydrolase. CD38, a differentiation antigen of B lymphoc ytes, has recently been shown to be a bifunctional enzyme catalyzing b oth the formation and hydrolysis of cADPR. The overall reaction cataly zed by CD38 is the formation of ADP-ribose and nicotinamide from NAD(), identical to that catalyzed by NADase. The difficulties in detectin g the formation of cADPR have led to frequent identification of CD38 a s a classical NADase. In this study, we show that both ADP-ribosyl cyc lase and CD38, but not NADase, can cyclize nicotinamide guanine dinucl eotide (NGD(+)) producing a new nucleotide. Analyses by high performan ce liquid chromatography and mass spectroscopy indicate the product is cyclic GDP-ribose (cGDPR) with a structure similar to cADPR except wi th guanine replacing adenine. Compared to cADPR, cGDPR is a more stabl e compound showing 2.8 times more resistance to heat-induced hydrolysi s. These results are consistent with a catalytic scheme for CD38 where the cyclization of the substrate precedes the hydrolytic reaction. Sp ectroscopic analyses show that cGDPR is fluorescent and has an absorpt ion spectrum different from both NGD(+) and GDPR, providing a very con venient way for monitoring its enzymatic formation. The use of NGD(+) as substrate for assaying the cyclization reaction was found to be app licable to pure enzymes as well as crude tissue extracts making it a u seful diagnostic tool for distinguishing CD38-like enzymes from degrad ative NADases.