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.
Highlights
Analyses by high ribosyl cyclase converts NAD+to Cyclic ADP-ribose (cADPR) stoichiometrically and performanceliquid chromatographyand mass spectros- its activity has been detected in a wide variety of tissues from copy indicate the product is cyclic GDP-ribose invertebrates to mammals(9-11)
We show that metabolic enzymesof cADPRcan
These results show that the Aplysia enzyme can use cADPR produced were so small that it was not apparent in the both NAD+and NGD' as substrate
Summary
(Received for publication, August 8, 1994, and in revised form, September 22, 1994). From the Departments of $Physiology and §Pharmacology, University of Minnesota, Minneapolis, Minnesota 55455. The overall reaction catalyzed by CD38 is the formation of ADP-ribose and nicotinamide from NAD', identical to that catalyzed by NADase. Show that both ADP-ribosyl cyclase and CD38, but not The synthesis and degradation ofcADPR are catalyzed by NADase, can cyclize nicotinamide guanine dinucleotide ADP-ribosyl cyclase and cADPR hydrolase, respectively. Analyses by high ribosyl cyclase converts NAD+to cADPR stoichiometrically and performanceliquid chromatographyand mass spectros- its activity has been detected in a wide variety of tissues from copy indicate the product is cyclic GDP-ribose (cGDPR) invertebrates to mammals(9-11). The degradation enzyme, cADPR hydrolase, which hydrolyzes cADPR to ADP-ribose (ADPR), is an ubiquitous enzyme (11).Recently, a novel class of enzymes has been identifiedthat catalyze boththe formation and hydrolysis of cADPR (13-15). ADP-ribosyl cyclase and CD38, but not NADase, cyclize NGD' to cyclic GDP-ribose (cGDPR).The structureof this
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