The cell surface antigen, CD38, is a 45-kDa transmembrane protein which is predominantly expressed on hematopoietic cells during differentiation. As a bifunctional ectoenzyme, it catalyzes the synthesis of cyclic ADP-ribose (cADPR) from NAD+ and hydrolysis of either NAD+ or cADPR to ADP-ribose. All-trans-retinoic acid (RA) is a potent and specific inducer of CD38 in myeloid cells. In this report, we demonstrate that the nuclei of RA-treated human HL-60 myeloblastic cells reveal enzymatic activities inherent to CD38. Thus, GDP-ribosyl cyclase and NAD+ glycohydrolase activities in the nuclear fraction increased very significantly in response to incubation with RA. With Western blotting, we detected in the nuclear protein fraction from RA-treated cells a ∼43-kDa protein band which was reactive with the CD38-specific monoclonal antibody OKT10. The expression of CD38 in HL-60 nuclei was also shown with FACScan analysis. RA treatment gave rise to an increase in in vitro ADP ribosylation of the ∼43-kDa nuclear protein. Moreover, nuclei isolated from RA-treated HL-60 cells revealed calcium release in response to cADPR, whereas a similar response was not observed in control nuclei. These results suggest that CD38 is expressed in HL-60 cell nuclei during RA-induced differentiation.

Cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) are two novel Ca(2+) messengers derived respectively from NAD and NADP. Since their discovery in sea urchin eggs, both have now been shown to serve messenger functions in a wide range of cells from plant to human. In this article, a series of fluorimetric assays for cADPR, NAADP and their metabolic enzymes is compiled. The enzyme assay makes use of an analog of NAD, nicotinamide guanine dinucleotide, which is non-fluorescent but is cyclized by the enzymes to a fluorescent analog of cADPR, cyclic GDP-ribose. Other NAD utilizing enzymes are not capable of catalyzing the cyclization and thus produce no interference. The fluorimetric assays for cADPR and NAADP make use of coupled-enzyme amplification and can readily detect nanomolar concentrations of either messenger. All the assays described can be performed in multi-well format, allowing ready automation and use in high throughput screening. An added advantage of these assays is that all the required reagents are commercially available, facilitating general adoption of the techniques by all those who are interested in the physiology and enzymology of the novel Ca(2+) signaling pathways mediated by cADPR and NAADP.

Recent studies have provided evidence for a role of cyclic ADP-ribose (cADPR) in the regulation of intracellular calcium in smooth muscles of the intestine, blood vessels and airways. We investigated the presence and subcellular localization of ADP-ribosyl cyclase, the enzyme that catalyzes the conversion of β-NAD+ to cADPR, and cADPR hydrolase, the enzyme that degrades cADPR to ADPR, in tracheal smooth muscle (TSM). Sucrose density fractionation of TSM crude membranes provided evidence that ADP-ribosyl cyclase and cADPR hydrolase activities were associated with a fraction enriched in 5′-nucleotidase activity, a plasma membrane marker enzyme, but not in a fraction enriched in either sarcoplasmic endoplasmic reticulum calcium ATPase or ryanodine receptor channels, both sarcoplasmic reticulum markers. The ADP-ribosyl cyclase and cADPR hydrolase activities comigrated at a molecular weight of approximately 40 kDa on SDS–PAGE. This comigration was confirmed by gel filtration chromatography. Investigation of kinetics yielded Km values of 30.4±1.5 and 695.3±171.2 μM and Vmax values of 330.4±90 and 102.8±17.1 nmol/mg/h for ADP-ribosyl cyclase and cADPR hydrolase, respectively. These results suggest a possible role for cADPR as an endogenous modulator of [Ca2+]i in porcine TSM cells.

Cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate were discovered >2 decades ago. That they are second messengers for mobilizing Ca2+ stores has since been firmly established. Separate stores and distinct Ca2+ channels are targeted, with cyclic ADP-ribose acting on the ryanodine receptors in the endoplasmic reticulum, whereas nicotinic acid adenine dinucleotide phosphate mobilizes the endolysosomes via the two-pore channels. Despite the structural and functional differences, both messengers are synthesized by a ubiquitous enzyme, CD38, whose crystal structure and catalytic mechanism have now been well elucidated. How this novel signaling enzyme is regulated remains largely unknown and is the focus of this minireview.

Cyclic ADP-ribose (cADPR) is a Ca2+-mobilizing cyclic nucleotide that functions as an endogenous modulator of the Ca2+-induced Ca2+ release mechanism in cells. Its synthesizing enzyme, ADP-ribosylcyclase, is widely distributed among animal tissues and is particularly abundant in Aplysia ovotestis. This chapter describes a procedure for a one-step large-scale purification of the Aplysia cyclase that is useful for biochemical analyses and crystallography. A lymphocyte antigen, CD38, which shares considerable sequence homology with the Aplysia cyclase, is shown to possess not only the cyclase activity but also to catalyze the hydrolysis of cADPR. A fluorimetric assay based on using nicotinamide guanine dinucleotide (NGD+), a guanine analog of NAD+, is proposed in the chapter. NGD is cyclized by CD38 to produce cyclic GDP-ribose (cGDPR), which is fluorescent. The product is also resistant to hydrolysis and accumulates, making this simple fluorimetric assay ideally suitable for monitoring the cyclize activity of CD38-like bifunctional enzymes in crude tissue extracts and during purification.

Cyclic ADP-ribose (cADPR) is a newly discovered cyclic nucleotide that functions by mobilizing intracellular Ca2+ stores. Unlike cAMP, cADPR is derived from NAD+ and its synthesis is catalyzed by ADP-ribosyl cyclase. Two modes of action are proposed for cADPR. First, cADPR can be a modulator constantly present in cells at some basal concentration that determines the Ca2+ sensitivity of the Ca2+-induced Ca2+ release mechanism. Substantial evidence from studies in sea urchin microsomes and Xenopus neurons supports this mode of action. Alternatively, cADPR can function as a Ca2+ messenger. The cADPR may play a messenger role in mediating the stimulatory action of glucose in pancreatic β cells and the Ca2+ mobilizing effect of a cholecystokinin octapeptide in intestinal longitudinal smooth muscle. The measurements of endogenous concentrations of cADPR are made by bioassays based on the Ca2+ release activity of cADPR. This chapter presents an improved radioimmunoassay (RIA) for cADPR using an antibody developed in chickens.

Mobilization of internal Ca+2 is an important signaling mechanism in cells. In addition to the inositol trisphosphate pathway, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide (NAADP) have been shown to mobilize Ca+2 via independent mechanisms. Although the structures of cADPR and NAADP are totally distinct, both nucleotides can be synthesized by ADP-ribosyl cyclase or CD38, a lymphocyte antigen. Both enzymes cyclize NAD to cADPR. In the presence of nicotinic acid the two enzymes catalyze a base exchange reaction resulting in the synthesis of NAADP from NADP. The switch between these two modes of catalysis is regulated by pH. Furthermore, both enzymes can also cyclize nicotinamide guanine dinucleotide (NGD) to produce a fluorescent product, cyclic GDP-ribose (cGDPR), which has a site of cyclization different from cADPR. A model is proposed to account for the multi-functionality of these enzymes. In order to be able to verify the model, a soluble ADP-ribosyl cyclase has been crystallized and X-ray diffraction shows that it is a dimer. Solution of the crystal structure of the cyclase should provide valuable insight into the structural features necessary for its multiple catalytic functions.

Publisher Summary This chapter describes a bioassay for cyclic ADP-ribose (ADPR) on the basis of its Ca 2+ -releasing activity in homogenates prepared from sea urchin eggs. Substances that could potentially interfere with the Ca 2+ release bioassay, such as Ca 2+ , inositol 1,4,5-triphosphate, and NAD + , must be removed from the sample. Cyclic ADP-ribose in the extracts is purified by a two-step system consisting of phenylboronate chromatography followed by anion-exchange chromatography on AG MP-1. Strongylocentrotus purpuratus eggs are obtained by stimulating ovulation of a female sea urchin with a 1-ml injection of 0.5 M KCI and washed once in artificial seawater, twice in Ca 2+ -free seawater containing 1 m M ethylene glycol tetraacetic acid (EGTA), twice in Ca 2+ -free seawater without EGTA, once with the homogenization buffer, and resuspended with the same medium to 25%. The sensitivity of the bioassay can be increased by pretreating the homogenate with caffeine, which is known to potentiate cADPR-induced Ca 2+ release.

Cyclic ADP-ribose (cADPR) is a Ca2+-mobilizing cyclic nucleotide derived from NAD(+) Accumulating evidence indicates that it is an endogenous modulator of the Ca2+-induced Ca2+ release mechanism in cells. In this study, we show that ADP-ribosyl cyclase catalyzes the cyclization of not only NAD(+) but also several of its analogs with various purine bases (guanine, hypoxanthine, or xanthine) substituting for adenine. Unlike cADPR, the resulting cyclic products are fluorescent. Comparisons with various model compounds indicate that only 7-methyl substituted purine nucleosides and nucleotides are fluorescent, and the pH-dependence of their UV spectra is most similar to that of the fluorescent cADPR analogs, indicating that the site of cyclization of these analogs is at the N7-position of the purine ring. This finding is novel since the site of cyclization is at the N1-position for cADPR as determined by X-ray crystallography. That a single enzyme can cyclize a variety of substrates at two different sites has important implications mechanistically, and a model is proposed to account for these novel catalytic properties. Among the analogs synthesized, cyclic GDP-ribose is highly resistant to hydrolysis, while cyclic IDP-ribose can be readily hydrolyzed by CD38, a bifunctional enzyme involved in the metabolism of cADPR. These unique properties of the analogs can be used to develop fluorimetric assays for monitoring separately the cyclization and hydrolytic reactions catalyzed by the metabolic enzymes of cADPR. The convenience of the method in measuring kinetic parameters, pH-dependence, and modulator activity of the metabolic enzymes of cADPR is illustrated.

Nicotinic acid adenine dinucleotide phosphate (NAADP(+)) is a recently identified metabolite of NADP(+) that is as potent as inositol trisphosphate (IP3) and cyclic ADP-ribose (cADPR) in mobilizing intracellular Ca2+ in sea urchin eggs and microsomes (Clapper, D. L., Walseth, T. F., Dargie, P. J., and Lee, H. C. (1987) J. Biol. Chem. 262, 9561-9568; Lee, H. C., and Aarhus, R. (1995) J. Biol, Chem, 270, 2152-2157). The mechanism of Ca2+ release activated by NAADP(+) and the Ca2+ stores it acts on are different from those of IP3 and cADPR. In this study we show that photolyzing caged NAADP(+) in intact sea urchin eggs elicits long term Ca2+ oscillations, On the other hand, uncaging threshold amounts of NAADP(+) produces desensitization. In microsomes, this self-inac tivation mechanism exhibits concentration and time dependence, Binding studies show that the NAADP(+) receptor is distinct from that of cADPR, and at subthreshold concentrations, NAADP(+) can fully inactivate subsequent binding to the receptor in a time-dependent manner, Thus, the NAADP(+)-sensitive Ca2+ release process has novel regulatory characteristics, which are distinguishable from Ca2+ release mediated by either IP3 or cADPR. This battery of release mechanisms may provide the necessary versatility for cells to respond to diverse signals that lead to Ca2+ mobilization.

ADP-ribosyl cyclase catalyzes the cyclization of NAD(+) to produce cyclic ADP-ribose (cADPR), which is emerging as an endogenous regulator of the Ca2+-induced Ca2+ release mechanism in cells, CD38 is a lymphocyte differentiation antigen which has recently been shown to be a bifunctional enzyme that can synthesize cADPR from NAD(+) as well as hydrolyze cADPR to ADP-ribose, In this study, we show that both the cyclase and CD38 can also catalyze the exchange of the nicotinamide group of NADP(+) with nicotinic acid (NA). The product is nicotinic acid adenine dinucleotide phosphate (NAADP(+)), a metabolite we have previously shown to be potent in Ca2+ mobilization (Lee, H. C., and Aarhus, R. (1995) J. Biol. Chem. 270, 2152-2157). The switch of the catalysis to the exchange reaction requires acidic pH and NA. The half-maximal effective concentration of NA is about 5 mM for both the cyclase and CD38. In the absence of NA or at neutral pH, the cyclase converts NADP(+) to another metabolite, which is identified as cyclic ADP-ribose 2'-phosphate, Under the same conditions, CD38 converts NADP(+) to ADP-ribose 2'-phosphate instead, which is the hydrolysis product of cyclic ADP-ribose 2'-phosphate. That two different products of ADP-ribosyl cyclase and CD38, cADPR and NAADP(+), are both involved in Ca2+ mobilization suggests a crucial role of these enzymes in Ca2+ signaling.

Cyclic ADP-ribose (cADPR) is emerging as an endogenous regulator of Ca2+-induced Ca2+ release (CICR), and we have recently demonstrated that its action is mediated by calmodulin (CaM) (Lee, H. C,, Aarhus, R,, Graeff, R., Gurnack, M. E,, and Walseth, T. F. (1994) Nature 370, 307-309). In this study we show by immunoblot analyses that the protein factor in sea urchin eggs responsible for conferring cADPR sensitivity to egg microsomes was CaM. This was further supported by the fact that bovine CaM was equally effective as the egg factor. In contrast, plant CaM was only partially active even at 10-20-fold higher concentrations. This exquisite specificity was also shown by binding studies using I-125-labeled bovine CaM. The effectiveness of various CaMs (bovine > spinach > wheat germ) in competing for the binding sites was identical to their potency in conferring cADPR sensitivity to the microsomes. A comparison between bovine and wheat germ CaM in competing for the sites suggests only 10-14% of the total binding was crucial for the activity, Depending on the CaM concentration, the sensitivity of the microsomes to cADPR could be changed by several orders of magnitude. The requirement for CaM could be alleviated by raising the divalent cation concentration with Sr2+. Results showed that CaM, cADPR, and caffeine all act synergistically to increase the divalent cation sensitivity of the CICR mechanism, The combined action of any of the three agonists was sufficient to sensitize the mechanism so much that even the nanomolar concentration of ambient Ca2+ was enough to activate the release. Unlike the CICR mechanism, the microsomal inositol 1,4,5-trisphosphate-sensitive Ca2+ release showed no dependence on CaM. Using an antagonist of CaM, W7, it was demonstrated that the cADPR(-) but not the inositol 1,4,5-trisphosphate-dependent release mechanism could be blocked in live sea urchin eggs, These results indicate cADPR can function as a physiological modulator of CICR and, together with CaM, can alter the sensitivity of the release mechanism to divalent cation by several orders of magnitude.

The pharmacology of the cyclic ADP-ribose (cADPR)-dependent Ca2+ release mechanism is very similar to that of the ryanodine receptor (RyR). Here we showed that MgCl2, a known inhibitor of RyR, blocked cADPR-induced Ca+2 release in sea urchin egg homogenates with a half maximal concentration of about 2.5 mM. The effect was specific since up to 10 mM Mg+2 had no effect on the Ca+2 release induced by inositol trisphosphate. K2ATP, another known modulator of RyR, at up to 10 mM did not affect the half-maximal concentration of cADPR, which remained at about 96 nM. These results indicate cADPR is a specific Ca+2 release activator and not merely an adenine nucleotide acting on the ATP-site. The inhibitory effects of Mg+2 further demonstrate the similarity between RyR and the cADPR-dependent Ca+2 release system.

Cyclic adenosine diphosphate (ADP)-ribose (cADPR), a metabolite of nicotinamide adenine dinucleotide (NAD+), mobilizes calcium from intracellular stores in many cells, The synthesis of cADPR from NAD(+) and its subsequent hydrolysis to ADPR is catalyzed by an ADP-ribosyl cyclase and a cADPR hydrolase, respectively, The ADP-ribosyl cyclase cloned from the ovotestis of the marine invertebrate Aplysia californica has amino acid sequence homology to the human lymphocyte surface antigen CD38, CD38 has been shown to catalyze both the formation and the hydrolysis of cADPR. In this study, we produced soluble, enzymatically active CD38 using recombinant expression techniques in bacteria and yeast, We engineered a gene coding for a soluble form of CD38 by excision of the region of the gene coding for the N-terminal amino acids representing the putative membrane spanning sequence and short putative intracellular sequence. For expression in bacteria (Escherichia coli), this construct was cloned into the pFlag-1 plasmid which allows induced, periplasmic expression and relatively simple purification of the soluble CD38, For expression in yeast (Pichia pastoris) the CD38 sequence was further modified to eliminate four putative N-linked glycosylation sites and the resulting construct was expressed as a secreted protein, Both systems produce soluble enzymes of approximately 30 kDa and both recombinant enzymes display similar cyclase and hydrolase activities.