Formation Of Cyclic ADP-ribose Research Articles

Impaired learning and memory in CD38 null mutant mice.

CD38 is an enzyme that catalyzes the formation of cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate, both of which are involved in the mobilization of Ca2+ from intracellular stores. Recently, CD38 has been shown to regulate oxytocin release from hypothalamic neurons. Importantly, CD38 mutations are associated with autism spectrum disorders (ASD) and CD38 knockout (CD38−/−) mice display ASD-like behavioral phenotypes including deficient parental behavior and poor social recognition memory. Although ASD and learning deficits commonly co-occur, the role of CD38 in learning and memory has not been investigated. We report that CD38−/− mice show deficits in various learning and memory tasks such as the Morris water maze, contextual fear conditioning, and the object recognition test. However, either long-term potentiation or long-term depression is not impaired in the hippocampus of CD38−/− mice. Our results provide convincing evidence that CD38−/− mice show deficits in various learning and memory tasks including spatial and non-spatial memory tasks. Our data demonstrate that CD38 is critical for regulating hippocampus-dependent learning and memory without modulating synaptic plasticity.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-016-0195-5) contains supplementary material, which is available to authorized users.

Probing the catalytic mechanism of bovine CD38/NAD+glycohydrolase by site directed mutagenesis of key active site residues

Bovine CD38/NAD+ glycohydrolase catalyzes the hydrolysis of NAD+ to nicotinamide and ADP-ribose and the formation of cyclic ADP-ribose via a stepwise reaction mechanism. Our recent crystallographic study of its Michaelis complex and covalently-trapped intermediates provided insights into the modalities of substrate binding and the molecular mechanism of bCD38. The aim of the present work was to determine the precise role of key conserved active site residues (Trp118, Glu138, Asp147, Trp181 and Glu218) by focusing mainly on the cleavage of the nicotinamide–ribosyl bond. We analyzed the kinetic parameters of mutants of these residues which reside within the bCD38 subdomain in the vicinity of the scissile bond of bound NAD+. To address the reaction mechanism we also performed chemical rescue experiments with neutral (methanol) and ionic (azide, formate) nucleophiles. The crucial role of Glu218, which orients the substrate for cleavage by interacting with the N-ribosyl 2′-OH group of NAD+, was highlighted. This contribution to catalysis accounts for almost half of the reaction energy barrier. Other contributions can be ascribed notably to Glu138 and Asp147 via ground-state destabilization and desolvation in the vicinity of the scissile bond. Key interactions with Trp118 and Trp181 were also proven to stabilize the ribooxocarbenium ion-like transition state. Altogether we propose that, as an alternative to a covalent acylal reaction intermediate with Glu218, catalysis by bCD38 proceeds through the formation of a discrete and transient ribooxocarbenium intermediate which is stabilized within the active site mostly by electrostatic interactions.

Heterogeneous CD38 expression in tumor tissues of patients with colorectal cancer

The CD38 gene encodes a membrane-bound protein that takes part in cell adhesion and catalyzes the formation of cyclic ADP-ribose. The presence of the full-size and alternative forms of CD38 mRNA in tumor tissue specimens from patients with colorectal cancer and tumor cell lines was analyzed by RT-PCR. It was shown that tumor specimens contained cells that constantly express CD38; full-size CD38 mRNA was detected more frequently than its alternative form. CD38 mRNA was found in Colo-205, T-84, HCT15, and HCT116 cells, but not in Caco-2 or SW-620 cells. CD38 expression was observed in all cases of stage-I colorectal cancer; at stages II, III, and IV, it was significantly less frequent. The frequency of CD38 mRNA detection did not depend on the localization or grade of the tumor, or the presence of metastases. An analysis using methylation-sensitive restriction endonucleases showed that CpG dinucleotides were methylated at the binding sites of the Sp1 transcription factor and retinoic acid receptor in all tumor specimens studied, regardless of the presence or absence of CD38 mRNA. The obtained data suggest that CD38 expression in tumor cells of colorectal cancer patients is heterogeneous.

Insights into the Mechanism of Bovine CD38/NAD+Glycohydrolase from the X-Ray Structures of Its Michaelis Complex and Covalently-Trapped Intermediates

Bovine CD38/NAD+glycohydrolase (bCD38) catalyses the hydrolysis of NAD+ into nicotinamide and ADP-ribose and the formation of cyclic ADP-ribose (cADPR). We solved the crystal structures of the mono N-glycosylated forms of the ecto-domain of bCD38 or the catalytic residue mutant Glu218Gln in their apo state or bound to aFNAD or rFNAD, two 2′-fluorinated analogs of NAD+. Both compounds behave as mechanism-based inhibitors, allowing the trapping of a reaction intermediate covalently linked to Glu218. Compared to the non-covalent (Michaelis) complex, the ligands adopt a more folded conformation in the covalent complexes. Altogether these crystallographic snapshots along the reaction pathway reveal the drastic conformational rearrangements undergone by the ligand during catalysis with the repositioning of its adenine ring from a solvent-exposed position stacked against Trp168 to a more buried position stacked against Trp181. This adenine flipping between conserved tryptophans is a prerequisite for the proper positioning of the N1 of the adenine ring to perform the nucleophilic attack on the C1′ of the ribofuranoside ring ultimately yielding cADPR. In all structures, however, the adenine ring adopts the most thermodynamically favorable anti conformation, explaining why cyclization, which requires a syn conformation, remains a rare alternate event in the reactions catalyzed by bCD38 (cADPR represents only 1% of the reaction products). In the Michaelis complex, the substrate is bound in a constrained conformation; the enzyme uses this ground-state destabilization, in addition to a hydrophobic environment and desolvation of the nicotinamide-ribosyl bond, to destabilize the scissile bond leading to the formation of a ribooxocarbenium ion intermediate. The Glu218 side chain stabilizes this reaction intermediate and plays another important role during catalysis by polarizing the 2′-OH of the substrate NAD+. Based on our structural analysis and data on active site mutants, we propose a detailed analysis of the catalytic mechanism.

Niacin Status Impacts Chromatin Structure 1–3

Niacin is required to form NAD and NADP, which are involved in many essential redox reactions in cellular metabolism. In addition, NAD+ acts as a substrate for a variety of ADP-ribosylation reactions, including poly- and mono-ADP-ribosylation of proteins, formation of cyclic ADP-ribose, and the generation of O-acetyl-ADP-ribose in deacetylation reactions. These nonredox reactions are critical in the regulation of cellular metabolism, and they are sensitive to dietary niacin status. There are 4 known mechanisms by which ADP-ribosylation reactions directly regulate chromatin structure. These include the covalent modification of histones with poly(ADP-ribose), the extraction of histones from chromatin by noncovalent binding to poly(ADP-ribose) on poly(ADP-ribose) polymerase-1, poly ADP-ribosylation of telomeric repeat-binding factor-1 within telomeres, and deacetylation of histones by the sirtuins. These reactions produce a variety of localized effects in chromatin structure, and altered function in response to changes in niacin status may have dramatic effects on genomic stability, cell division and differentiation, and apoptosis.

Niacin Status, NAD Distribution and ADP-Ribose Metabolism

Dietary niacin deficiency, and pharmacological excesses of nicotinic acid or nicotinamide, have dramatic effects on cellular NAD pools, ADP-ribose metabolism, tissue function and health. ADP-ribose metabolism is providing new targets for pharmacological intervention, and it is important to consider how the supply of vitamin B3 may directly influence ADP-ribosylation reactions, or create interactions with other drugs designed to influence these pathways. In addition to its redox roles, NAD+ is used as a substrate for mono-, poly- and cyclic ADP-ribose formation. During niacin deficiency, not all of these processes can be maintained, and dramatic changes in tissue function and clinical condition take place. Conversely, these reactions may be differentially enhanced by pharmacological intakes of vitamin B3, and potentially by changing expression of specific NAD generating enzymes. A wide range of metabolic changes can take place following pharmacological supplementation of nicotinic acid or nicotinamide. As niacin status decreases towards a deficient state, the function of other types of pharmaceutical agents may be modified, including those that target ADP-ribosylation reactions, apoptosis and inflammation. This article will explore what is known and yet to be learned about the response of tissues, cells and subcellular compartments to excessive and limiting supplies of niacin, and will discuss the etiology of the resulting pathologies.

Decreased cADPR and increased NAD + in the Cd38−/− mouse

CD38 is a type II glycoprotein that catalyzes the formation of cyclic ADP-ribose (cADPR), an intracellular calcium signalling molecule, from nicotinamide adenine dinucleotide (NAD +). Using a modified version of the fluorimetric cycling assay for cADPR which reduces between-subject variability, we report significant decreases in brain and lung cADPR, which although similar to previously published values, showed much less individual variation. The reduced variation within each group suggests that the range of cADPR is narrower than previously thought, and that the regulatory mechanisms controlling these levels are more finely tuned. We also report significant increases in brain, lung, and kidney NAD + in the Cd38 −/− mouse, and provide the first experimental demonstration of the proximate relationship between CD38 and NAD +.

ADP Ribosyl Cyclase and Ryanodine Receptors Mediate G‐Protein Coupled Receptor‐Induced Renal Vasoconstriction in vivo

Renal vasoconstriction is mediated by an increase in cytosolic calcium concentration ([Ca2+]i). ADP ribosyl cyclase (ADPR cyclase) activation leads to the formation of cyclic ADP ribose (cADPR) or nicotinic acid ADP (NAADP), both of which result in activation of ryanodine receptors (RYR) on the sarcoplasmic reticulum to cause Ca2+ release. We tested the hypothesis that interruption of the ADPR cyclase / RyR pathway inhibits angiotensin II (Ang II)-, norepinephrine (NE)-, and endothelin-1 (ET-1)- induced renal vasoconstriction. Using an ultrasonic flow transducer, we continuously measured renal blood flow in anesthetized Sprague-Dawley rats. Inhibitory agents were infused into the renal artery 2 min before a 10 μl injection of Ang II, NE, ET-1, or the ETB receptor agonist sarafotoxin (S6c). Infusion of ruthenium red, a RyR inhibitor (50 μg/kg/min), reduced Ang II,-, NE-, and S6c- induced renal vasoconstriction by 60 ± 8% and ET-1- induced constriction by 43 ± 12% (n ≥ 5/ group). Nicotinamide, an ADPR cyclase inhibitor (8 mg/kg/min), reduced Ang II-, NE-, and S6c- induced constriction by 75 ± 2% and ET-1- induced constriction by 43 ± 7%. These results show that Ang II, NE, and ET-1 activate ADPR cyclase and RyR signaling to produce renal vasoconstriction. ET-1 activation of this pathway in the renal microcirculation is likely mediated predominantly by ETB receptors. (This work was funded by NIH research grant HL-02334)

MJ0917 in Archaeon Methanococcus jannaschii Is a Novel NADP Phosphatase/NAD Kinase

NAD kinase phosphorylates NAD(+) to form NADP(+). Conversely, NADP phosphatase, which has not yet been identified, dephosphorylates NADP(+) to produce NAD(+). Among the NAD kinase homologs, the primary structure of MJ0917 of hyperthermophilic archaeal Methanococcus jannaschii is unique. MJ0917 possesses an NAD kinase homologous region in its C-terminal half and an inositol-1-phosphatase homologous region in its N-terminal half. In this study, MJ0917 was biochemically shown to possess both NAD kinase and phosphatase activities toward NADP(+), NADPH, and fructose 1,6-bisphosphate, but not toward inositol 1-phosphate. With regard to the phosphatase activity, kinetic values indicated that NADP(+) is the preferred substrate and that MJ0917 would function as a novel NADP phosphatase/NAD kinase showing conflicting dual activities, viz. synthesis and degradation of an essential NADP(+). Furthermore, in vitro analysis of MJ0917 showed that, although MJ0917 could supply NADP(+), it prevented excess accumulation of NADP(+); thus, it has the ability to maintain a high NAD(+)/NADP(+) ratio, whereas 5'-AMP would decrease this ratio. The evolutionary process during which MJ0917 arose is also discussed.

Hypoxia stimulates Ca 2+ release from intracellular stores in astrocytes via cyclic ADP ribose-mediated activation of ryanodine receptors

The ability of O 2 levels to regulate Ca 2+ signalling in non-excitable cells is poorly understood, yet crucial to our understanding of Ca 2+-dependent cell functions in physiological and pathological situations. Here, we demonstrate that hypoxia mobilizes Ca 2+ from an intracellular pool in primary cultures of cortical astrocytes. This pool can also be mobilized by bradykinin, which acts via phospholipase C and inositol trisphosphate production. By contrast, hypoxic Ca 2+ mobilization utilizes ryanodine receptors, which appear to be either present on the same intracellular pool, or on a separate but functionally coupled pool. Hypoxic activation of ryanodine receptors requires formation of cyclic ADP ribose, since hypoxic Ca 2+ mobilization was fully prevented by nicotinamide (which inhibits ADP ribosyl cyclase) or by 8-Br-cADP ribose, an antagonist of cyclic ADP ribose. Our results demonstrate for the first time the involvement of cyclic ADP ribose in hypoxic modulation of Ca 2+ signalling in the central nervous system, and suggest that this modulator of ryanodine receptors may play a key role in the function of astrocytes under conditions of fluctuating O 2 levels.

Multiple signal transduction pathways mediated by 5-HT receptors.

Among human serotonin (5-HT) receptor subtypes, each G protein-coupled receptor subtype is reported to have one G protein-signaling cascade. However, the signaling may not be as simple as previously thought to be. 5-HT5A receptors are probably the least well understood among the 5-HT receptors, but the authors found that 5-HT5A receptors couple to multiple signaling cascades. When the 5-HT5A receptors were expressed in undifferentiated C6 glioma cells, they modulated the level of second messengers. For example, activation of 5-HT5A receptors inhibited the adenylyl cyclase activity and subsequently reduced the cAMP level, as previously reported. In addition to this known signaling via Gi/Go, 5-HT5A receptors are coupled to the inhibition of ADP-ribosyl cyclase and cyclic ADP ribose formation. On the other hand, activation of 5-HT5A receptors transiently opened the K+ channels, presumably due to the increase in intracellular Ca2+ after formation of inositol (1,4,5) trisphosphate. The K+ currents were inhibited by both heparin and pretreatment with pertussis toxin, suggesting the cross-talk between Gi/Go protein and phopholipase C cascade. Thus, the authors results indicate that 5-HT5A receptors couple to multiple second messenger systems and may contribute to the complicated physiological and pathophysiological states. Although this multiple signaling has been reported only for 5-HT5A/5-HT1 receptors so far, it is possible that other 5-HT receptor subtypes bear similar complexity. As a result, in addition to the wide variety of expression patterns of each 5-HT receptor subtype, it is possible that multiple signal transduction systems may add complexity to the serotonergic system in brain function. The investigation of these serotonergic signaling and its impairment at cellular level may help to understand the symptoms of brain diseases.

Activation of ryanodine receptor/Ca2+ release channels downregulates CD38 in the Namalwa B lymphoma

CD38 is a multifunctional ectoenzyme that catalyses formation of cyclic ADP ribose (cADPr), a second messenger that opens ryanodine receptor (RyR) Ca2+ channels. Despite its importance in signal transduction processes, little is known about the mechanisms regulating CD38 expression levels. In the current study, ryanodine stimulation of Ca2+ release in Namalwa cells decreased both CD38 protein abundance and cyclase activity. Reductions in cyclase activity were prevented by RyR antagonists, by lysosomal blockers, though not by calpain or proteasomal inhibitors. These findings indicate a novel negative feedback mechanism between RyR channel activity and CD38 abundance acts in cADPr signal transduction.

Pathophysiological aspects of cellular pyridine nucleotide metabolism: focus on the vascular endothelium. Review.

In recent years, pyridine nucleotides NAD(H) and NADP(H) have been established as an important molecules in physiological and pathophysiological signaling and cell injury pathways. Protein modification is catalyzed by ADP-ribosyl transferases that attach the ADP-ribose moiety of NAD+ to specific aminoacid residues of the acceptor proteins, with significant changes in the function of these acceptors. Mono(ADP-ribosyl)ation reactions have been implicated to play a role both in physiological responses and in cellular responses to bacterial toxins. Cyclic ADP-ribose formation also utilizes NAD+ and primarily serves as physiological, signal transduction mechanisms regulating intracellular calcium homeostasis. In pathophysiological conditions associated with oxidative stress (such as various forms of inflammation and reperfusion injury), activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) occurs, with subsequent, substantial fall in cellular NAD+ and ATP levels, which can determine the viability and function of the affected cells. In addition, NADPH oxidases can significantly affect the balance and fate of NAD+ and NADP in oxidatively stressed cells and can facilitate the generation of various positive feedback cycles of injury. Under severe oxidant conditions, direct oxidative damage to NAD+ has also been reported. The current review focuses on PARP and on NADPH oxidases, as pathophysiologically relevant factors in creating disturbances in the cellular pyridine nucleotide balance. A separate section describes how these mechanisms apply to the pathogenesis of endothelial cell injury in selected cardiovascular pathophysiological conditions.

Membrane-bound form of ADP-ribosyl cyclase in rat cortical astrocytes in culture.

ADP-ribosyl cyclase activities in cultured rat astrocytes were examined by using TLC for separation of enzymatic products. A relatively high rate of [3H]cyclic ADP-ribose production converted from [3H]NAD+ by ADP-ribosyl cyclase (2.015+/-0.554 nmol/min/mg of protein) was detected in the crude membrane fraction of astrocytes, which contained approximately 50% of the total cyclase activity in astrocytes. The formation rate of [3H]ADP-ribose from cyclic ADP-ribose by cyclic ADP-ribose hydrolase and/or from NAD+ by NAD glycohydrolase was low and enriched in the cytosolic fraction. Although NAD+ in the extracellular medium was metabolized to cyclic ADP-ribose by incubating cultures of intact astrocytes, the presence of Triton X-100 in the medium for permeabilizing cells increased cyclic ADP-ribose production three times as much. Isoproterenol and GTP increased [3H]cyclic ADP-ribose formation in crude membrane-associated cyclase activity. This isoproterenol-induced stimulation of membrane-associated ADP-ribosyl cyclase activity was confirmed by cyclic GDP-ribose formation fluorometrically. This stimulatory action was blocked by prior treatment of cells with cholera toxin but not with pertussis toxin. These results suggest that ADP-ribosyl cyclase in astrocytes has both extracellular and intracellular actions and that signals of beta-adrenergic stimulation are transduced to membrane-bound ADP-ribosyl cyclase via G proteins within cell surface membranes of astrocytes.

Sympathetic Potentiation of Cyclic ADP-ribose Formation in Rat Cardiac Myocytes

We examined the role of cyclic ADP-ribose (cADP-ribose) as a second messenger downstream of adrenergic receptors in the heart after excitation of sympathetic neurons. To address this question, ADP-ribosyl cyclase activity was measured as the rate of [(3)H]cADP-ribose formation from [(3)H]NAD(+) in a crude membrane fraction of rat ventricular myocytes. Isoproterenol at 1 microM increased ADP-ribosyl cyclase activity by 1.7-fold in ventricular muscle; this increase was inhibited by propranolol. The stimulatory effect on the cyclase was mimicked by 10 nM GTP and 10 microM guanosine 5'-3-O-(thio)triphosphate, whereas 10 microM GTP inhibited the cyclase. Cholera toxin blocked the activation of the cyclase by isoproterenol and GTP. The above effects of isoproterenol and GTP in ventricular membranes were confirmed by cyclic GDP-ribose formation fluorometrically. These results demonstrate the existence of a signal pathway from beta-adrenergic receptors to membrane-bound ADP-ribosyl cyclase via G protein in the ventricular muscle cells and suggest that increased cADP-ribose synthesis is involved in up-regulation of cardiac function by sympathetic stimulation.

Cyclic ADP-ribose-mediated insulin secretion and Reg, regenerating gene.

Glucose is the primary stimulus of insulin secretion in pancreatic beta-cells of the islets of Langerhans. CD38 has both ADP-ribosyl cyclase, which catalyzes the formation of cyclic ADP-ribose from NAD+, and cyclic ADP-ribose hydrolase, which converts cyclic ADP-ribose to ADP-ribose. ATP, produced by glucose metabolism, inhibits the cyclic ADP-ribose hydrolase of CD38 and therefore causes cyclic ADP-ribose accumulation in beta-cells. Then, cyclic ADP-ribose acts as a second messenger for Ca2+ mobilization from the endoplasmic reticulum to secrete insulin. The mechanism of insulin secretion as described above is completely different from the conventional hypothesis in which Ca2+ influx from extracellular sources was assumed to play a role in insulin secretion by glucose. On the other hand, strategies for influencing the replication of islet beta-cells and the growth of the beta-cell mass may be more important for ameliorating diabetes. Reg, regenerating gene, is involved in the growth of the beta-cell mass, and Reg protein has been shown to increase the beta-cell mass in a 90% depancreatized diabetic rat model, thereby ameliorating the diabetes. CD38 is involved in the formation of cyclic ADP-ribose and is essential for the glucose sensitivity of beta-cells for insulin secretion. Therefore, CD38 gene and Reg gene will become targets for genetic engineering for diabetic beta-cells.

"The CD38-cyclic ADP-ribose signal system": molecular mechanism and biological significance

Glucose induces an increase in the intracellular Ca2+ concentration in pancreatic beta-cells to secrete insulin. CD38 exists in beta-cells and has both ADP-ribosyl cyclase, which catalyzes the formation of cyclic ADP-ribose (cADPR) from NAD+, and cADPR hydrolase, which converts cADPR to ADP-ribose. ATP, produced by glucose metabolism, competes with cADPR for the binding site, Lys-129, of CD38, resulting in the inhibition of the hydrolysis of cADPR and thereby causing cADPR accumulation in beta-cells. cADPR then binds to FK506-binding protein 12.6 (FKBP 12.6) in the islet type of the ryanodine receptor (RyR), dissociating the binding protein from RyR to induce the release of Ca2+ from the endoplasmic reticulum. Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) phosphorylates RyR to sensitize and activate the Ca2+ channel. Ca2+, released from the RyR, further activates CaM kinase II and amplifies this process. Thus, cADPR acts as a second messenger for Ca2+ mobilization to secrete insulin. The novel mechanism of insulin secretion described above is different from the conventional hypothesis in which Ca2+ influx from extracellular sources plays a role in insulin secretion by glucose. Furthermore, many physiological and pathological phenomena in various tissues and cells such as cardiac muscles, cerebellum, neuronal cells, pancreatic acinar cells, alveolar macrophages and immune B-cells become understandable in terms of "the CD38-cADPR signaling system" that sometimes acts in cooperation with other signal systems.

Stimulation of ADP-ribosyl cyclase activity of the cell surface antigen CD38 by zinc ions resulting from inhibition of its NAD+ glycohydrolase activity.

The lymphocyte cell surface antigen, CD38, which has an amino acid sequence similar to Aplysia ADP-ribosyl cyclase, catalyzes not only the hydrolysis of NAD+ and 1-(5-phospho-beta-D-ribosyl) adenosine 5'-phosphate cyclic anhydride (cyclic ADP-ribose) but also the formation of cyclic ADP-ribose from NAD+. To characterize the bifunctional enzyme properties, we produced the recombinant CD38 fused with a maltose-binding protein (MBP-CD38). Zinc ions stimulated the ADP-ribosyl cyclase activity of MBP-CD38, but inversely inhibited its NAD+ glycohydrolase activity which was approximately 100-fold dominant to the cyclase activity in the absence of Zn2+. Such dual effects of Zn2+ were also observed in the native membrane-bound CD38 of HL-60 cells which had been caused to differentiate by retinoic acid. Zinc ions inhibited the NAD+ glycohydrolase reaction catalyzed by MBP-CD38 in an uncompetitive manner, whereas they enhanced the ADP-ribosyl cyclase reaction without affecting the Km value for NAD+. There was an increase in the fluorescence intensity of a hydrophobic fluorescent probe, 8-anilino-1-naphthalenesulfonate, in the presence of MBP-CD38. The fluorescence increase was further enhanced by the addition of Zn2+ with a shift in the maximum emission wavelength from 484 nm to 470 nm, suggesting that Zn2+ caused conformational changes of MBP-CD38. These results indicate that Zn2+ directly interacts with CD38 to stimulate its ADP-ribosyl cyclase with inhibition of its NAD+ glycohydrolase, probably due to prevention of the access of water molecule to an intermediate of the enzymesubstrate complex.

The Kinetics of Cyclic ADP-Ribose Formation in Heart Muscle

The kinetics of NAD glycohydrolase activity in cardiac muscle homogenates were investigated using HPLC and TLC techniques to identify reaction products. NAD was found to undergo enzymatic hydrolysis yielding ADP-ribose (ADPR) as well as a cyclase reaction resulting in the formation of cyclic ADP-ribose (cADPR). Both ADPR and cADPR were further converted into adenine and ADPR, respectively. The kinetics of the formation reactions, that manifested a clearly distinguishable initial lag phase in the time course of cADPR formation, suggest that the hydrolytic reaction yielding ADPR and the cyclase reaction yielding cADPR probably represent alternative catalytic functions of the same enzyme, which might be controlled by endogenous ADP-ribosylation reactions.

Mechanistic Implications of Cyclic ADP-Ribose Hydrolysis and Methanolysis Catalyzed by Calf Spleen NAD+Glycohydrolase

Calf spleen NAD+glycohydrolase, besides its well known reactions, was shown to catalyze hydrolysis and methanolysis (with retention of configuration) of cyclic ADP-ribose, indicating that this classical NADase also belongs to the class of cyclic ADP-ribose hydrolases. No formation of cyclic ADP-ribose could be detected during the hydrolysis of β-NAD+; moreover, the kinetic parameters of cyclic ADP-ribose seem to rule out that it is a kinetically competent reaction intermediate in the conversion of NAD+ into ADP-ribose by this enzyme.