Determination of ADP-Ribosyl Cyclase Activity, Cyclic ADP-Ribose, and Nicotinic Acid Adenine Dinucleotide Phosphate in Tissue Extracts

The novel Ca2+-mobilizing second messengers cADPr (cyclic ADP-ribose) and NAADP (nicotinic acid–adenine dinucleotide phosphate) are both synthesized by ADP-ribosyl cyclases. Using HSR (heavy sarcoplasmic reticulum) fractions from rabbit skeletal muscle, NAADP-induced Ca2+ release was observed. In the present paper, we show in HSR membranes the formation of authentic cADPr, cGDPr (cyclic GDP-ribose) and NAADP. The cyclization reaction to form cADPr and cGDPr as well as the base-exchange reaction to form NAADP were strictly dependent on pH. Although the formation of cGDPr is optimized at pH 6, the synthesis of NAADP was most pronounced at a pH below 5. A novel regulation mechanism is provided for nicotinic acid, the co-substrate for NAADP synthesis. Nicotinic acid had virtually no influence on the cyclization reaction, but increased the affinity of NADP at an acidic pH and had the opposite effect at alkaline pH. Nicotinamide, the side product of cADPr synthesis, is an inhibitor of the cyclization reaction (IC50, 0.7±0.1 mM) and was 30-fold more potent at suppressing the base-exchange reaction. Although the synthesis of NAADP was highly sensitive to nicotinamide inhibition, this was not via a competition with the nicotinic-acid-binding site. In contrast with the ecto-ADP-ribosyl cyclase (CD38), the cyclization and base-exchange reaction of the skeletal muscle isoform was inhibited by Cu2+ and Zn2+, while other bivalent cations such as Ca2+, Mg2+ and Mn2+ had virtually no effect. These findings allow for the prediction of a novel ADP-ribosyl cyclase isoform in skeletal muscle HSR, other than CD38. Hence the enzymic prerequisite for cADPr- and NAADP-mediated Ca2+ signalling is present.

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Oscillation of ADP-ribosyl cyclase activity during the cell cycle and function of cyclic ADP-ribose in a unicellular organism, Euglena gracilis

FEBS Letters ◽

10.1016/s0014-5793(97)00168-3 ◽

1997 ◽

Vol 405 (1) ◽

pp. 104-106 ◽

Cited By ~ 31

Author(s):

Wataru Masuda ◽

Shigeo Takenaka ◽

Kiyoshi Inageda ◽

Hiroshi Nishina ◽

Katsunobu Takahashi ◽

...

Keyword(s):

Cell Cycle ◽

Euglena Gracilis ◽

Cyclase Activity ◽

Unicellular Organism ◽

Adp Ribosyl Cyclase ◽

Cyclic Adp Ribose ◽

And Function

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Catalytic properties of the retinal rod outer segment disk ADP-ribosyl cyclase

Visual Neuroscience ◽

10.1017/s0952523810000404 ◽

2011 ◽

Vol 28 (2) ◽

pp. 121-128 ◽

Cited By ~ 3

Author(s):

ANDREA FABIANO ◽

ISABELLA PANFOLI ◽

DANIELA CALZIA ◽

MAURIZIO BRUSCHI ◽

SILVIA RAVERA ◽

...

Keyword(s):

Divalent Cations ◽

Cyclase Activity ◽

The Novel ◽

Intracellular Membrane ◽

Adp Ribosyl Cyclase ◽

Retinal Rod ◽

Cyclic Adp Ribose ◽

Response To Change ◽

Shed Light

AbstractCyclic ADP-ribose (cADPR) is a second messenger modulating intracellular calcium levels. We have previously described a cADPR-dependent calcium signaling pathway in bovine rod outer segments (ROS), where calcium ions play a pivotal role. ROS ADP-ribosyl cyclase (ADPR-cyclase) was localized in the membrane fraction. In the present work, we examined the properties of the disk ADPR-cyclase through the production of cyclic GDP-ribose from the NAD+ analogue NGD+. The enzyme displayed an estimated Km for NGD+ of 12.5 ± 0.3 μM, a Vmax of 26.50 ± 0.70 pmol cyclic GDP-ribose synthesized/min/mg, and optimal pH of 6.5. The effect of divalent cations (Zn2+, Cu2+, and Ca2+) was also tested. Micromolar Zn2+ and Cu2+ inhibited the disk ADPR-cyclase activity (half maximal inhibitory concentration, IC50 = 1.1 and 3.6 μM, respectively). By contrast, Ca2+ ions had no effect. Interestingly, the properties of the intracellular membrane–associated ROS disk ADPR-cyclase are more similar to those of the ADPR-cyclase found in CD38-deficient mouse brain, than to those of CD38 or CD157. The novel intracellular mammalian ADPR-cyclase would elicit Ca2+ release from the disks at various rates in response to change in free Ca2+ concentrations, caused by light versus dark adaptation, in fact there was no difference in disk ADPR-cyclase activity in light or dark conditions. Data suggest that disk ADPR-cyclase may be a potential target of retinal toxicity of Zn2+ and may shed light to the role of Cu2+ and Zn2+ deficiency in retina.

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Identification of bovine liver mitochondrial NAD+ glycohydrolase as ADP-ribosyl cyclase

Biochemical Journal ◽

10.1042/bj3260401 ◽

1997 ◽

Vol 326 (2) ◽

pp. 401-405 ◽

Cited By ~ 42

Author(s):

Mathias ZIEGLER ◽

Dierk JORCKE ◽

Manfred SCHWEIGER

Keyword(s):

Calcium Release ◽

Bovine Liver ◽

Enzymic Activity ◽

Cyclase Activity ◽

Partial Recovery ◽

Nad Glycohydrolase ◽

Adp Ribosyl Cyclase ◽

Cyclic Adp Ribose ◽

Hydrolysis Of ◽

Synthesis And Degradation

The present investigation identifies bovine liver mitochondrial NADase (NAD+ glycohydrolase) as a member of the class of bifunctional ADP-ribosyl cyclases/cyclic ADP-ribose hydrolases, known to be potential second messenger enzymes. These enzymes catalyse the synthesis and degradation of cyclic ADP-ribose, a potent intracellular calcium-mobilizing agent. The mitochondrial enzyme utilized the NAD+ analogues nicotinamide guanine dinucleotide (NGD+) and nicotinamide hypoxanthine dinucleotide (NHD+) to form fluorescent cyclic purine nucleoside diphosphoriboses. ADP-ribosyl cyclase activity was also demonstrated using 32P-labelled NAD+ as substrate. The identity of NADase and ADP-ribosyl cyclase was supported by their co-migration in SDS/polyacrylamide gels. Cyclase activity was visualized directly within the gel by detecting the formation of fluorescent cyclic IDP-ribose from NHD+. The enzyme catalysed the hydrolysis of cyclic ADP-ribose to ADP-ribose. Moreover, in the presence of nicotinamide and cyclic ADP-ribose the enzyme synthesized NAD+. Both the ADP-ribosyl cyclase and NADase activities of the enzyme were strongly inhibited by reducing agents. Treatment of the NADase with dithiothreitol caused the apparent inactivation of the enzyme. Subsequent removal of the reducing agent and addition of oxidized glutathione led to a partial recovery of enzymic activity. The results support a model for pro-oxidant-induced calcium release from mitochondria involving cyclic ADP-ribose as a specific messenger, rather than the non-enzymic modification of proteins by ADP-ribose.

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Angiotensin II stimulates cyclic ADP-ribose formation in neonatal rat cardiac myocytes

Biochemical Journal ◽

10.1042/bj3520197 ◽

2000 ◽

Vol 352 (1) ◽

pp. 197-202 ◽

Cited By ~ 19

Author(s):

Haruhiro HIGASHIDA ◽

Jia-Sheng ZHANG ◽

Minako HASHII ◽

Miyuki SHINTAKU ◽

Chiharu HIGASHIDA ◽

...

Keyword(s):

Angiotensin Ii ◽

Ventricular Myocytes ◽

Receptor Activation ◽

Neonatal Rat ◽

Cyclase Activity ◽

Ventricular Muscle ◽

Ang Ii ◽

Adult Rats ◽

Adp Ribosyl Cyclase ◽

Cyclic Adp Ribose

To examine the role of cyclic ADP-ribose (cADP-ribose) as a second messenger downstream of angiotensin II (Ang II) receptor activation in the heart, ADP-ribosyl cyclase activity was measured in a crude membrane fraction of ventricular myocytes. Ang II at 10–100nM increased ADP-ribosyl cyclase activity by 40–90% in the ventricular muscle of neonatal (2–4-day-old) rats, but not in fetal or adult hearts. This increase was inhibited by the Ang II antipeptide. Stimulation of ADP-ribosyl cyclase was reproduced by GTP and guanosine 5´-[γ-thio]triphosphate, and prevented by guanosine 5´-[β-thio]diphosphate. Prior treatment of the rats with cholera toxin A and B subunits also blocked the Ang II-induced activation. The density of Ang II receptors detected as [3H]Ang II binding was higher in neonatal than adult rats. These results demonstrate the existence of a signalling pathway from Ang II receptors to membrane-bound ADP-ribosyl cyclase in the ventricular muscle cell and suggest that the Ang II-induced increase in cADP-ribose synthesis is involved in the regulation of cardiac function and development.

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A Single Residue in a Novel ADP-ribosyl Cyclase Controls Production of the Calcium-mobilizing Messengers Cyclic ADP-ribose and Nicotinic Acid Adenine Dinucleotide Phosphate

Journal of Biological Chemistry ◽

10.1074/jbc.m110.105312 ◽

2010 ◽

Vol 285 (26) ◽

pp. 19900-19909 ◽

Cited By ~ 8

Author(s):

Latha Ramakrishnan ◽

Hélène Muller-Steffner ◽

Christophe Bosc ◽

Victor D. Vacquier ◽

Francis Schuber ◽

...

Keyword(s):

Nicotinic Acid ◽

Adp Ribosyl Cyclase ◽

Cyclic Adp Ribose

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CD38-dependent ADP-ribosyl cyclase activity in developing and adult mouse brain

Biochemical Journal ◽

10.1042/bj20020604 ◽

2003 ◽

Vol 370 (1) ◽

pp. 175-183 ◽

Cited By ~ 40

Author(s):

Claire CENI ◽

Nathalie POCHON ◽

Virginie BRUN ◽

Hélène MULLER-STEFFNER ◽

Annie ANDRIEUX ◽

...

Keyword(s):

Developmental Stages ◽

The Body ◽

Adult Brain ◽

Cyclase Activity ◽

Wild Type ◽

Intracellular Location ◽

Nad Glycohydrolase ◽

Adp Ribosyl Cyclase ◽

Activity Measurements ◽

Cyclic Adp Ribose

CD38 is a transmembrane glycoprotein that is expressed in many tissues throughout the body. In addition to its major NAD+-glycohydrolase activity, CD38 is also able to synthesize cyclic ADP-ribose, an endogenous calcium-regulating molecule, from NAD+. In the present study, we have compared ADP-ribosyl cyclase and NAD+-glycohydrolase activities in protein extracts of brains from developing and adult wild-type and Cd38-/- mice. In extracts from wild-type brain, cyclase activity was detected spectrofluorimetrically, using nicotinamide—guanine dinucleotide as a substrate (GDP-ribosyl cyclase activity), as early as embryonic day 15. The level of cyclase activity was similar in the neonate brain (postnatal day 1) and then increased greatly in the adult brain. Using [14C]NAD+ as a substrate and HPLC analysis, we found that ADP-ribose is the major product formed in the brain at all developmental stages. Under the same experimental conditions, neither NAD+-glycohydrolase nor GDP-ribosyl cyclase activity could be detected in extracts of brains from developing or adult Cd38-/- mice, demonstrating that CD38 is the predominant constitutive enzyme endowed with these activities in brain at all developmental stages. The activity measurements correlated with the level of CD38 transcripts present in the brains of developing and adult wild-type mice. Using confocal microscopy we showed, in primary cultures of hippocampal cells, that CD38 is expressed by both neurons and glial cells, and is enriched in neuronal perikarya. Intracellular NAD+-glycohydrolase activity was measured in hippocampal cell cultures, and CD38-dependent cyclase activity was higher in brain fractions enriched in intracellular membranes. Taken together, these results lead us to speculate that CD38 might have an intracellular location in neural cells in addition to its plasma membrane location, and may play an important role in intracellular cyclic ADP-ribose-mediated calcium signalling in brain tissue.

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ADP-ribosyl cyclase: an enzyme that cyclizes NAD+ into a calcium-mobilizing metabolite.

Cell Regulation ◽

10.1091/mbc.2.3.203 ◽

1991 ◽

Vol 2 (3) ◽

pp. 203-209 ◽

Cited By ~ 255

Author(s):

H C Lee ◽

R Aarhus

Keyword(s):

Sea Urchin ◽

Exchange Chromatography ◽

Reaction Products ◽

Sea Urchin Eggs ◽

Tissue Extracts ◽

Adp Ribosyl Cyclase ◽

Sea Urchin Egg ◽

Sds Page ◽

Cyclic Adp Ribose ◽

Charge Analysis

Cyclic ADP-ribose (cADPR) is a metabolite of NAD+ that is as active as inositol trisphosphate (IP3) in mobilizing intracellular Ca2+ in sea urchin eggs. The activity of the enzyme responsible for synthesizing cADPR is found not only in sea urchin eggs but also in various mammalian tissue extracts, suggesting that cADPR may be a general messenger for Ca2+ mobilization in cells. An aqueous soluble enzyme, thought to be an NADase, has been purified recently from the ovotestis of Aplysia californica (Hellmich and Strumwasser, 1991). This paper shows that the Aplysia enzyme catalyzes the conversion of NAD+ to cADPR and nicotinamide. The Aplysia enzyme was purified by fractionating the soluble extract of Aplysia ovotestis on a Spectra/gel CM column. The purified enzyme appeared as a single band of approximately 29,000 Da on SDS-PAGE but could be further separated into multiple peaks by high-resolution, cation-exchange chromatography. All of the protein peaks had enzymatic activity, indicating that the enzyme had multiple forms differing by charge. Analysis of the reaction products of the enzyme by anion-exchange high-pressure liquid chromatography (HPLC) indicated no ADP-ribose was produced; instead, each mole of NAD+ was converted to equimolar of cADPR and nicotinamide. The identification of the product as cADPR was further substantiated by proton NMR and also by its Ca(2+)-mobilizing activity. Addition of the product to sea urchin egg homogenates induced Ca2+ release and desensitized the homogenate to authentic cADPR but not to IP3. Microinjection of the product into sea urchin eggs elicited Ca2+ transients as well as the cortical exocytosis reaction. Therefore, by the criteria of HPLC, NMR, and calcium-mobilizing activity, the product was identical to cADPR. To distinguish the Aplysia enzyme from the conventional NADases that produce ADP-ribose, we propose to name it ADP-ribosyl cyclase.

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