Calcium mobilization by dual receptors during fertilization of sea urchin eggs.

Fertilization is accompanied by a transient increase in the concentration of intracellular Ca2+, which serves as a signal for initiating development. Some of the Ca2+ appears to be released from intracellular stores by the binding of inositol trisphosphate (IP3) to its receptor. However, in sea urchin eggs, other mechanisms appear to participate. Cyclic adenosine diphosphate--ribose (cADPR), a naturally occurring metabolite of nicotinamide adenine dinucleotide, is as potent as IP3 in mobilizing Ca2+ in sea urchin eggs. Experiments with antagonists of the cADPR and IP3 receptors revealed that both Ca2+ mobilizing systems were activated during fertilization. Blockage of either of the systems alone was not sufficient to prevent the sperm-induced Ca2+ transient. This study provides direct evidence for a physiological role of cADPR in the Ca2+ signaling process.

Wide distribution of an enzyme that catalyzes the hydrolysis of cyclic ADP-ribose.

Cyclic ADP-ribose (cADPR) is a metabolite of NAD+ that is as effective as inositol trisphosphate in mobilizing intracellular-Ca2+ stores. Its synthesizing enzyme, ADP-ribosyl cyclase, has been shown to be present in mammalian and invertebrate tissues. In this study we identify another widely-distributed enzyme that can hydrolyze cADPR to ADP-ribose. Incubation of cADPR with brain extracts resulted in progressive decrease in its Ca2+ mobilizing activity. The degradation of cADPR was catalyzed by a heat-labile protein factor in the brain extracts. Analysis by HPLC indicated a single degradation product was produced in equal molar quantity and that it has identical elution time as ADP-ribose. Proton NMR confirmed that the product was ADP-ribose. The degradation enzyme had a Michaelis constant of 0.16 mM and a broad pH maximum around neutrality. Centrifugation studies of the total brain extracts showed that the degradation activity was membrane-bound. Survey of tissues from various animals established that both the degradation and the synthesizing enzyme of cADPR were widely distributed from mammals to invertebrates. Since the degradation enzyme hydrolyzes an unique linkage between the adenine group and the terminal ribosyl moiety of cADPR, we propose to call it cyclic ADP-ribose hydrolase.

Fluorescent analogs of NAADP with calcium mobilizing activity.

Nicotinic acid adenine dinucleotide phosphate (NAADP) mobilizes Ca2+ through a mechanism totally independent of cyclic ADP-ribose or inositol trisphosphate. Fluorescent analogs of NAADP were synthesized in this study to facilitate further characterization of this novel Ca2+ release mechanism. The base-exchange reaction catalyzed by ADP-ribosyl cyclase was utilized to convert nicotinamide 1,N6-ethenoadenine dinucleotide phosphate to a fluorescent product, nicotinic acid 1,N6-ethenoadenine dinucleotide phosphate (etheno-NAADP). The excitation spectrum of the product showed two maxima at 275 nm and 300 nm and an emission maximum at 410 nm. An aza derivative of etheno-NAADP was also synthesized by sequential treatments with NaOH and nitrite. The product, nicotinic acid 1,N6-etheno-2-aza-adenine dinucleotide phosphate (etheno-aza-NAADP) had excitation maxima at 280 nm and 360 nm and an emission maximum at 470 nm. The fluorescence of both analogs was sensitive to polarity and exhibited a 3-4-fold enhancement going from an aqueous buffer to an organic solvent. Proton-NMR measurements confirmed the presence of the etheno ring in both analogs. In the aza derivative the proton at the 2-position of the adenine ring was absent, consistent with the conversion of the 2-carbon to a nitrogen. Both analogs could activate Ca2+ release from sea urchin egg homogenates and the half-maximal concentrations for etheno-aza-NAADP and etheno-NAADP were at about 2.5 microM and 5 microM, respectively. At sub-threshold concentrations, both analogs could also function as antagonists, inactivating the NAADP-sensitive Ca2+ release with a half-maximal concentration of 60-80 nM. Microinjection of etheno-aza-NAADP into live eggs activated Ca2+ increase and triggered a cortical exocytotic reaction confirming its effectiveness in vivo. These fluorescent analogs are potentially useful for visualizing the novel Ca2+ stores that are sensitive to NAADP in live cells.

Cyclic 3-deaza-adenosine diphosphoribose: a potent and stable analog of cyclic ADP-ribose.

Cyclic 3-deaza-adenosine diphosphoribose (3-deaza-cADPR), an analog of cyclic adenosine diphosphoribose (cADPR) was synthesized. 3-deaza-cADPR differs from cADPR by only the substitution of carbon for nitrogen at the 3-position of the purine ring. Similar to cADPR, the analog has potent calcium releasing activity in sea urchin egg homogenates and was able to induce calcium release at concentrations as low as 0.3 nM. The EC(50) value for 3-deaza-cADPR-induced calcium release was 1 nM, which is about 70 times more potent than cADPR. The properties of calcium release induced by 3-deaza-cADPR in all other respects were similar to those of cADPR. Thus, 3-deaza-cADPR and cADPR were capable of cross-desensitizing each other and their calcium releasing activities were potentiated by Sr(2+) as well as caffeine. 8-amino-cADPR, a selective antagonist of cADPR, was also able to inhibit 3-deaza-cADPR induced calcium release. Taken together, these data suggest that 3-deaza-cADPR releases calcium through the same mechanism as cADPR. 3-deaza-cADPR was found to be resistant to both heat and enzymatic hydrolysis. Only 15% of 3-deaza-cADPR was destroyed after boiling this compound for 2 h. No loss of 3-deaza-cADPR was observed when treated with CD38 under conditions where cADPR was completely hydrolyzed. Thus, 3-deaza-cADPR is a potent and stable analog of cADPR. These properties should make 3-deaza-cADPR a useful probe in studies focused on the mechanism of cADPR action.

Determination of endogenous levels of cyclic ADP-ribose in rat tissues.

Cyclic ADP-ribose (cADPR) is a potent mediator of calcium mobilization in sea urchin eggs. The cADPR synthesizing enzyme is present not only in the eggs but also in various mammalian tissue extracts. The purpose of this study was to ascertain whether cADPR is a naturally occurring nucleotide in mammalian tissues. Rat tissues were frozen and powdered in liquid N2, followed by extraction with perchloric acid at -10 degrees C. [32P]cADPR was prepared and used as a tracer. The acid extracts were chromatographed on a Mono-Q column and cADPR in the fractions were determined by its ability to release Ca2+ from egg homogenates. That the release was mediated by cADPR and not inositol trisphosphate (IP3) in the extracts was shown by the fact that the homogenates, subsequent to Ca2+ release induced by active fractions, were desensitized to authentic cADPR but not to IP3. Furthermore, the Ca2+ release activity was shown to co-elute with [32P]cADPR. The endogenous level of cADPR determined in rat liver is 3.37 +/- 0.64 pmol/mg, in heart is 1.04 +/- 0.08 pmol/mg and in brain is 2.75 +/- 0.35 pmol/mg. These results indicate cADPR is a naturally occurring nucleotide and suggest that it may be a general second messenger for mobilizing intracellular Ca2+.

Thio-NADP is not an antagonist of NAADP.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a metabolite of NADP, which can release Ca2+ from stores that are distinct from those activated by either cyclic ADP-ribose or inositol 1,4,5-trisphosphate (IP3). It has previously been suggested that thio-NADP is a specific antagonist of NAADP (Chini et al. [1995] J. Biol. Chem. 270, 3216-3223). Its effects in sea-urchin egg homogenates were investigated. At 50 microM, thio-NADP activates partial Ca2+ release and totally inhibits subsequent challenge with a saturating concentration of NAADP. Purification by HPLC eliminates the Ca2+ releasing activity of 50 microM thio-NADP and reduces the subsequent inhibition by 73.7 +/- 1.3%. The residual inhibitory effect is no more than that exerted by 50 microM of either NADP itself or nicotinic acid adenine dinucleotide (NAAD). These results are confirmed by 32P-NAADP binding studies. Unpurified thio-NADP inhibits the specific 32P-NAADP binding to egg microsomes with an IC50 of 40 microM. After HPLC purification, only 20% inhibition is seen at a concentration as high as 50 microM, similar to the extent of inhibition effected by 40 microM NADP. These results indicate the inhibitory substance in thio-NADP is a contaminant. The partial Ca2+ release activity of unpurified thio-NADP suggests the contaminant is NAADP itself. This is supported by the fact that pretreatment with a subthreshold concentration of only 2 nM NAADP totally desensitizes the egg homogenates such that no Ca2+ response is seen with saturating NAADP. Estimation from the binding studies shows that a contamination of 0.012% of NAADP in the unpurified thio-NADP samples is sufficient to account for the inhibitory effects. These results indicate thio-NADP is not an antagonist of NAADP.

Functional visualization of the separate but interacting calcium stores sensitive to NAADP and cyclic ADP-ribose.

Cells possess multiple Ca(2+) stores and their selective mobilization provides the spatial-temporal Ca(2+) signals crucial in regulating diverse cellular functions. Except for the inositol trisphosphate (IP(3))-sensitive Ca(2+) stores, the identities and the mechanisms of how these internal stores are mobilized are largely unknown. In this study, we describe two Ca(2+) stores, one of which is regulated by cyclic ADP-ribose (cADPR) and the other by nicotinic acid adenine dinucleotide phosphate (NAADP). We took advantage of the large size of the sea urchin egg and stratified its organelles by centrifugation. Using photolysis to produce either uniform or localized increases of cADPR and NAADP from their respective caged analogs, the two separate stores could be visually identified by Ca(2+) imaging and shown to be segregated to the opposite poles of the eggs. The cADPR-pole also contained the IP(3)-sensitive Ca(2+) stores, the egg nucleus and the endoplasmic reticulum (ER); the latter was visualized using Bodipy-thapsigargin. On the other hand, the mitochondria, as visualized by rhodamine 123, were segregated to the opposite pole together with the NAADP-sensitive calcium stores. Fertilization of the stratified eggs elicited a Ca(2+) wave starting at the cADPR-pole and propagating toward the NAADP-pole. These results provide the first direct and visual evidence that the NAADP-sensitive Ca(2+) stores are novel and distinct from the ER. During fertilization, communicating signals appear to be transmitted from the ER to NAADP-sensitive Ca(2+) stores, leading to their activation.

Structural determinants of nicotinic acid adenine dinucleotide phosphate important for its calcium-mobilizing activity.

Nicotinic acid adenine dinucleotide phosphate (NAADP) mobilizes Ca2+ through a mechanism totally independent of cyclic ADP-ribose or inositol trisphosphate. The structural determinants important for its Ca2+ release activity were investigated using a series of analogs. It is shown that changing the 3-carboxyl group of the nicotinic acid (NA) moiety in NAADP to either an uncharged carbinol or from the 3-position to the 4-position of the pyridine ring totally eliminates the Ca2+ release activity. Conversion of the 3-carboxyl to other negatively charged groups, either 3-sulfonate, 3-acetate, or 3-quinoline carboxylate, retains the Ca2+ release activity, although their half-maximal effective concentrations (EC50) are 100-200-fold higher. Changing the 6-amino group of the adenine to a hydroxyl group results in more than a 1000-fold decrease in the Ca2+ release activity. Conversion of the 2'-phosphate to 2',3'-cyclic phosphate or 3'-phosphate likewise increases the EC50 by about 5- and 20-fold, respectively. Similar to NAADP, all of the active analogs can also desensitize the Ca2+ release mechanism at subthreshold concentrations, suggesting that this novel property is intrinsic to the release mechanism. The series of analogs used was produced by using ADP-ribosyl cyclase to catalyze the exchange of the nicotinamide group of various analogs of NADP with various analogs of NA. An important determinant in NA that is crucial to the base exchange reaction was shown to be the 2-position of the pyridine ring. Neither pyridine-2-carboxylate nor 2-methyl-NA support the exchange reaction. The negative charge and the position of the 3-carboxyl group are nonessential since both pyridine-3-carbinol and pyridine-4-carboxylate support the base exchange reaction. In addition to the information on the structure-activity relationships of NAADP and NA, this study also demonstrates the utility of the base exchange reaction as a general approach for synthesizing NAADP analogs.

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

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.

A derivative of NADP mobilizes calcium stores insensitive to inositol trisphosphate and cyclic ADP-ribose.

We have previously shown that alkaline treatment of NADP generates a derivative which can mobilize Ca2+ from sea urchin egg homogenates (Clapper, D. L., Walseth, T. F., Dargie, P. J., and Lee, H. C. (1987) J. Biol. Chem. 262, 9561-9568). In this study, the active derivative was purified and shown by high pressure liquid chromatography to be distinct from NADP and NADPH. However, its proton NMR spectrum was virtually identical to that of NADP. The mass of its molecular ion was measured by high resolution mass spectrometry to be 743.0510, one mass unit larger than the corresponding ion of NADP. These results are consistent with the active derivative being nicotinic acid adenine dinucleotide phosphate (NAADP). Ca2+ release induced by NAADP was saturable with a half-maximal concentration of about 30 nM. The release was specific since NADP and nicotinic acid adenine dinucleotide were ineffective even at 10-40-fold higher concentrations. The NAADP-dependent Ca2+ release showed desensitization and was insensitive to heparin and a specific antagonist of cyclic ADP-ribose (cADPR), 8-amino-cADPR. The release mechanism did not require calmodulin. This is similar to the inositol trisphosphate-sensitive release but distinct from that of cADPR. That the NAADP-sensitive Ca2+ stores were different from those sensitive to inositol trisphosphate- or cADPR was further indicated by their differences in distribution on Percoll density gradients. Microinjection of NAADP into live sea urchin eggs induced transient elevation of intracellular Ca2+ and triggered the cortical reaction, indicating the NAADP-dependent mechanism is operative in intact cells.

Sensitization of calcium-induced calcium release by cyclic ADP-ribose and calmodulin.

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 125I-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.

Caged cyclic ADP-ribose. Synthesis and use.

Cyclic ADP-ribose (cADPR) is a recently discovered cyclic nucleotide with Ca2+ mobilizing activity. Caged cADPR was synthesized by reacting cADPR with 2-nitrophenethyldiazoethane. Elemental analyses, 1H NMR, and extinction coefficient measurements indicate that the product contains only one caging group. Anion exchange high pressure liquid chromatography separated caged cADPR into two forms, which most likely represent isomers. Both forms could be uncaged with equal efficiency by UV exposure to regenerate cADPR. Photolysis of caged cADPR was accomplished effectively with a spectrofluorimeter. The efficiency of uncaging depended on wavelength with UV light shorter than about 320 nm being the most effective. Caged cADPR was biologically inactive and could induce Ca2+ release from sea urchin egg homogenates only after photolysis. Specificity of the Ca2+ release was shown by inhibition by 8-amino-cADPR, a specific antagonist of cADPR. To demonstrate its utility in live cells, caged cADPR was microinjected into sea urchin eggs. Photolysis using a mercury light source effectively regenerated cADPR and resulted in Ca2+ mobilization and activation of cortical exocytosis in the eggs.

Identification of cyclic ADP-ribose-binding proteins by photoaffinity labeling.

We have synthesized 8-azido-cyclic ADP-ribose (8N3-cADPR) and [32P]8-azido-cyclic ADP-ribose ([32P]8N3-cADPR) in order to characterize cyclic ADP-ribose-(cADPR) binding sites in sea urchin egg homogenates. 8N3-cADPR was an antagonist of cADPR since it did not induce Ca2+ release from egg microsomes but did inhibit the ability of cADPR to do so. The effect of 8N3-cADPR was reversible and could be overcome by high concentrations of cADPR, suggesting that both were acting on the same site. This was supported by the fact that 8N3-cADPR effectively competed for [32P]cADPR binding to microsomes. Reciprocally, binding of [32P]8N3-cADPR could also be selectively displaced by cADPR and 8N3-cADPR, but not by ADP-ribose. These results indicate that 8N3-cADPR binds specifically to the cADPR-binding sites and inhibits cADPR from releasing Ca2+. Photolysis of microsomes preincubated with [32P]8N3-cADPR resulted in specific labeling of proteins of 140 and 100 kDa, which could be prevented by 8N3-cADPR or nanomolar concentrations of cADPR, but not by micromolar concentrations of ADP-ribose, AMP, ADP, ATP, cyclic AMP or inositol 1,4,5-trisphosphate. Caffeine, an agonist of Ca(2+)-induced Ca2+ release, preferentially inhibited the labeling of the 100 kDa as compared to the 140-kDa protein. These results suggest that cADPR may not interact directly with the ryanodine receptor, but may instead, exert its effect through intermediate proteins.

Identification of the enzymatic active site of CD38 by site-directed mutagenesis.

CD38 is a ubiquitous protein originally identified as a lymphocyte antigen and recently also found to be a multifunctional enzyme participating in the synthesis and metabolism of two Ca(2+) messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate. It is homologous to Aplysia ADP-ribosyl cyclase, where the crystal structure has been determined. Residues of CD38 corresponding to those at the active site of the Aplysia cyclase were mutagenized. Changing Glu-226, which corresponded to the catalytic residue of the cyclase, to Asp, Asn, Gln, Leu, or Gly eliminated essentially all enzymatic activities of CD38, indicating it is most likely the catalytic residue. Photoaffinity labeling showed that E226G, nevertheless, retained substantial NAD binding activity. The secondary structures of these inactive mutants as measured by circular dichroism were essentially unperturbed as compared with the wild type. Other nearby residues were also investigated. The mutants D147V and E146L showed 7- and 19-fold reduction in NADase activity, respectively. The cADPR hydrolase activity of the two mutants was similarly reduced. Asp-155, on the other hand, was crucial for the GDP-ribosyl cyclase activity since its substitution with either Glu, Asn, or Gln stimulated the activity 3-15-fold, whereas other activities remained essentially unchanged. In addition to these acidic residues, two tryptophans were also important, since all enzyme activities of W125F, W125Y, W189G and W189Y were substantially reduced. This is consistent with the two tryptophans serving a substrate positioning function. A good correlation was observed when the NADase activity of all the mutants was plotted against the cADPR hydrolase activity. Homology modeling revealed all these critical residues are clustered in a pocket near the center of the CD38 molecule. The results indicate a strong structural homology between the active sites of CD38 and the Aplysia cyclase.

Cyclic ADP ribose activation of the ryanodine receptor is mediated by calmodulin.

Cyclic ADP-ribose (cADPR) is a newly identified nucleotide which can release calcium from a variety of cells, suggesting it is a messenger for mobilizing internal Ca2+ stores. Its cyclic structure has now been confirmed by X-ray crystallography. Available results are consistent with it being a modulator of Ca(2+)-induced Ca2+ release. Here we report that sea urchin egg microsomes purified by Percoll gradients lose sensitivity to cADPR, but the response can be restored by a soluble protein in the supernatant. Purification and characterization of the protein indicate that it is calmodulin. It appears to be sensitizing the Ca2+ release mechanism because caffeine and strontium, agonists of Ca(2+)-induced Ca2+ release, can also mimic calmodulin in conferring cADPR-sensitivity. Although evidence indicates that cADPR may be an activator of the ryanodine receptor, present results point to the importance of accessory proteins such as calmodulin in modulating its activity.

Caged nicotinic acid adenine dinucleotide phosphate. Synthesis and use.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a metabolite of NADP with Ca2+ mobilizing activity. The Ca2+ release mechanism activated by NAADP as well as the Ca2+ stores that it acts on are different from those activated by either cyclic ADP-ribose or inositol 1,4,5-trisphosphate (IP3) (Lee, H. C., and Aarhus, R. (1995) J. Biol. Chem. 270, 2152-2157). In order to demonstrate unambiguously that NAADP can mobilize Ca2+ stores in live cells, a caged analog was synthesized by reacting NAADP with 1-(2-nitrophenyl)diazoethane. Anion exchange high pressure liquid chromatography (HPLC) was used to purify one particular caged form from the mixture of products. Phosphate analyses following specific enzymatic cleavage indicate that the caging group is on the 2'-phosphate. This is confirmed by 31P NMR spectroscopy, showing that the 2'-phosphate of the caged compound exhibits an altered chemical shift of -2.6 ppm as compared with 2.3 ppm determined for the 2'-phosphate of NAADP. Caged NAADP had no Ca2+ releasing activity at a concentration as high as 1 micro;M when tested on sea urchin egg microsomes. After photolysis, it released Ca2+, was effective in nanomolar range, and was indistinguishable from authentic NAADP. The regeneration of NAADP after photolysis was also confirmed by HPLC analyses. The analog is particularly susceptible to UV and can be efficiently photolyzed using a spectrofluorimeter. To demonstrate its utility in live cells, caged NAADP was microinjected into sea urchin eggs. Photolysis effectively regenerated NAADP and activated Ca2+ oscillations in the eggs. Removal of external Ca2+ did not prevent the Ca2+ oscillations but only delayed the second Ca2+ peak by about 45 s, indicating that the oscillations are due to release from internal stores and not caused by Ca2+ influx. A mechanism based on sensitization of the Ca2+ release by Ca2+ loading is proposed to account for the Ca2+ oscillation observed.

A single residue at the active site of CD38 determines its NAD cyclizing and hydrolyzing activities.

CD38 is a multifunctional enzyme involved in metabolizing two Ca(2+) messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). When incubated with NAD, CD38 predominantly hydrolyzes it to ADP-ribose (NAD glycohydrolase), but a trace amount of cADPR is also produced through cyclization of the substrate. Site-directed mutagenesis was used to investigate the amino acid important for controlling the hydrolysis and cyclization reactions. CD38 and its mutants were produced in yeast, purified, and characterized by immunoblot. Glu-146 is a conserved residue present in the active site of CD38. Its replacement with Phe greatly enhanced the cyclization activity to a level similar to that of the NAD hydrolysis activity. A series of additional replacements was made at the Glu-146 position including Ala, Asn, Gly, Asp, and Leu. All the mutants exhibited enhanced cyclase activity to various degrees, whereas the hydrolysis activity was inhibited greatly. E146A showed the highest cyclase activity, which was more than 3-fold higher than its hydrolysis activity. All mutants also cyclized nicotinamide guanine dinucleotide to produce cyclic GDP. This activity was enhanced likewise, with E146A showing more than 9-fold higher activity than the wild type. In addition to NAD, CD38 also hydrolyzed cADPR effectively, and this activity was correspondingly depressed in the mutants. When all the mutants were considered, the two cyclase activities and the two hydrolase activities were correlated linearly. The Glu-146 replacements, however, only minimally affected the base-exchange activity that is responsible for synthesizing NAADP. Homology modeling was used to assess possible structural changes at the active site of E146A. These results are consistent with Glu-146 being crucial in controlling specifically and selectively the cyclase and hydrolase activities of CD38.

Magnesium ions but not ATP inhibit cyclic ADP-ribose-induced calcium release.

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.