©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ADP-ribosyl Cyclase and CD38 Catalyze the Synthesis of a Calcium-mobilizing Metabolite from NADP(*)

(Received for publication, June 28, 1995; and in revised form, August 30, 1995)

Robert Aarhus (1) Richard M. Graeff (1) Deborah M. Dickey (2)(§) Timothy F. Walseth (2) Hon Cheung Lee (1)(¶)

From the  (1)Departments of Physiology and (2)Pharmacology, University of Minnesota, Minneapolis, Minnesota 55455

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

ADP-ribosyl cyclase catalyzes the cyclization of NAD to produce cyclic ADP-ribose (cADPR), which is emerging as an endogenous regulator of the Ca-induced Ca 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 Ca 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 Ca mobilization suggests a crucial role of these enzymes in Ca signaling.


INTRODUCTION

Mobilization of intracellular Ca is a major signaling mechanism in cells and the best known agonist for this process is inositol 1,4,5-trisphosphate (IP(3)) (^1)(reviewed in (1) ). Most cells, however, appear to contain Ca stores insensitive to IP(3), suggesting the existence of other Ca signaling molecules. One possible candidate is cyclic ADP-ribose (cADPR)(2, 3) . It is a cyclic nucleotide derived from NAD by linking the N-1 position of the adenine ring to the anomeric carbon of the terminal ribose and displacing the nicotinamide moiety(3) . cADPR is highly effective in mobilizing Ca from internal stores of cells. This was first demonstrated in an invertebrate cell, the sea urchin egg, where cADPR was shown to be as effective as IP(3)(4) . In addition, amphibian neurons(5) , a variety of mammalian cells (reviewed in (6) ), and, most recently, vacuoles from plant cells have all been shown to be responsive to cADPR (7) . Accumulating evidence indicates the action of cADPR is to increase the Ca sensitivity of the Ca-induced Ca release mechanism(8, 9, 10) , a major pathway for Ca mobilization.

The synthesis of cADPR is catalyzed by ADP-ribosyl cyclase, which was first described in sea urchin egg extracts (4) and later shown to be ubiquitous among animal tissues(11, 12) . Metabolism of cADPR is also catalyzed by a novel bifunctional enzyme purified from spleen, which can both synthesize cADPR from NAD and hydrolyze cADPR to ADP-ribose(13) . CD38 is a lymphocyte differentiation antigen which not only shares considerable sequence homology with ADP-ribosyl cyclase (14) but also was found to be a bifunctional enzyme with catalytic properties similar to the spleen enzyme(15, 16) . Recent mutagenesis studies show that CD38 can be converted to a monofunctional enzyme exhibiting only the ADP-ribosyl cyclase activity by altering two specific cysteine residues in its amino acid sequence(17) . The two enzymes are therefore closely related enzymatically and structurally.

In addition to cADPR, alkaline treatment of NADP generates a derivative which can also mobilize internal Ca from live sea urchin eggs as well as egg homogenates(4, 18) . Subsequent structural determination showed that the active derivative is nicotinic acid adenine dinucleotide phosphate (NAADP)(18) . Cross-desensitization and inhibitor studies indicate that the Ca release mechanism activated by NAADP is totally independent from that activated by cADPR and IP(3)(4, 18) . Furthermore, the NAADP-sensitive Ca stores can be separated from those sensitive to cADPR and IP(3) by Percoll density centrifugation, indicating they are distinctive organelles(4, 18) . In addition to alkaline treatment, NAADP can also be enzymatically synthesized by exchanging the base, nicotinamide, of NADP with nicotinic acid, a reaction catalyzed by spleen extracts(19, 20) . In this study, we show that ADP-ribosyl cyclase and CD38 both catalyze the synthesis of NAADP by the base-exchange reaction. The two catalytic pathways of ADP-ribosyl cyclase and CD38 are differentially regulated by pH and nicotinic acid. That two different products of ADP-ribosyl cyclase and CD38, cADPR, and NAADP, are involved in Ca mobilization suggests a crucial role of these enzymes in Ca signaling.


EXPERIMENTAL PROCEDURES

Purification of ADP-ribosyl Cyclase

ADP-ribosyl cyclase was purified from Aplysia ovotestes according to procedures published previously(12) , with some modifications. Frozen ovotestes (100 g) were purchased from Marinus (Long Beach, CA), thawed, and rinsed with the homogenization buffer containing 250 mM sucrose, 0.1 mM EDTA, 0.2 ml/liter beta-mercaptoethanol, 20 mM HEPES, pH 7.3. The minced tissues were added to 300 ml of the homogenization buffer containing protease inhibitors (17 µg of soybean trypsin inhibitor, 10 µg/ml leupeptin, 1.3 µg/ml aprotinin). The tissues were homogenized with 5-10-s pulses of an electrical tissue mincer, followed by 2-5 strokes with a Teflon pestle connected to an electric drill. The homogenate was diluted to about 400 ml with the homogenization buffer containing protease inhibitors and centrifuged for 30 min at 50,000 rpm in a Ti50.2 rotor (Beckman, Palo Alto, CA). About 330 ml of supernatant were recovered after centrifugation and stored at -80 °C until used.

The soluble extract (100 ml) was thawed and loaded at 1 ml/min onto a 2.5 times 20-cm Econo column (Bio-Rad) packed with the resin CM Hiflow (Sterogene, Arcadia, CA). The column was equilibrated with buffer A containing 1 mM EDTA, 0.2 ml/liter of beta-mercaptoethanol, and 20 mM HEPES, pH 7.3. After loading, the column was washed with 250 ml of buffer A at 2 ml/min. The bound ADP-ribosyl cyclase was eluted with 200 ml of 0.5 M NaCl in buffer A. The enzyme activity was measured by either of the two following assays: a bioassay based on the Ca release activity of cADPR as described below or by a recently developed fluorimetric assay based on measuring the synthesis of fluorescent cyclic GDP-ribose from nicotinamide guanine dinucleotide(21) . For the nicotinamide guanine dinucleotide assay; 1 µl of each fraction was added to 0.2 ml of 0.1 mM nicotinamide guanine dinucleotide in 20 mM Tris, pH 7.0, and the increase in cyclic GDP-ribose fluorescence was measured at 410 nm with excitation at 300 nm. All enzyme assays were done at room temperature. The final purification was conducted with a 300SW gel-filtration column (Waters, Milford, MA). The cyclase was eluted with 50 mM NaCl in buffer A listed above and the flow rate was 0.75 ml/min.

Production and Purification of Recombinant Human CD38

A full-length cDNA clone of human CD38 (22) was generously provided by Dr. D. G. Jackson (John Radcliffe Hospital, Oxford, UK). The details of the expression of the extracellular domain of human CD38 in yeast is as described previously(23) . Briefly, the putative intracellular and transmembrane portions of human CD38 were deleted and the four potential N-linked glycosylation sites removed by conservative amino acid substitutions. The remaining DNA sequence was spliced into Pichia expression vector pHIL-S1 (Invitrogen Corporation, San Diego, CA) and expressed as a soluble, secreted protein in Pichia pastoris according to protocols provided by Invitrogen. The soluble CD38 in the concentrated and dialyzed media from the yeast expression experiments was further purified to homogeneity on a cationic ion exchange column (SP 5PW, Waters). The purified preparation appears as a single band on silver-stained gels in SDS-polyacrylamide gel electrophoresis. In all respects it behaves the same as the preparation we have used and published previously(15) .

Enzyme Assays

Purified ADP-ribosyl cyclase (17-83 ng/ml) or CD38 (0.3-1.8 µg/ml) was incubated for 30 min at room temperature (20-23 °C) with 1 mM substrate, NADP or NAD, 0-100 mM nicotinic acid, pH 5.0, with Tris base. Bovine serum albumin (0.1 mg/ml) was included in the mixture to prevent inactivation of the cyclase due to nonspecific adsorption to tubes. Preliminary results indicate the reaction is linear for the first 30 min as long as at least 60% of the substrate is present. The reaction was terminated by 100-fold dilution with an ice-cold buffer containing 15 mM Tris, pH 10.5-11. The products were analyzed by HPLC and quantified by comparison with standards. The nucleotides of interest were separated on a 15 times 0.3-cm column packed with AG MP-1 (Bio-Rad) similar to that described previously(2, 12, 21) . The flow rate was maintained at 1 ml/min, and the nucleotides were eluted with a gradient of trifluoroacetic acid starting at 0% solvent B (solvent B is 150 mM trifluoroacetic acid in water, solvent A is water) increased to 1% solvent B in 1 min, increased linearly to 2% from 1 to 2 min, increased linearly to 4% from 2 to 5 min, increased linearly to 8% from 5 to 9 min, increased linearly to 16% from 9 to 13 min, increased linearly to 32% from 13 to 17 min, increased linearly to 100% from 17 to 18 min, and held at 100% until 22 min. A different trifluoroacetic acid gradient was used to analyze metabolites of NAD. The gradient started at 0% solvent B, increased linearly to 1% solvent B in 1 min, increased linearly to 2% from 1 to 6 min, increased linearly to 4% from 6 to 11 min, increased linearly to 8% from 11 to 16 min, increased linearly to 16% from 16 to 21 min, increased linearly to 32% from 21 to 26 min, stepped to 100% at 26.1 min, and maintained at 100% from 26.1 to 31 min. The column was calibrated with nicotinamide, NAD, NADP, cADPR, ADP-ribose (ADPR), ADP-ribose 2`-phosphate (ADPRP or ATP-ribose, Sigma) as standards. NAD and NADP were purchased from Sigma and further purified by using the same HPLC conditions described above. The HPLC analyses were done using a Gilson HPLC equipped with an autoinjector. The following millimolar extinction coefficients at 254 nm were used for standard calibration: 17.4 for NADP and NAD and 14.0 for cADPR and cADPRP.

Calcium Release Assay

Homogenates of sea urchin egg (Strongylocentrotus purpuratus) were prepared as described previously(9, 10, 18) . Frozen egg homogenates (25%) were thawed at 17 °C for 20 min and sequentially diluted to 1.25% as described previously(9, 10, 18) . Ca release was measured spectrofluorimetrically at 17 °C using Fluo 3 as a Ca indicator.


RESULTS

ADP-ribosyl Cyclase and CD38 Both Can Catalyze the Base Exchange Reaction

ADP-ribosyl cyclase was discovered as an enzyme that catalyzes the cyclization of NAD to cADPR(4, 11, 12) . Fig. 1A shows a HPLC chromatograph of the reaction products. NAD, the remaining substrate, eluted at about 7.1 min. The product, cADPR, eluted at about 13.3 min. The nicotinamide group (Nic) of NAD was released during the cyclization and eluted as a broad peak at about 1.5 min. The Ca releasing activity of the cADPR peak was verified using sea urchin egg homogenates (data not shown). The catalysis of ADP-ribosyl cyclase can be changed to base exchange by the addition of nicotinic acid as shown in Fig. 1B. Incubation of the cyclase with 1 mM NAD and 30 mM nicotinic acid resulted in the production of an additional product eluting at 16.33 ± 0.04 min (n = 6, ±S.D.). This product had no Ca releasing activity and was identified as NAAD by its virtually identical retention time as that of authentic NAAD (16.23 ± 0.08 min, n = 6). These results show that, under the appropriate conditions, the enzyme can catalyze the exchange of the nicotinamide group of NAD with nicotinic acid leading to the formation of NAAD.


Figure 1: Cyclization and base-exchange reactions catalyzed by ADP-ribosyl cyclase. NAD (1 mM) was incubated with Aplysia ADP-ribosyl cyclase (17 ng/ml) at room temperature (20-22 °C) and the products analyzed by HPLC. The column was calibrated with standards: nicotinamide (Nic), cADPR, and NAAD. A, the incubation was done at pH 7.0 for 30 min. B, the incubation was done at pH 4.0 with 30 mM nicotinic acid (NA) for 45 min.



That ADP-ribosyl cyclase can catalyze the base exchange reaction suggests that it may also be able to produce NAADP from NADP in a manner similar to the spleen extracts reported previously(19) . A chromatograph of the reaction products after incubation of the cyclase with NADP and nicotinic acid is shown in the inset of Fig. 2. In addition to peaks corresponding to nicotinamide (Nic), nicotinic acid (NA) and NADP, there are two additional peaks labeled 1 and 2. All peaks were collected and assayed for Ca release activity. Only peak 1 was found to be active (Fig. 2) and the Ca release was rapid, characteristic of that induced by NAADP(4, 18) . We have previously shown that prior exposure of sea urchin egg microsomes to NAADP can make them unresponsive to further challenge of NAADP(4, 18) . To show that peak 1 was indeed NAADP, the microsomes were tested subsequently for desensitization and, as shown in Fig. 2, were found to be refractory to authentic NAADP but remained responsive to cADPR.


Figure 2: Synthesis of NAADP by ADP-ribosyl cyclase. NADP (1 mM) was incubated with the cyclase (83 ng/ml) at pH 5.5 in the presence of 30 mM nicotinic acid (NA) for 30 min at room temperature. The products were analyzed by HPLC and the resulting chromatograph is shown in the inset. Fractions corresponding to various peaks of the chromatograph were collected and tested for Ca release activity using sea urchin egg homogenates. Fluo 3 was used as an indicator for the Ca release. All fractions were added to a final concentration of 80 nM, except cADPR, which was 50 nM. Concentrations of various compounds in the fractions were determined by absorbence at 254 nm and calibrated with the respective extinction coefficients.



It is reasonable to expect that the unknown product (peak 2) shown in Fig. 2may be the cyclic form of NADP, or cADPRP, since the cyclase is known to cyclize NAD to produce cADPR (see also Fig. 1A). Indeed, the UV spectrum of peak 2 is indistinguishable from that of cADPR (data not shown). Various treatments were performed on the unknown product (peak 2/cADPRP) to convert it to known substances and the results are summarized in Table 1. We have previously shown that cADPR can be quantitatively hydrolyzed to ADP-ribose by boiling(21) . Similar treatment of the unknown converted it to a product with a retention time 0.53 min later than the original unknown. The product was ADP-ribose 2`-phosphate (ADPRP) since it had a retention time of 20.35 ± 0.01 min, which is virtually identical to that of authentic ADPRP (20.31 ± 0.01 min). Also, when the product of the boiled unknown and ADPRP standard were subsequently treated with nucleotide pyrophosphatase, both were converted to products (2`,5`-ADP) having similar retention times of about 14.1 min.



If the unknown were indeed the phosphorylated form of cADPR, one would expect the removal of the 2`-phosphate should covert it to cADPR. Treatment of cADPRP with alkaline phosphatase (Table 1) converted it to a product with a retention time of 8.44 ± 0.03 min, virtually identical to that of authentic cADPR (8.36 ± 0.03 min). The Ca release assay was used to further demonstrate that the product was cADPR as shown in Fig. 3. A 10 µM aliquot of cADPRP was treated with alkaline phosphatase (APase). Immediately after starting the reaction (0`) no Ca release activity was detected, consistent with the results in Fig. 2showing that cADPRP has no Ca release activity. The Ca release activity developed maximally after 10 min of incubation (10`). The Ca release activity of the mixture after 27 min (27`) of incubation was completely blocked by a specific antagonist of cADPR, 8-amino-cADPR(24) , indicating the Ca release was mediated by cADPR. To quantify the conversion of the unknown compound to cADPR by the alkaline phosphatase, the Ca release activity was converted to production of cADPR by comparison with a calibration curve constructed with authentic cADPR. As shown in the inset of Fig. 3, by 10 min of incubation, all of the starting cADPRP (10 µM) was converted to cADPR by the alkaline phosphatase. These results indicate that cADPRP is identical to cADPR except for the additional 2`-phosphate. The proposed structure of cADPRP is shown in Fig. 4.


Figure 3: Conversion of cADPRP to cADPR. Alkaline phosphatase (APase, 1 unit/ml) was used to remove the 2`-phosphate from cADPRP (10 µM) and convert it to cADPR. Immediately (0`) after starting the reaction and at 5, 10, 21 and 27 min (`) during the reaction, a 2-µl aliquot of the mixture was added to 0.2 ml of egg homogenate (1.25%), and the resulting Ca release was measured by the indicator Fluo 3. The Ca release activity was calibrated with authentic cADPR, and the resulting time course of cADPR produced is shown in the inset. 8-Amino-cADPR (8-NH), a specific antagonist of cADPR, completely blocked the Ca release indicating that the release was due to cADPR produced during the 27 min (27`) of incubation.




Figure 4: The proposed structures of cADPRP and ADPRP.



Similar to the cyclase, CD38 can also catalyze the base-exchange reaction. The inset of Fig. 5shows a chromatograph of the reaction products after incubation of CD38 with NADP and nicotinic acid. Peak 1 in the chromatograph was identified as NAADP by its Ca release activity. Peak 2 was not cADPRP since they could be separated by HPLC (see Fig. 6A and inset of Fig. 8). Its elution time of 20.35 ± 0.01 min (n = 10, S.D.) was virtually identical to authentic ADPRP (Table 1). Treatment of the substance in peak 2 (Fig. 5) with nucleotide pyrophosphatase converted it to 2`,5`-ADP, which is similar to that observed with authentic ADPRP (Table 1). The product is thus identified as ADPRP and its structure is also shown in Fig. 4.


Figure 5: Synthesis of NAADP by CD38. NADP (1 mM) was incubated with CD38 (1.8 µg/ml) at pH 5.5 in the presence of 30 mM nicotinic acid (NA) for 30 min at room temperature. The products were analyzed by HPLC and the resulting chromatograph is shown in the inset. Fractions corresponding to various peaks of the chromatograph were collected and tested for Ca release activity using sea urchin egg homogenate as described in the legend of Fig. 2. All fractions were added to a final concentration of 80 nM, except cADPR, which was 50 nM. Concentrations of various compounds in the fractions were determined by absorbence at 254 nm and calibrated with the respective extinction coefficients.




Figure 6: Synthesis of cADPRP by ADP-ribosyl cyclase and ADPRP by CD38. A, NADP (1 mM) was incubated for 4 h with either the cyclase (20 ng/ml) at room temperature or with CD38 (200 ng/ml) at 37 °C at pH 7.0 in a medium containing 30 mM nicotinic acid, 20 mM Tris, and 0.1 mg/ml bovine serum albumin. Aliquots of 50 µl of the reaction mixture were analyzed by HPLC. The products from the cyclase reaction are shown in the upper chromatograph and those from the CD38 reaction are shown in the lower chromatograph. For the sake of clarity, only regions of interest of the chromatographs are shown. To avoid superposition, the chromatograph of the cyclase reaction is displaced along the vertical axis. B, The peaks (in A) corresponding to cADPRP and ADPRP were collected, lyophilized, and reconstituted with 1 ml of 20 mM Tris, pH 10.5. The samples were then treated with 1 unit of alkaline phosphatase for 30 min at 37 °C. The resulting products were analyzed by HPLC. The two insets show Ca release activity of the samples before and after the phosphatase treatment. Equal amounts of the samples before and after the treatment were added to sea urchin homogenates and the resulting Ca release measured by the increase in Fluo 3 fluorescence.




Figure 8: Dependence of the synthesis of NAADP and cADPRP on pH. NADP (1 mM) was incubated with A, ADP-ribosyl cyclase (25 ng/ml) or B, CD38 (0.3 µg/ml) at the various pH values indicated and in the presence of 30 mM nicotinic acid for 30 min at room temperature. The amounts of NAADP, cADPRP, and ADPRP produced were measured by HPLC. Results shown are mean ± S.D., n = 3. The upper HPLC chromatograph in the inset is that of 6.9 nmol of cADPRP and the lower one is that of a mixture of 3.8 nmol each of cADPRP and ADPRP. To improve the separation of the two compounds, a shallow trifluoroacetic acid gradient was used. For the sake of clarity, only regions of interest of the chromatographs are shown. To avoid superposition, the chromatograph of the cADPRP is displaced along the vertical axis.



Fig. 6provides more evidence that ADPRP is different from cADPRP. Mixtures of NADP and nicotinic acid were incubated with either the cyclase (the upper chromatograph in Fig. 6A) or CD38 (the lower chromatograph in Fig. 6A) and analyzed by HPLC. Comparison of the two chromatographs in Fig. 6A indicates cADPRP elutes about 0.5 min earlier than ADPRP. The peak corresponding to cADPRP was collected and treated with alkaline phosphatase to convert it to cADPR (cf. Fig. 3). The upper inset of Fig. 6B shows that Ca release activity was produced after the phosphatase treatment. The product was further analyzed by HPLC and 91% of the product was found to be cADPR, while 6.5% was ADP-ribose (upper chromatograph in Fig. 6B). Therefore, very little ADPRP is produced by the cyclase after incubation with NADP and nicotinic acid. The majority of the product is cADPRP. The peak corresponding to ADPRP produced by CD38 (lower chromatograph of Fig. 6A) was similarly analyzed. No Ca release activity was generated after the alkaline phosphatase treatment (lower inset in Fig. 6B). 92% of the product was ADPR as shown in the lower chromatograph of Fig. 6B and only 0.7% was cADPR. It is thus clear that the enzymatic properties of the cyclase and CD38 are quite different. The cyclase cyclizes NADP to produce cADPRP, while CD38 hydolyzes it to ADPRP. However, both enzymes can also catalyze the base-exchange reaction, resulting in the formation of NAADP.

Regulation of the Catalysis of the Base Exchange Reaction

The shift of the catalysis of the ADP-ribosyl cyclase from cyclization of NAD to base exchange depends critically on the pH of the reaction as shown in Fig. 7. The cyclase was incubated with 1 mM NAD at pH 4 or 7, with or without 30 mM nicotinic acid. The production of cADPR and NAAD under each condition was determined by HPLC and the product identities are similar to that described in Fig. 1. In the absence of nicotinic acid, only the synthesis of cADPR was observed at both pH. In the presence of nicotinic acid and at acidic pH, the reaction catalyzed by the cyclase shifted from cyclization to base exchange and the product produced was mainly NAAD. At neutral pH, even in the presence of nicotinic acid, only the production of cADPR was observed.


Figure 7: Regulation of the cyclization and the base-exchange reactions by pH and nicotinic acid. ADP-ribosyl cyclase was incubated with 1 mM NAD at pH 4 or 7, with (+) or without(-) 30 mM nicotinic acid (NA) as described in the legend of Fig. 1. Results shown are mean ± S.E., n = 6.



Similar to the results obtained with NAD as a substrate, the cyclization of NADP and the base-exchange reaction catalyzed by the cyclase are also pH-dependent. This is expected since both reactions are catalyzed by the same enzyme. As shown in the inset of Fig. 2, when the reaction of the cyclase was performed at pH 5.5, the amount of NAADP produced was about equal to cADPRP. When the reaction was performed at pH 7.0 (upper chromatograph in Fig. 6A) the majority of the product was cADPRP. A more detailed pH dependence is shown in Fig. 8A. The amount of cADPRP produced was determined by HPLC. The validity of the HPLC assay is depicted in the inset in Fig. 8B which shows that cADPRP can easily be distinquished from ADPRP by HPLC. At acidic pH, the cyclase catalyzes mainly the base-exchange reaction resulting in production of NAADP, while the cyclization reaction dominates at pH values above neutrality and produces cADPRP. NAADP production is maximal at pH 4 to 5, whereas cADPRP production displays a broad pH optimum from pH 6 to 9. Fig. 8B shows a similar dependence of the catalysis of CD38 on pH. The product was mainly NAADP in acidic pH but changed to exclusively ADPRP at or above neutrality. The pH optimum of NAADP production by CD38 was about 4 and ADPRP generation showed a broad optimum from pH 6 to 8.

In addition to pH, nicotinic acid is also required for the switching between the two catalytic pathways of the cyclase. NADP (1 mM) was incubated with the cyclase at pH 5.0 in the presence of various concentrations of nicotinic acid for 30 min and the remaining NADP and the products formed were quantified by HPLC. The results are shown in Fig. 9A. In the absence of nicotinic acid, only the cyclization reaction can occur even at an acidic pH of 5.0, which, as shown in Fig. 8A, should switch the catalysis of the cyclase to the base-exchange reaction. With increasing concentrations of nicotinic acid, the amounts of cADPRP produced decreased. Inversely, the amounts of NAADP synthesized showed a corresponding increase. The amounts of the remaining substrate, NADP, were constant. It thus appears that as the concentrations of nicotinic acid increase, the substrate utilization is unchanged, but the cyclase simply switches quantitatively from cyclization to base-exchange. The half-maximal effective concentration of nicotinic acid was about 5 mM.


Figure 9: Dependence of the synthesis of NAADP and cADPRP on nicotinic acid. NADP (1 mM) was incubated with A, ADP-ribosyl cyclase (25 ng/ml) or B, CD38 (0.3 µg/ml) at pH 5.0 and in the presence of various concentrations of nicotinic acid for 30 min at room temperature. The amounts of NAADP, cADPRP, and ADPRP produced were measured by HPLC.



Similar results were obtained using CD38. The amounts of substrate and products present after 30 min incubation of CD38 with 1 mM NADP at pH 5 and various concentrations of nicotinic acid are shown in Fig. 9B. Unlike the amounts of cADPRP produced by the cyclase (Fig. 9A), the amounts of ADPRP produced were relatively independent of the concentration of nicotinic acid. In contrast, the amounts of NAADP synthesized by the base-exchange reaction were critically dependent on nicotinic acid concentration and its half-maximal effect was observed at about 5 mM. The extent of the decrease of the amounts of the remaining NADP appeared to be directly correlated with the increase in the synthesis of NAADP. The production of ADPRP and NAADP by CD38 does not seem to be directly related, at least as far as their dependence on nicotinic acid is concerned. This behavior of CD38 is distinct from the cyclase and appears to be consistent with the bifunctional nature of CD38. In any case, these results show that the mode of catalysis of the cyclase and CD38 can be effectively regulated by pH and nicotinic acid.


DISCUSSION

The presence of ADP-ribosyl cyclase in sea urchin egg homogenates and its ability to convert NAD to a Ca mobilizing metabolite led to the discovery of cADPR(2, 4) . Since NAD is known to be sensitive to base, alkaline treatments were used to produce derivatives of NAD. Although the yield was low, alkaline treatments could, indeed, produce cADPR(4) . Similar treatment of NADP also produced a Ca mobilizing derivative(4) . However, the Ca release mechanism activated by this derivative was shown to be independent of the cADPR-sensitive release(4) . The derivative was later identified as NAADP(18) . Although similar chemical treatments can produce cADPR and NAADP, their structural differences and the independence of their Ca mobilizing action suggest that different enzymatic pathways may be involved in their synthesis. This is shown not to be the case in this study. At acidic pH and in the presence of nicotinic acid, ADP-ribosyl cyclase can efficiently catalyze the exchange of the nicotinamide group of NADP with nicotinic acid and produce NAADP. Furthermore, the cyclase can also cyclize NADP in the absence of nicotinic acid and at neutral pH. The product was identified as the phosphorylated form of cADPR, or cADPRP. Not only is this multifunctionality of ADP-ribosyl cyclase novel, but also that the two products of the cyclase, cADPR and NAADP, produced under different conditions are intimately related to Ca mobilization suggests that this enzyme may play a pivotal role in Ca signaling.

There are three different types of ADP-ribosyl cyclase known so far. In addition to the Aplysia enzyme, the CD38-like bifunctional enzymes and a cGMP-sensitive enzyme in sea urchin eggs are all members of the family (reviewed in (25) ). Here we show that CD38 can also catalyze the base-exchange reaction with dependence on pH and nicotinic acid similar to that observed with the cyclase, further strengthening the notion that the two enzymes are not only structurally but also enzymatically related. As for the possible role of nicotinic acid and pH as physiological regulators of the cyclase, the half-maximal concentration of nicotinic acid of about 5 mM and the acidic pH required for switching of the cyclase to the base-exchange reaction appear to be somewhat out of the physiological range. However, it should be pointed out that the half-maximal effective concentration of NAADP in mobilizing Ca is about 30 nM(18) . Therefore, the cyclase does not have to operate anywhere near its maximal rate to synthesize physiologically relevant amounts of NAADP. Also, the Aplysia cyclase is believed to be present inside vesicular organelles in the oocytes(26) , and its characteristics may reflect its in vivo location. This is particularly relevant for CD38. It has previously been proposed that internalization of CD38, a surface antigen, may be part of the signal transduction mechanism (reviewed in (25) ). This would bring CD38 into the acidic environment of the endosomes, an environment condusive for the base-exchange reaction. Whether this would result in synthesis of NAADP remains to be determined. The coincidence between the transit of CD38 through an acidic environment and the requirement of acidic pH for the base-exchange reaction is certainly very suggestive.

The ability of the cyclase to catalyze the base exchange reaction strongly suggests the formation of an enzyme intermediate is part of the enzymatic mechanism. The first step of the catalysis is likely to be the release of the nicotinamide group and the formation of an intermediate involving ADP-ribose, or ADP-ribose phosphate if NADP is used as a substrate. The anomeric carbon of the ADP-ribosyl intermediate (or ADPRP) may well be in an activated state poised to react with various substances. If nicotinic acid is present, the reaction with the activated intermediate will lead to the formation of NAAD (or NAADP). In the absence of nicotinic acid, the anomeric carbon can react intramolecularly with N1 of the adenine group resulting in the formation of cADPR (or cADPRP). Nicotinamide, if present, can also react with the intermediate and the result is the reformation of NAD. This reversal of the reaction has been observed to be catalyzed by a bifunctional ADP-ribosyl cyclase purified from the spleen(13) . That the formation of an intermediate is part of the catalysis of the cyclase is also consistent with the crystal structure of cADPR(3) . The cycle linkage between the anomeric carbon and the adenine in cADPR is in the beta-conformation, the same as in NAD (see also Fig. 4). A double inversion at the anomeric carbon resulting from, first, the formation of the intermediate with the cyclase and, second, the subsequent cyclization with the N1 of the adenine, would be consistent with the observed conservation of the conformation of the linkage. The model can also accommodate the enzymatic properties of CD38. One needs only to postulate that, in the case of CD38, the enzyme intermediate is much more accessible to the attack by water. Reacting with water instead of the N-1 of the adenine would result in the formation of ADP-ribose (or ADPDP) instead of cADPR (or cADPRP). The mechanism described so far is similar to that proposed for the bifunctional ADP-ribosyl cyclase (13) and the NAD glycohydrolase(27) . Although the Aplysia ADP-ribosyl cyclase is not an NAD glycohydrolase since it does not synthesize ADP-ribose from NAD, the similarity in the enzymatic mechanism suggests they may all belong to the same family of enzymes. NAD glycohydrolases have been known for more than 50 years but their functions have been an enigma(27) . Accumulating evidence from this and previous studies on cADPR has pointed to the likelihood that this family of enzymes may be important players in Ca signaling.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants HD17484 (to H. C. L.) and DA08131 (to T. F. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a National Institutes of Health Training Grant T32GM07994.

To whom correspondence should be addressed: Dept. of Physiology, 6-255 Millard Hall, 435 Delaware St., S.E., University of Minnesota, Minneapolis, MN 55455. Tel.: 612-625-7120/4641; Fax: 612-625-0991/5149.

(^1)
The abbreviations use are: IP(3), inositol 1,4,5-trisphosphate; cADPR, cyclic ADP-ribose; cADPRP, cyclic ADP-ribose 2`-phosphate; ADPR, ADP-ribose; ADPRP, ADP-ribose 2`-phosphate (also known as 2`-phosphate ADP-ribose); NAAD, nicotinic acid adenine dinucleotide; NADP, nicotinamide adenine dinucleotide phosphate; NAADP, nicotinic acid adenine dinucleotide phosphate; HPLC, high pressure liquid chromatography.


REFERENCES

  1. Berridge, M. J. (1993) Nature 361, 315-325 [Medline]
  2. Lee, H. C., Walseth, T. F., Bratt, G. T., Hayes, R. N., and Clapper, D. L. (1989) J. Biol. Chem. 264, 1608-1615 [Medline]
  3. Lee, H. C., Aarhus, R., and Levitt, D. (1994) Nature Struct. Biol. 1, 143-144
  4. Clapper, D. L., Walseth, T. F., Dargie, P. J., and Lee, H. C. (1987) J. Biol. Chem. 262, 9561-9568, [Medline]
  5. Hua, S. Y., Tokimasa, T., Takasawa, S., Furuya, Y., Nohmi, M., Okamoto, H., and Kuba, K. (1994) Neuron 12, 1073-1079 [Medline]
  6. Lee, H. C., Galione, A., and Walseth, T. F. (1994) Vitam. Horm. 48, 199-258 [Medline]
  7. Allen, G. J., Muir, S. R., and Sanders, D. (1995) Science 268, 735-737 [Medline]
  8. Galione, A., Lee, H. C., and Busa, W. B. (1991) Science 253, 1143-1146 [Medline]
  9. Lee, H. C. (1993) J. Biol. Chem. 268, 293-299 [Medline]
  10. Lee, H. C., Aarhus, R., and Graeff, R. (1995) J. Biol. Chem. 270, 9060-9066 [JBC][Medline]
  11. Rusinko, N., and Lee, H. C. (1989) J. Biol. Chem. 264, 11725-11731 [Medline]
  12. Lee, H. C., and Aarhus, R. (1991) Cell Regul. 2, 203-209
  13. Kim, H., Jacobson, E. L., and Jacobson, M. K. (1993) Science 261, 1330-1333 [Medline]
  14. States, D. J., Walseth, T. F., and Lee, H. C. (1992) Trends Biochem. Sci. 17, 495 [Medline]
  15. Howard, M., Grimaldi, J. C., Bazan, J. F., Lund, F. E., Santos-Argumedo, L., Parkhouse, R. M. E., Walseth, T. F., and Lee, H. C. (1993) Science 262, 1056-1059 [Medline]
  16. Takasawa, S., Tohgo, A., Noguchi, N., Koguma, T., Nata, K., Sugimoto, T., Faruya, H., Yonekura, H., and Okamoto, H. (1993) J. Biol. Chem. 268, 26052-26054 [Medline]
  17. Tohgo, A., Takasawa, S., Noguchi, N., Koguma, T., Nata, K., Sugimoto, H., and Okomoto, H. (1994) J. Biol. Chem. 269, 28555-28557 [Medline]
  18. Lee, H. C., and Aarhus, R. (1995) J. Biol. Chem. 270, 2152-2157 [Medline]
  19. Bernofsky, C. (1980) Methods Enzymol. 66, 105-112 [Medline]
  20. Chini, E. N., Beers, K. W., and Dousa, T. P. (1995) J. Biol. Chem. 270, 3216-3223 [Medline]
  21. Graeff, R., Walseth, T. F., Fryxell, K., Branton, D., and Lee, H. C. (1994) J. Biol. Chem. 269, 30260-30267 [Medline]
  22. Jackson, D. G., and Bell, J. I. (1990) J. Immunol. 144, 2811-2815 [Medline]
  23. Fryxell, K. B., O'Donoghue, K., Graeff, R. M., Lee, H. C., and Branton, W. D. (1995) Protein Expression Purif. 6, 329-336
  24. Walseth, T. F., and Lee, H. C. (1993) Biochim. Biophys. Acta 1178, 235-242
  25. Lee, H. C., Graeff, R., and Walseth, T. F. (1995) Biochimie (Paris) 77, 345-355
  26. Hellmich, M. R., and Strumwasser, F. (1991) Cell Regul. 2, 193-202
  27. Price, S. R., and Pekala, P. H. (1987) in Coenzymes and Cofactors (Dolphin, D., Poulson, R., and Avramovic, O., eds) Vol. 2, Part B, pp. 513-548

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.