(Received for publication, June 28, 1995; and in revised form, August 30, 1995)
From the
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.
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
) (
)(reviewed in (1) ). Most cells, however, appear to
contain Ca
stores insensitive to IP
,
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
(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
(4, 18) . Furthermore, the
NAADP
-sensitive Ca
stores can be
separated from those sensitive to cADPR and IP
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.
The
soluble extract (100 ml) was thawed and loaded at 1 ml/min onto a 2.5
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
-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.
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
.
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.
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
-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.