A covalent intermediate in CD38 is responsible for ADP-ribosylation and cyclization reactions

Article abstract of DOI:10.1021/ja001139c

Human CD38 is an ectoenzyme expressed on the surface of B-cells that makes cyclic-ADP-ribose (cADPR) and ADP-ribose from NAD⁺ (nicotinamide diphosphate ribose, oxidized form). The compound cADPR is a potent second messenger for calcium release inside cells. Nicotinamide guanine dinucleotide (NGD⁺) is also cyclized by CD38 to form cGDPR (cyclic guanosine diphosphate ribose) and hydrolyzed to form GDPR (guanosine diphosphate ribose). Kinetic isotope effect studies in the presence of 20 mM nicotinamide gave 1'-³H and 1'-¹⁴C isotope effects of 1.02 ± 0.01 and 1.00 ± 0.01, respectively, for the cyclization reaction and 1.23 ± 0.01 and 1.02 ± 0.01, respectively, for the hydrolysis reaction. These values support a covalent intermediate. The existence of a covalent intermediate was established by reaction of ara-F-NMN⁺ (arabinosyl-2'-fluoro-2'-deoxynicotinamide mononucleotide) with the enzyme. This compound reacted to release 1 mol of nicotinamide/mole of CD38 monomer and to form an inactive covalent intermediate. Reaction with excess nicotinamide rescued catalytic activity with an apparent K(m) of 17 ± 5 mM and a V(max) of 0.023 ± 0.003 s^-1. Proof of covalent labeling of the enzyme by this inhibitor was obtained by MS analysis. Treatment of CD38 with ara-F-NMN⁺ increased mass by 215 amu, consistent with formation of CD38-fluoro-sugar monophosphate. Tryptic digestion in urea, phosphatase treatment, and purification of peptides in combination with MALDI-PSD permitted identification of Glu226 as the amino acid nucleophile. This residue is highly conserved across all ADP-ribosyl (adenosine diphosphate ribosyl) cyclases. The covalent intermediate inherent to the catalytic mechanism of human CD38 provides chemical precedent for related NAD⁺ glycosyltransferases.

Full text of DOI:10.1021/ja001139c

VOLUME 122, NUMBER 33
AUGUST 23, 2000
© Copyright 2000 by the
American Chemical Society
A Covalent Intermediate in CD38 Is Responsible for
ADP-Ribosylation and Cyclization Reactions
Anthony A. Sauve, HaiTeng Deng, Ruth H. Angeletti, and Vern L. Schramm*
Contribution from the Department of Biochemistry, Albert Einstein College of Medicine,
Bronx, New York, 10461
ReceiVed March 31, 2000
Abstract: Human CD38 is an ectoenzyme expressed on the surface of B-cells that makes cyclic-ADP-ribose
(cADPR) and ADP-ribose from NAD+ (nicotinamide diphosphate ribose, oxidized form). The compound cADPR
is a potent second messenger for calcium release inside cells. Nicotinamide guanine dinucleotide (NGD+) is
also cyclized by CD38 to form cGDPR (cyclic guanosine diphosphate ribose) and hydrolyzed to form GDPR
(guanosine diphosphate ribose). Kinetic isotope effect studies in the presence of 20 mM nicotinamide gave
1′-3H and 1′-14C isotope effects of 1.02 ( 0.01 and 1.00 ( 0.01, respectively, for the cyclization reaction and
1.23 ( 0.01 and 1.02 ( 0.01, respectively, for the hydrolysis reaction. These values support a covalent
intermediate. The existence of a covalent intermediate was established by reaction of ara-F-NMN+ (arabinosyl-
2′-fluoro-2′-deoxynicotinamide mononucleotide) with the enzyme. This compound reacted to release 1 mol of
nicotinamide/mole of CD38 monomer and to form an inactive covalent intermediate. Reaction with excess
nicotinamide rescued catalytic activity with an apparent Km of 17 ( 5 mM and a Vmax of 0.023 ( 0.003 s-1
.
Proof of covalent labeling of the enzyme by this inhibitor was obtained by MS analysis. Treatment of CD38
with ara-F-NMN+ increased mass by 215 amu, consistent with formation of CD38-fluoro-sugar monophosphate.
Tryptic digestion in urea, phosphatase treatment, and purification of peptides in combination with MALDI-
PSD permitted identification of Glu226 as the amino acid nucleophile. This residue is highly conserved across
all ADP-ribosyl (adenosine diphosphate ribosyl) cyclases. The covalent intermediate inherent to the catalytic
mechanism of human CD38 provides chemical precedent for related NAD+ glycosyltransferases.
Introduction
synthesize cyclic-ADP-ribose (cADPR) from NAD+.9,10 The
compound cADPR is formed by intramolecular ADP-ribosyla-
tion at the N1 position of the adenine ring,11 and is a potent
agent for Ca2+ release from intracellular Ca2+ stores.12-20 An
Human CD381-3 and Aplysia californica ADP ribosyl-
cyclase4-7 share 68% homology in primary sequence8 and
* To whom correspondence should be addressed: Department of
Biochemistry, Albert Einstein College of Medicine, Bronx, New York,
10461. Telephone: (718) 430-2813. FAX: (718) 430-8565. E-mail:
vern@aecom.yu.edu.
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10.1021/ja001139c CCC: $19.00 © 2000 American Chemical Society
Published on Web 08/03/2000

7856 J. Am. Chem. Soc., Vol. 122, No. 33, 2000
SauVe et al.
Scheme 1. Catalytic Mechanism for Reaction of CD38 with
NAD+ at pH 7.5 and 37 °C
Scheme 2. Catalytic Mechanism for Reaction of CD38 with
NGD+a
increasing volume of evidence indicates that these enzymes
regulate important physiological processes in invertebrates,12-19
plants,21 and mammals1,2,22-25 via their synthesis of cADPR.
In mammals, CD38 and cADPR have been implicated in the
regulation of cellular processes including insulin release,22
lymphocyte activation,2,23 bone homeostasis,24 and synaptic
plasticity.25 These biological studies have made the cyclases
attractive targets for inhibitor design26,27 and have increased
interest in the details of their catalytic mechanism. The catalytic
mechanism of the ADP-ribosyl cyclases and CD38 are thought
to be similar,28-32 proceeding through double displacement
mechanisms where NAD+ forms an enzyme-bound ADP-ribosyl
electrophile with departure of nicotinamide. Subsequent reaction
of this intermediate with nucleophiles, including N1 of adenine,
H2O, or the freely exchangeable nicotinamide, completes the
chemical steps leading to formation of products or reformation
of substrate (Scheme 1). The atomic structure of this intermedi-
ate has not been determined, although it has been proposed to
be a noncovalent enzyme-stabilized oxo-carbenium ion.33-35 The
alternative species is a covalent intermediate, attached to an
active site residue, possibly a Glu or Asp.31,35
a The KIEs were determined in the presence of 20 mM nicotinamide.
The khyd/kcyc at pH 7.5 and 37 °C is 0.38.28
Scheme 3. Reaction Catalyzed by CD38 for Formation of
cADPR and the Reaction Mechanism of ara-F-NMN+
Inhibition of CD38 and Rescue by Nicotinamide
Results and Discussion
Our previous mechanistic study of CD38 included measure-
ment of the microscopic rate constants for the reaction of the
substrate NGD+ (the guanine analogue of NAD+) to hydrolytic
and cyclized products.28 The nicotinamide bond-breaking step
is fast relative to the forward reaction and product release (k1
> khyd + kcyc Scheme 1). The intermediate is relatively long-
lived in the catalytic cycle (t1/2 ) 6 ms). This longevity explains
an NGD+ T nicotinamide base-exchange reaction at elevated
nicotinamide concentration.28 The substrate NGD+ reforms
readily from the intermediate and forward reaction progress in
the presence of 20 mM nicotinamide is diminished (reverse/
forward ) 35:1, Km ) 7 mM).28 Under these conditions, near
equilibration of the intermediate with free substrate and free
nicotinamide is achieved during the steady state (Scheme 2).
Kinetic isotope effects (KIEs, 1′-3H and 1′-14C) were obtained
by the competitive method36 under these conditions for the
reaction of NGD+ to both GDPR and cGDPR. The method
reports the KIE on kcat/Km, encompassing all catalytic steps from
free substrate to the first irreversible step.36 Irreversible steps
are the hydrolysis or cyclization of the intermediate. The 1′-3H
and 1′-14C isotope effects obtained at 20 mM nicotinamide are
small for cyclization (Scheme 2). These KIEs indicate that kcyc
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Nature 1994, 307-309.
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(22) Okamoto, H. Mol. Cell. Biochem. 1999, 193, 115-118.
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H.; Randall, T. D.; Lund, F. E.; Murray, R.; Schuber, F.; Howard, M. C.
Blood 1998, 92, 1324-1333.
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datheerthavarada, H. K.; Biswas, G.; Arakawa, T.; Hakeda, Y.; Koval, A.;
Sodam, B.; Bevis, P. J. R.; Moser, A. J.; Lai, F. A.; Epstein, S.; Troen, B.
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P. K. Proc. Natl. Acad. Sci. 1999, 96, 4061-4066.
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for NGD+ involves rate limiting conformational change (t1/2
)
4-5 ms) since either nucleophilic or dissociative chemistry as
a rate-limiting step would cause larger KIEs.37 Motion that
brings the purine ring to the appropriate attack geometry is
consistent with the KIEs. The large KIEs for NGD+ hydrolysis
establish that chemistry is rate-limiting (t1/2 ) 1-2 ms).28 In
consequence, entry of a water molecule to the site appropriate
(28) Sauve, A. A.; Munshi, C.; Lee, H. C.; Schramm, V. L. Biochemistry
1998, 37, 13239-13249.
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12078.

CoValent InactiVation of CD38
J. Am. Chem. Soc., Vol. 122, No. 33, 2000 7857
Figure 1. Plot of released [carbonyl-14C]nicotinamide as a function
of time where 228 nM CD38 is incubated with 1.8 µM [carbonyl-14C]-
ara-F-NMN+. Free nicotinamide is isolated by HPLC separation and
the radioactivity counted. Counts of nicotinamide were fit according
Figure 2. Plot of residual enzymatic activity as a function of time
when 228 nM CD38 is incubated with 1.8 µM [carbonyl-14C]ara-F-
NMN+. Time points reflect residual catalytic activity that converts
NGD+ to cGDPR. The solid curve is best fit to the equation A(t) )
to N(1 - e(-k t)) + C ) N(t) where N(t) is counts measured in cpm at
Ae(-k t) for decay of activity, where A is 100% activity, A(t) is activity
obs
obs
a given time, C is counts at time ) 0, and kobs is the rate constant.
measured at a given time t, and kobs is the rate constant.
for the hydrolytic reaction is fast (t1/2 , 1-2 ms) since KIEs
would be diminished if water entry is partially rate-limiting.
The observed KIEs (KIEobserved) are not intrinsic to the irrevers-
ible steps (KIEirrev ), but are the products of the isotope effect
on the equilibrium of the intermediate with solution substrate,
iso
i.e., KIEobserved
)
K ‚KIEirrev (Scheme 2).36 Because the
eq
cyclization is rate-limited by a nonchemical process, KIEirrev
for cyclization is expected to be near unity, thus isoKeq must be
near unity as well. Calculations for a proposed oxo-carbenium
ion intermediate (E•I in Schemes 1 and 2) predict larger values
for isoKeq, approximately 1.02 for 1′-14C and 1.20 for 1′-3H.37,38
The constraint on isoKeq for this enzyme implies that a covalent
intermediate exists at the active site during catalysis.
Support for a covalent intermediate at the active site of CD38
was obtained by the mechanism of inactivation of CD38 by
ara-F-NMN+,39 a truncated relative of ara-F-NAD+ (Scheme
3).39 ara-F-NAD+ is a potent inhibitor of CD38 (Ki ) 169 nM),
and has been reported to function by a slow-binding mecha-
nism.30,40 Under the conditions of our studies, ara-F-NMN+ was
shown to inhibit CD38 with a Ki ) of 61 nM (kon) 410 M-1
s-1, koff ) 2.5 × 10-5 s-1 at 310 K). Incubation of 200 µM
ara-F-NMN+ with 1 µM CD38 in the presence of 10 mM
[carbonyl-14C]nicotinamide (50 µCi/µmol) resulted in the
complete exchange of radiolabel into the inhibitor over a 24 h
period. This result establishes that CD38 catalyzes the exchange
reaction with the ara-F-NMN+ molecule as the substrate. The
radiolabeled [carbonyl-14C]ara-F-NMN+ was used to demon-
strate that decomposition of the nicotinamide-sugar bond by
the enzyme leads directly to inhibition. Treatment of CD38 with
[carbonyl-14C]ara-F-NMN+ releases [14C]nicotinamide estab-
lishing covalent reaction with ara-F-NMN+. The rate of [14C]-
nicotinamide release could be fit to a single-exponential curve
(kobs ) 0.042 ( 0.006 min-1, Figure 1) that reached completion
at 1:1 mol nicotinamide/mol CD38 subunit. Aliquots of this
inactivation mixture were assayed for activity and activity
declined in an exponential fashion with a rate of kobs of 0.038
( 0.004 min-1 (Figure 2). Inactivation of CD38 by ara-F-
NMN+ is therefore due to formation of an intermediate with
Figure 3. Rate of rescue of catalytic activity of ara-F-NMN+ inhibited
CD38 as a function of added nicotinamide. The solid line is the best
fit of points to the Michaelis-Menten. equation. The value of Vmax is
0.023 ( 0.003 s-1 and the apparent Km for nicotinamide is 17 ( 5 mM.
release of nicotinamide. Rescue of CD38 activity occurs with
added nicotinamide. The maximum rate of rescue is 0.023 (
0.003 s-1 with an apparent Km for nicotinamide of 17 ( 5 mM
(Figure 3). Based on the exchange reaction, rescue occurs
through the reverse reaction to reform ara-F-NMN+. A chemi-
cally similar rescue of catalytic activity after covalent reaction
of deoxy-nucleoside transferase with ara-F-adenosine is effected
by additions of adenine.41 Adenine rescue exhibits a saturable
rate with an appropriate Km for adenine.41 Mechanism-based
inhibition of deoxy-nucleoside transferase and glycosidases by
2′-F-substituted compounds along with identification of a stable
covalent moiety has been used to support the existence of a
covalent intermediate along the reaction coordinate of the
catalytic mechanism.42-45
Proof of covalent modification of CD38 by ara-F-NMN+ was
obtained by MS analysis. The MW of underivatized CD38
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G. J. Biol. Chem. 1998, 273, 34057-34062.
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C. Biochim. Biophys. Acta 1998, 1388, 428-436.

7858 J. Am. Chem. Soc., Vol. 122, No. 33, 2000
SauVe et al.
Figure 5. Mass spectrum of the fragmentation in the MALDI-PSD
mode of the tryptic peptide derivatized with the 2-fluoroarabinosyl
group. The fragments yn represent charged species whose amino acid
residues increase in length starting from the C-terminal end (K). The
mass 134 represents the mass increase due to the presence of the 2-F-
arabinose moiety on the peptide fragment.
cyclase by X-ray crystallography and is proposed to be an
important catalytic residue.6,31,46 The present result clearly
identifies Glu226 as the attachment point of the intermediate
with the enzyme, via a ribosyl-ester type bond.
Conclusion
Evidence from KIE data and from the covalent modification
of CD38 by ara-F-NMN+ establishes the mechanism of CD38
via a covalent intermediate. Covalency stabilizes an ADP-ribosyl
moiety on the enzyme that is formed by displacement of
nicotinamide by an enzyme glutamate (E226). These results are
inconsistent with a stabilized oxo-carbenium ion as the active
site electrophile. However, the values of KIE are consistent with
an oxo-carbenium transition state for hydrolysis of the covalent
intermediate. ADP-ribosyl cyclases and NAD+ glycohydrolases
from a variety of species have high homology, catalyze similar
reactions, and conserve this active site residue. It is likely that
the ADP-ribosyl cyclases and NAD+ glycohydrolases proceed
through covalent intermediates to achieve their respective
chemistries.
Figure 4. Upper panel: Comparison of MALDI-MS of CD38 before
and after treatment with ara-F-NMN+. Charge +1 species is the
monomer. Lower panel: Electrospray mass spectroscopy of CD38 not
treated (a) and treated (b) with ara-F-NMN+. The inset represents the
mass of the species determined by deconvolution of the raw signal
shown in the main figure. The mass difference of 216 is the mass
difference expected from covalent attachment of the 2-fluoro-5-
phosphoarabinosyl moiety to the monomer.
Experimental Section
monomer was determined to be 29516 (Figure 4). The major
species in the derivatized sample has a mass of 29732 consistent
with addition of the 2-fluoro-5-phosphoarabinosyl moiety (215
amu) to the protein. The MS study shows that most monomers
are labeled, consistent with the results of the radiochemical
titration. The amino acid that undergoes covalent modification
was determined by subjecting the derivatized protein to trypsin
digestion in 5 M urea followed by HPLC purification of peptide
fragments. The labeled peptide was treated with alkaline
phosphatase to remove the 5′-phosphate, repurified, and then
examined by MALDI-PSD. A mass analysis demonstrates the
mass of peptide fragments at different places along the amino
acid chain (Figure 5). Fragmentation analysis identifies a Glu
(E226) as the modified amino acid based on the presence of
Glu in all fragments containing an extra mass of 134 (the mass
addition resulting from the 2-fluoroarabinosyl modification).
Homology studies reveal E226 to be highly conserved across
all known ADP-ribosyl cyclase sequences,31 and has been
mutagenized resulting in abolition of catalytic activity (Figure
6).31 It has been localized at the active site of ADP-ribosyl
General Experimental. Solvents and compounds were obtained in
the highest purity available from commercial vendors. The inhibitor
ara-F-NMN+ was made as previously described.39 Human recombinant
CD38 was made and purified according to published procedures.46
HPLC purifications used a Waters 600/626 pump fitted with a 996
PDA detector controlled by computer using the Millenium32 software
package. UV/vis measurements were obtained on either a HP8453 diode
array instrument or a Varian Cary100Bio with temperature control. All
enzymatic measurements were done at 37 °C. Radioactivity was counted
on a Wallac 1414 scintillation counter.
Kinetic Isotope Effects Measurements. Radiolabeled substrates [1′-
3H]NGD+, [1′-14C]NGD+, [5′-3H]NGD+, and [5′-14C]NGD+ were
synthesized as previously reported.28 KIE studies were done by reacting
200 µL of [1′-3H]NGD+ and [5′-14C]NGD+ or [1′-14C]NGD+ and [5′-
3H]NGD+ in the presence of CD38 in 50 mM potassium phosphate at
pH 7.0 and 20 mM nicotinamide. The reactions were permitted to reach
(46) Munshi, C. B.; Fryxell, K. B.; Lee, H. B.; Branton, W. D. Methods
Enzymol. 1997, 280, 318-330. E226 is the glutamate residue in the full-
length CD38 sequence. Recombinant CD38 used in this study lacks the
N-terminal membrane spanning domain, which makes the derivatized residue
E189 of the recombinant sequence.

CoValent InactiVation of CD38
J. Am. Chem. Soc., Vol. 122, No. 33, 2000 7859
Figure 6. Homology plot of ADP-ribosyl-cyclases and NAD+ glycohydrolases. The sequences are akADPRC, Aplysia kurodai ADPribosyl-
cyclase; acADPRC, Aplysia californica ADPribosyl-cyclase; ratBST1, rat bone stromal cell antigen; mouBST1, mouse bone stromal cell antigen;
humBST1, human bone stromal cell antigen; mouCD38, mouse CD38; ratCD38, rat CD38; humCD38, human CD38. The residues marked by an
asterisk correspond to the conserved glutamate covalently modified by ara-F-NMN+ in the human CD38 protein. The numbers above the sequences
correspond to the residue numbers for the full length human CD38 sequence. Recombinant human CD38 has substitutions for N at sites 100, 164,
209 and 219.
20% completion based upon the consumption of total NGD+. From
the reaction mixture 150 µL aliquots were removed and GDPR and
cGDPR were separated by HPLC,28 and fractions were collected and
separately counted after addition of scintillation fluid in a 9:1 ratio to
aqueous volume. 100% reactions were obtained by allowing CD38 to
react with the remaining substrate overnight and then purified cGDPR
and GDPR fractions were combined and counted. KIE observed was
determined using the equation KIEobs ) (3H/14C at 100% conversion)/
(3H/14C at 20% conversion). The reported KIE values are observed
values and are not corrected for isotopic depletion.
Rescue of CD38 Activity by Nicotinamide. A solution of CD38
(10 mL of 0.050 µM) inhibited by ara-F-NMN+ was prepared by
incubation of the enzyme with 1 µM inhibitor for 6 h at 37 °C in 50
mM potassium phosphate, pH 7.0. Solutions of 200 µM NGD+ and 0,
2, 10, 20, 30, 40, 60, and 200 mM nicotinamide were prepared in the
identical buffer. For each nicotinamide solution pH was adjusted to
7.0 using concentrated NaOH or HCl. In a stopped flow apparatus fitted
with two syringes the solutions containing inhibited enzymes and
NGD+/nicotinamide solutions were mixed and fluorescence (360 nm
filter) measured as a function of time. The resulting curves were fit
using the equation E(t) ) Vt + (V - b)(1 - e(-kt))/k + d, where E(t) is
emission at time t, V is the initial rate of emission change, b is the final
rate of emission change, k is the observed rate constant for rescue, and
d is the initial emission intensity. A plot of k versus concentration of
nicotinamide was fit to the Michaelis equation to derive values for Vmax
and Km.
Exchange of [carbonyl-14C]Nicotinamide into ara-F-NMN+. The
compound ara-F-NMN+ was purified as reported,39 dissolved in 0.5
mL of potassium phosphate, and determined by UV/vis to be at a
concentration of 200 µM using 4600 M-1 cm-1 for the extinction
coefficient at 266 nm. CD38 was added to reach a concentration of 1
µM and the total sample was added to 5 µmol of [carbonyl-14C]-
nicotinamide possessinga specific activity of 50 µCi/µmol. After 24 h
at room temperature the reaction mixture was separated by HPLC using
50 mM ammonium acetate, pH 5.0, as eluant on a Waters C-18
µBondapak column. The peaks for ara-F-NMN+ and for nicotinamide
were collected. Specific activity was determined by taking an aliquot
of ara-F-NMN+ and counting radioactivity after addition of a scintil-
lation cocktail. Concentration was determined by UV/vis measurement
of 266 nm absorbance. Similar measurements were done for eluted
nicotinamide. Specific activity incorporated into ara-F-NMN+ was
found to be at least 95% of theoretical based upon complete incorpora-
tion of radiolabel.
Mass Spectrum of CD38. A solution of CD38 (50 µM in 10 mM
Tris buffer, pH 7.0) was incubated for several hours with 200 µM ara-
F-NMN+ and then submitted for MS analysis in either MALDI or
electrospray modes. A control lacking inhibitor was similarly incubated
and analyzed.
Mass Spectrum of CD38 Peptides. A solution was prepared as
above and after 5 h 50 µL was removed and 50 mg of solid urea was
added to denature protein. After 15 min 50 µL of trypsin (60 µg) was
added in 10 mM Tris/acetic acid buffer. The final pH was adjusted to
6 using pH paper and microliter additions of 100 mM Tris, pH 7.0.
After 3 h of room-temperature incubation 10 µL aliquots were injected
onto a C-8 column equilibrated with 17 mM ammonium acetate, pH
6.0, in 5% acetonitrile. A gradient of 1 mL/min was run that changed
acetonitrile composition to 70% over 60 min. Peaks containing
derivatized peptide eluted at 25-32 min. Two peptides were found,
one of mass 2720, a 22mer + sugar, the other of mass 2505, a 20mer
+ sugar, representing different extents of tryptic digestion. The mixture
of 2505 and 2720 peaks was digested with alkaline phosphatase
overnight at pH 6.0 (1 µL of enzyme, 0.5 units), then repurified by
HPLC as above. The 25-32 min fractions were concentrated and
analyzed by MALDI-PSD to identify the site of modification. A control
similarly treated in all respects but not inhibited did not show evidence
for sugar modification.
Radiotitration of CD38 with [carbonyl-14C]ara-F-NMN+ and
Enzyme Activity Measurements. Samples of 250 µL containing 1.8
µM [carbonyl-14C]ara-F-NMN+ and 228 nM CD38 (1.5 µL CD38 at
38 µM determined by UV/vis using 280 nm measurement and an
extinction coefficient of 1.70 mg-1 mL) in 50 mM potassium phosphate,
pH 7.0, were incubated for times 5, 10, 15, 20, 30, 45, 60, 90, and 120
min at 37 °C. After these times 2 µL was removed and added to a
cuvette containing 200 µM NGD+ in 0.5 mL of 50 mM potassium
phosphate, pH 7.0. The residual catalytic activity was determined by
monitoring 295 nm absorbance over 5 min. A control was also prepared
that did not contain ara-F-NMN+ and it was assayed to determine the
100% catalytic activity. A volume of 10 µL of 5 mM nicotinamide
was added to the residual 248 µL and the total sample separated as
above by HPLC. The peak containing nicotinamide was collected and
radioactivity counted in separate vials each containing 1 mL of eluant
and 9 mL of cocktail. Plots of activity were fit to a single-exponential
curve versus time using the program Kaleidagraph and the equation
Acknowledgment. This work was supported by NIH post-
doctoral fellowship GM19335 and research grant AI34342 from
the NIH. The authors thank Eddie Nieves, Linda Siconolfi-Baez,
and Dr. Lisa Mints for MS characterization of CD38 and
peptides. We also thank Drs. Hon-Cheung Lee and Cyrus
Munshi for the yeast strain expressing recombinant human
CD38.
A(t) ) Ae(-k t) for decay of activity, where A is 100% activity, A(t) is
obs
activity measured at a given time t, and kobs is the rate constant. Counts
of nicotinamide were fit according to N(1 - e(-k t)) + C ) N(t), where
obs
N(t) is counts measured in cpm at a given time, C is counts at time )
0, and kobs is the rate constant.
JA001139C