In-gel activity staining of oxidized nicotinamide adenine dinucleotide kinase by blue native polyacrylamide gel electrophoresis.

Abstract

Oxidized nicotinamide adenine dinucleotide (NAD(+)) kinase (NADK, E.C. 2.7.1.23) plays an instrumental role in cellular metabolism. Here we report on a blue native polyacrylamide gel electrophoretic technique that allows the facile detection of this enzyme. The product, oxidized nicotinamide adenine dinucleotide phosphate (NADP(+)), formed following the reaction of NADK with NAD(+) and adenosine 5'-triphosphate was detected with the aid of glucose-6-phosphate dehydrogenase or NADP(+)-isocitrate dehydrogenase, iodonitrotetrazolium chloride, and phenazine methosulfate. The bands at the respective activity sites were excised and subjected to native and denaturing two-dimensional electrophoresis for the determination of protein levels. Hence this novel electrophoretic method allows the easy detection of NADK, a critical enzyme involved in pyridine homeostasis. Furthermore, this technique allowed the monitoring of the activity and expression of this kinase in various biological systems.

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phoresis; G6PDH, glucose-6-phosphate dehydrogenase; INT, iodonitro-
tetrazolium chloride; NADP+-ICDH, NADP+-dependent isocitrate
dehydrogenase; PMS, phenazine methosulfate; NADK, nicotinamide ade-
nine dinucleotide kinase; ROS, reactive oxygen species; CSB, cell storage
buVer; CFE, cell-free extract; MEM, minimal essential medium; 2D, two-
dimensional.
ANALYTICAL
BIOCHEMISTRY
Analytical Biochemistry 359 (2006) 210–215
www.elsevier.com/locate/yabio
0003-2697/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.ab.2006.09.019
In-gel activity staining of oxidized nicotinamide adenine dinucleotide
kinase by blue native polyacrylamide gel electrophoresis
Ryan J. Mailloux, Ranji Singh, Vasu D. Appanna¤
Department of Chemistry and Biochemistry, Laurentian University, Sudbury, ON, Canada P3E 2C6
Received 30 July 2006
Available online 17 October 2006
Abstract
Oxidized nicotinamide adenine dinucleotide (NAD+) kinase (NADK, E.C. 2.7.1.23) plays an instrumental role in cellular metabolism.
Here we report on a blue native polyacrylamide gel electrophoretic technique that allows the facile detection of this enzyme. The product,
oxidized nicotinamide adenine dinucleotide phosphate (NADP+), formed following the reaction of NADK with NAD+ and adenosine
5?-triphosphate was detected with the aid of glucose-6-phosphate dehydrogenase or NADP+–isocitrate dehydrogenase, iodonitrotetrazo-
lium chloride, and phenazine methosulfate. The bands at the respective activity sites were excised and subjected to native and denaturing
two-dimensional electrophoresis for the determination of protein levels. Hence this novel electrophoretic method allows the easy detec-
tion of NADK, a critical enzyme involved in pyridine homeostasis. Furthermore, this technique allowed the monitoring of the activity
and expression of this kinase in various biological systems.
© 2006 Elsevier Inc. All rights reserved.
Keywords: NAD+ kinase; BN–PAGE; Activity bands; NAD+; NADP+
Aerobic organisms are subjected to constant bombard-
ment of reactive oxygen species (ROS)1 as a consequence of
their regular metabolism [1]. These ROS are highly oxida-
tive moieties that need to be detoxiWed if the cell is to sur-
vive. Although catalase, superoxide dismutase, and
glutathione peroxidase play pivotal roles in this process,
their eVectiveness is dependent on the reductive environ-
ment generated by the steady supply of NADPH [2].
Enzymes such as ICDH, malic enzyme, and G6PDH have
been shown to participate in the production of NADPH
[3–6]. However, these NADPH-generating enzymes require
NADP+ to maintain their function. NADK is the key
enzyme which catalyzes the phosphorylation of NAD+ to
NADP+ [7]. Thus, the regulation of the cellular concentra-
tions of NADP+ mediated by NADK may play a critical
role in the generation of a reductive environment and hence
the detoxiWcation of ROS [8]. NADK has been regarded the
sole participant in the de novo synthesis of NADP+. The
maintenance of NADP+ levels is crucial for anabolism,
photosynthesis, and cell signaling [9]. NADK has been
found in both prokaryotes and eukaryotes, suggesting that
this enzyme is required for nicotinamide homeostasis in a
number of organisms [8,10,11].
This enzyme is usually monitored by spectrophotometric
techniques that involve enzyme-coupled assays [12]. In
these methods, an enzyme such as G6PDH is employed to
quantitate the formation of NADP+. Interfering reactions,
especially in the cell-free (CFEs), extracts can hinder the
accuracy of the assay. In addition high quantities of protein
are normally required and the sensitivity is relatively low
[3,13–16]. To quantitate the expression of the NADK,
antibodies that are expensive and/or not readily available
have to be used. The procedure described here allows the
*Corresponding author. Fax: +1 705 675 4844.
E-mail address: vappanna@laurentian.ca (V.D. Appanna).
1Abbreviations used: BN–PAGE, blue native polyacrylamide gel electro-

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mented with 5% fetal bovine serum (v/v) and 1% antibiotics
(v/v). Cells were routinely seeded at 1£105 cells/ml, cul-
tured in 175cm2 Xasks with loosened caps, and incubated
with 5% CO2 (v/v) in a humidiWed atmosphere at 37°C.
Upon reaching 70% conXuency the medium was removed,
replaced with serum-free ?-MEM containing citrate
(2.5mM) or aluminum (Al)–citrate (0.5mM Al:2.5mM
citrate) and exposed for a 24-h period. Cell viability was
In-gel activity of NADK / R.J. Mailloux et al. / Anal. Biochem. 359 (2006) 210–215
211
monitoring of both the enzyme activity and the relative
protein expression without necessitating the use of antibod-
ies. BN–PAGE provides a facile and inexpensive method to
analyze NADK. Although BN–PAGE has been widely uti-
lized to study mitochondrial membrane enzymes, we have
recently adapted this technique to monitor a wide variety of
soluble and membrane enzymes [17,18]. In this report we
describe the in-gel detection and monitoring of NADK
activity with the aid of either G6PDH or NADP+–ICDH.
This readily accessible procedure enables the quick identiW-
cation and quantitation of this nicotinamide-sensitive
kinase in various biological systems.
Materials and methods
Cell culture and subcellular fractionation
The strain of Pseudomonas Xuorescens (ATCC 13525)
was grown in a mineral medium containing Na2HPO4
(6.0g), KH2PO4 (3.0g), MgSO4 (0.2g), NH4Cl (0.8g), and
citric acid (4.0g) per liter of distilled and deionized water.
Trace elements were present as previously described [19].
The pH was adjusted to 6.8 with dilute NaOH. The
medium was dispensed in aliquots of 200ml in 500ml
Erlenmeyer Xasks and autoclaved for 20min. Menadione
experiments were performed by the addition of the mena-
dione (100?M) to the sterilized citrate media. The
medium was inoculated with 1ml of stationary-phase cells
grown in a medium unamended with menadione and aer-
ated on a gyratory water bath shaker (Model 76; New
Brunswick ScientiWc) at 26°C at 140rpm. The Bradford
protein assay was performed in triplicate to determine the
amount of cells in the culture by measuring the solubilized
cellular proteins [20]. The cells were treated with 1M
NaOH to solubilize the protein (cells obtained from 10ml
culture Xuid were heated for 5min in 1ml NaOH). The
bacterial cells were harvested at various growth intervals
and suspended in a cell storage buVer (CSB; 50mM Tris–
HCl pH 7.3, 5mM MgCl2, 1mM phenylmethylsulfonyl
Xuoride, and 1mM dithiothreitol). The cells were homog-
enized by sonication and centrifuged at 3000g for 30min
at 4°C to remove intact bacteria. The cell homogenate
was then centrifuged at 180,000g for 1h to aVord a soluble
CFE and a membrane CFE. The soluble fraction was fur-
ther centrifuged at 180,000g for 2h to ensure the removal
of any residual membrane fraction. The CFEs were kept
at 4°C for up to 5 days.
HepG2 cells were a gift from Dr. D. Templeton (Univer-
sity of Toronto) and were maintained in ?-MEM supple-
monitored using the Trypan blue exclusion assay [21]. Fol-
lowing the exposure, the cell monolayer was trypsinized
and the cells were pelleted by centrifugation at 250g for
10min at 4°C. The cellular pellet was washed twice in phos-
phate-buVered saline, resuspended in mammalian CSB
(mCSB), and stored at ¡86°C until needed. The CSB used
for the HepG2 cells contained 250mM sucrose, 0.6mM
MnCl2, 1mg/ml pepstatin, and 0.1mg/ml leupeptin. Cells
were thawed and centrifuged at 250g for 10min at 4°C and
resuspended in a minimal amount of ice-cold CSB. The cell
suspension was ultrasonically disrupted on ice using a
Brunswick sonicator for 20s operating at 2-s intervals. The
cell homogenate was then centrifuged at 12,000g for 30min
at 4°C to aVord a clean cytoplasmic fraction devoid of
whole cells, mitochondria, and nuclei [22, 23]. The purity of
the cytoplasm was conWrmed by immunoblot using anti-
bodies directed toward voltage-dependent anion channel,
histone 2A, and F-actin.
Blue native PAGE and two-dimensional (2D)
electrophoresis
BN–PAGE was performed as described [17] with the fol-
lowing modiWcations. P. Xuorescens and HepG2 proteins
were prepared in a blue native buVer (50mM Bis–Tris,
500mM six-amino hexanoic acid (pH 7.0)) at Wnal concen-
trations of 4 and 2mg/ml, respectively. Proteins (20–80?g)
from bacterial and HepG2 cell free extract were loaded into
each lane and electrophoresed under native conditions;
80V was used for the stacking and this was increased to
200V when the proteins reached the separating gel. The
blue cathode buVer (50mM Tricine, 15mM Bis–Tris, 0.02%
(w/v) Coomassie G-250 (pH 7.0) at 4°C) was changed to
colorless cathode buVer (50mM Tricine, 15mM Bis–Tris,
(pH 7.0) at 4°C) when the running front was half way
through the separating gel. Electrophoresis was subse-
quently stopped once the running front had reached the
bottom of the gel. The gel slab was removed from the
chamber and equilibrated for 15min in a gel equilibration
buVer (25mM Tris, 5mM MgCl2 (pH 7.4)). Immediately
following the incubation the gel slab was placed in a reac-
tion mixture consisting of equilibration buVer, NAD+
(0.5mM), ATP (3mM), 5mM glucose-6-phosphate, 5 units
of G6PDH, INT (0.4mg/ml), and PMS (0.2mg/ml). Con-
trols consisted of the reaction mixture without the substrate
(ATP or NADH) or coupling enzyme or in the presence of
the inhibitor iodoacetate (2mM) and the nucleotide CTP
(3mM). A similar reaction mixture was utilized in the
detection of NADK from HepG2 cells except that 5mM
isocitrate and 10 units of NADP+–ICDH substituted for
G6PDH and glucose-6-phosphate. HepG2 control experi-
ments were also performed as described above. The activity
band was excised and subjected to analysis by 2D BN–
PAGE as described above. 2D SDS–PAGE using a discon-
tinuous buVer system was performed according to the
method of Laemmli [24]. The activity gel was equilibrated
in the electrophoresis buVer (24mM Tris, 191mM glycine,

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Results and discussion
212
In-gel activity of NADK / R.J. Mailloux et al. / Anal. Biochem. 359 (2006) 210–215
1% SDS (w/v)) containing 1% mercaptoethanol (v/v) for
30min at room temperature. The band was then excised
and electrophoresed at a constant 200V. Gel slabs were
Wxed and destained according to standard procedures. Pro-
tein levels were ascertained by silver stain. Band intensity
was determined using Scion Imaging for Windows (Scion
Corp., Frederick, MD).
SDS–PAGE and immunoblot
SDS–PAGE was performed as described above. Samples
were solubilized in 62.5mM Tris–HCl (pH 6.8), 2% SDS
(w/v), and 2% ?-mercaptoethanol (v/v) at 100°C for 5min.
The proteins were transferred to a Hybond-P polyvinyli-
dene diXuoride membrane electrophoretically for immuno-
blotting. NonspeciWc binding sites were blocked by
incubating the membrane in 5% non fat skim milk in TTBS
(20mM Tris–HCl (pH 7.6), 0.8% NaCl (w/v), 1% Tween 20
(w/v)) for 1h. The antibody raised against NADK from
rabbits was generously supplied by Dr. Snedden (Queen’s
university, Kingston, ON, Canada). The secondary anti-
body consisted of a horseradish-peroxidase-conjugated
antirabbit (Santa Cruz). Detection of the desired antigen
was achieved using Chemiglow (Alpha Innotech). The
immunoblots were subsequently documented using the
ChemiDoc XRS system (BioRad Imaging Systems). Band
intensity was quantiWed using Alpha Innotech Software
(Alpha Innotech Corp.).
Metabolite analysis
NADK activity was monitored by the consumption and
production of NAD+ and NADP+, respectively. In a phos-
phate reaction buVer (10mM Na2HPO4, 5mM MgCl2 (pH
7.3)) consisting of 3mM ATP and 0.5mM NAD+, 2mg of
soluble CFE was added and allowed to react for 30min.
Reaction mixtures without ATP were utilized as controls.
The reactions were stopped with cold 0.5% perchlorate and
the resulting precipitate was removed by centrifugation at
12,000g for 10min. HPLC analysis was carried out on a
Waters Alliance HPLC equipped with a Waters model 2487
UV–vis dual wavelength detector and a C18 reverse-phase
column (3.5?m, amide cap, 4.6£150-mm inside diameter,
Symmetry Column, Phenomenex, Torrance, CA). A mobile
phase consisting of 20mM KH2PO4 (dissolved in millipore
water) was used operating at a Xow rate of 0.7ml/min at
ambient temperature. The eluants were measured by UV
absorption at 254nm.
NADK is a pivotal metabolic enzyme that allows living
organisms to modulate the intracellular concentrations of
NAD+ and NADP+. An increase in the former favors
energy production while an increment in the latter
promotes the synthesis of NADPH, an anabolic reductant.
Owing to its instrumental role in metabolism, it is
important to identify analytical tools that will allow
the facile monitoring of this enzyme. Here, we show a
BN–PAGE technique that allows the evaluation of the
activity and expression of NADK. This procedure main-
tains the enzyme in its active state and allows the visualiza-
tion of the migrating bands. The charge provided by the
dye coupled with the enzymatic stability provided by the
6-aminohexanoic acid via preferential hydration may be a
contributing factor that leads to the eVectiveness of this
method. Bands are more intense and sharper than those
provided by regular native PAGE (data not shown). The
dye, Coomassie blue G-250, also appears to contribute to
the stability of the enzymes [25]. The ability of NADK
to produce NADP+ in the presence of ATP was employed
to obtain an activity band. With the aid of the appropriate
substrate and NADP+-dependent enzyme (ICDH or
G6PDH), the resulting NADPH interacts with INT to gen-
erate a purple precipitate at the site where the enzyme is
immobilized. As shown in Fig. 1(I), an activity band was
observed only when ATP was present in the reaction mix-
ture. The two diVerent coupling enzymes, G6PDH and
ICDH, were utilized in an eVort to ascertain the suitability
and eVectiveness of these two enzymes to detect NADP+
formation as a consequence of NAD+ kinase activity. Since
G6PDH is a predominantly cytoplasmic enzyme, it may be
more suitable to monitor NAD+ kinase activity in mem-
brane fractions. Interfering bands due to ICDH and
G6PDH activities in the CFE were observed when higher
concentrations of substrates were utilized and when the
reaction was kept for prolonged periods. The activity band
was excised and analyzed by 2D BN–PAGE. The activity
band was more pronounced following 2D electrophoresis
Fig. 1. In-gel detection of NADK activity. Soluble CFE (60?g protein
equivalent) obtained from P. Xuorescens grown in citrate (control)
medium was utilized. (I) NADK activity. (II) 2D BN–PAGE NADK
activity (activity bands from (I) were excised and loaded). Lanes A, NAD+
(0.5mM), ATP (3mM), isocitrate (2mM) NADP+–ICDH (5U) (reaction
mixture); lane B, reaction mixture–ATP.

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In-gel activity of NADK / R.J. Mailloux et al. / Anal. Biochem. 359 (2006) 210–215
213
(Figs. 1 and 2). Fig. 2 depicts the dependence of activity
band intensity on protein concentration. A faint band was
discernable when 40?g protein equivalent of CFE was uti-
lized. However, the intensity increased very signiWcantly
when 80?g protein equivalent to CFE was loaded. These
bands can be quantiWed by using appropriate computer
software [4,6]. It is important to note that up to 10?g of
protein can be analyzed. However, the reaction time to
observe the activity band was longer and interfering bands
due to the activities ICDH and G6PDH in the CFE were
also evident. Fig. 3 shows the inXuence of CTP and iodoac-
etate on the activity of NADK. The enzyme appeared to
have a higher aYnity for ATP than for CTP. It was also
severely inhibited by iodoacetate. Indeed, it has been
reported that the activity of the enzyme decreases in the fol-
lowing nucleotide order: ATP>CTP>UTP>GTP [26]. As
the enzyme does possess an essential sulfydryl moiety, the
presence of iodoacetate in the reaction mixture did have a
negative eVect on the activity of NADK. In Fig. 4(I), the 2D
BN–PAGE of NADK in control and menadione cultures is
depicted. In the cells isolated from the menadione cultures,
the activity was more intense than in the control. When the
2D SDS–PAGE was performed, a prominent band at
»47kDa was evident. No such band was observed with the
cells from control cultures (Fig. 4(II)). However a faint
band was observed when the gel was stained with silver
staining solution instead of Coomassie blue. The ability of
NADK to produce NADP+ from NAD+ and ATP was fur-
ther conWrmed by HPLC. As shown in Fig. 5, NADK was
much more active in the cells grown in the presence of men-
adione as revealed by the intense NADP+ peak. In compar-
ison, the signal corresponding to NAD+, the reactant, was
far more intense in control cultures. The identities of these
peaks (NAD+, NADP+) were conWrmed by running
commercial standards individually and simultaneously.
The reaction mixtures were also spiked to conWrm the pres-
ence of these nucleotides (data not shown). These data con-
Wrmed the presence of NADK in P. Xuorescens.
Fig. 2. In-gel detection of NADK activity and dependence on protein con-
centration. Lanes A, B, and C correspond to 40, 60, and 80?g, respec-
tively, of soluble CFE obtained from P. Xuorescens grown in a citrate
medium.
Fig. 3. InXuence of various nucleotides and inhibitors on NADK activity.
Soluble CFE from P. Xuorescens (60?g) was utilized. (I) Lane A, reaction
with ATP; lane B, Reaction with CTP; lane C, reaction without ATP (note
the absence of NADK activity). (II) Lane A, reaction mixture; lane B,
reaction mixture+iodoacetate (2mM).
Fig. 4. PuriWcation of NADK. (I) 2D BN–PAGE of NADK activity. Lane
A, soluble CFE (control); lane B, soluble CFE (menadione culture). (II)
2D SDS–PAGE. Lane A, soluble CFE (control); lane B, soluble CFE
(menadione culture). Coomassie staining was utilized to detect the
protein.

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tein levels of NADK were further conWrmed by immuno-
blot analysis. Antibodies directed against NADK were able
to reveal the higher expression of NADK in the Al-stressed
cultures (Fig. 6(III)). However, HepG2 cells exposed to
control did not exhibit any detectable levels of NADK.
This discrepancy between the silver stain and the immuno-
blot may be due to the increased sensitivity of the silver
stain. Accordingly, the higher activity and expression of
214
In-gel activity of NADK / R.J. Mailloux et al. / Anal. Biochem. 359 (2006) 210–215
The activity and expression of NADK in the human
liver cell line, HepG2, was also detected using the same
technique. The cytoplasmic fraction of the HepG2 exposed
to Al had higher levels of NADK activity than the control
(Fig. 6(I)). In addition, the presence of iodoacetate in the
reaction mixture had an inhibitory inXuence on NADK
activity, thus conWrming the signiWcance of sulfydryl moie-
ties in this enzyme. Following the activity stain, the bands
were excised and subjected to 2D BN–PAGE and silver
stain. Higher levels of NADK were detected in the HepG2
cells exposed to Al-stressed conditions (Fig. 6(II)). The pro-
NADK in the Al-stressed HepG2 cells suggests the impor-
tance of this enzyme in maintaining high levels of NADP+.
Indeed, Al is known to trigger oxidative stress and this
enzyme may play a key role in promoting a reductive envi-
ronment. Hence the BN–PAGE technique coupled with 2D
electrophoresis is a very useful tool to monitor activity and
expression of NADK in diVerent cellular systems.
In conclusion, the BN–PAGE technique proved to be
invaluable in the detection of NADK activity and expres-
sion. The versatility and sensitivity of this assay allowed the
selective characterization of this enzyme in two separate
organisms. Finally, this novel approach in detecting
Fig. 5. HPLC analysis of NADK activity in P. Xuorescens. (I) Soluble CFE
(control). (II) Soluble CFE (menadione culture). Peak 1 corresponds to
the NADP+ retention time and peak 2 corresponds to the NAD+ retention
time.
Fig. 6. In-gel detection of the activity and expression of NADK. HepG2
cells were grown to 70% conXuency and exposed to ?-MEM supple-
mented with lane A, citrate and lane B, Al–citrate for 24h and soluble
CFE was analyzed. (I) In-gel activity stain. (II) Analysis of protein levels
by 2D BN–PAGE. The activity band was electrophoresed and protein
levels were detected by silver staining. (III) detection of NADK protein
levels by immunoblot.

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In-gel activity of NADK / R.J. Mailloux et al. / Anal. Biochem. 359 (2006) 210–215
215
NADK will allow the monitoring of this pivotal enzyme in
various biological systems.
Acknowledgments
This project was supported by funding from Material
and Manufacturing Ontario and Industry Canada.
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