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Purification and characterization of nucleoside diphosphate kinase from the brain of Bombyx mori.

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Archives of Insect Biochemistry and Physiology 49:147–155 (2002)
Purification and Characterization of Nucleoside
Diphosphate Kinase From the Brain of Bombyx mori
Tomohide Uno,* Mayumi Ueno, Michiko Kikuchi, and Yasuo Aizono
Nucleoside diphosphate kinase in the brain of Bombyx mori was purified by ammonium sulfate fractionation, and a sequence
of chromatographies on DEAE-Cellulofine, hydroxyapatite, Mono-S, and Mono-Q column. The purified enzyme preparation was
found to be electrophoretically homogeneous on SDS-PAGE, and its molecular mass was determined to be 18 kDa. The
purified protein was digested and the amino acid sequences of resulting peptides were determined. The enzyme showed high
similarity to the amino acid sequences of the Drosophila NDP kinase. The enzyme showed NDP kinase activity and mediated
the phosphorylation of myelin basic protein. Gel filtration and Hill plot analysis indicate that the purified NDP kinase forms a
tetramer and shows little interaction among substrates. Dephosphorylation of NDP kinase by bacterial alkaline phosphatase
increased NDP kinase activity. This result indicates that phosphorylation of NDP kinase represses NDP kinase activity. Arch.
Insect Biochem. Physiol. 50:147–155, 2002. © 2002 Wiley-Liss, Inc.
KEYWORDS: NDP kinase; brain; Bombyx mori
INTRODUCTION
Nucleoside diphosphate kinase (NDP kinase,
EC 2.7.4.6) catalyzes the transfer of a phosphoryl
group from nucleoside triphosphate to nucleoside
diphosphate [N1TP+N2DP «N1DP+N2TP] (Parks
and Agarwal, 1973). NDP kinase is not substrate
specific and can use purine and pyrimidine, riboor deoxy ribonucleotides as substrate.
Previously, the primary role of NDP kinase in
the cell was considered as the maintenance of a
pool of nucleoside triphosphates required for biosynthesis, but lately several NDP kinases encoded
by the genes of the nm23 family have been shown
to play important roles in tumor metastasis, morphogenesis, cell proliferation, differentiation, and
transcriptional regulation (Lacombe et al., 1992;
Rosengard et al., 1989: Biggs et al., 1990; Keim et
al., 1992; Lakso et al., 1993; Okabe-Kado et al.,
1988; Postel et al., 1993). For example, nm23-H1
has been reported to be down regulated in some
metastic cell lines (Wallet et al., 1990). The gene,
nm23-H2, was identified as a transcriptional factor for c-myc, a well-known regulator of cell proliferation and differentiation (Stahl et al., 1991;
Postel et al., 1993). In insects; the product of the
abnormal wing disc (awd) developmental gene,
which is responsible for normal wing development
in Drosophila melanogaster, exhibits NDP kinase activity (Dearolf et al., 1988). Little is known about
the biochemical and functional features of NDP
kinase of insects except Drosophila melanogaster
(Inoue et al., 1996).
In this study, NDP kinase was purified to homogeneity from the brain of Bombyx mori. The purified protein was demonstrated to be NDP kinase,
as judged from the partial amino acid sequence.
The purified protein showed NDP kinase activity
Laboratory of Biological Chemistry, Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Nada-ku, Kobe Hyogo, Japan
Abbreviations used: NDP kinase = nucleoside diphosphate kinase; BSA = bovine serum albumin; PTTH = prothoracicotropic hormone; MBP = myelin basic
protein; GTPgS = guanosine 5,-O-(3-thiotri) phosphate.
Grant sponsor: Ministry of Education and JSPS Research for the Future Program. Grant number: 14760033.
*Correspondence to: Laboratory of Biological Chemistry, Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Nada-ku, Kobe Hyogo
657-8501, Japan. E-mail: unotom@kobe-u.ac.jp
Received 19 April 2001; Accepted 16 March 2002
© 2002 Wiley-Liss, Inc.
DOI: 10.1002/arch.10037
Published online in Wiley InterScience (www.interscience.wiley.com)
148
Uno et al.
and phosphorylated myelin basic protein (MBP)
in vitro. Dephosphorylation of the purified NDP
kinase increased NDP kinase activity.
MATERIALS AND METHODS
Materials
Pupae of the silkworm, Bombyx mori, was purchased from Nikko Shoji. DEAE-Cellulofine
A-500m and hydroxyapatite were donated by Seikagaku Kogyo Co., Ltd. Lysyl endopeptidase was purchased from Wako Pure Chemicals Industries.
Mono-Q HR5/5 and Superdex 75 PC 3.2/30 were
purchased from Pharmacia LKB. [35S] GTPgS (1,000–
1,500 Ci/m mol) and [g-32P] ATP (3,000 Ci/m mol)
were from ICN Biochemicals. Other chemicals used
were of analytical grade.
Determination of Protein
Proteins were determined using BSA (Fraction
V, Sigma) as the standard by the method of Lowry
et al. (1951).
SDS-PAGE
SDS-polyacrylamide gel analysis was performed
according to the method of Laemmli (1970), using a 4.5% stacking gel and a 15% separating gel,
at a constant current of 16 mA. The proteins in a
gel were stained by the silver-staining method
(Morissey 1981).
GTPgS Binding Assay
The incubation mixture included 50 mM TrisHCl (pH 8.0), 40 mM [35S] GTPgS, and 10 mM
MgCl2 in a volume of 100 ml. The reaction was allowed to proceed for 30 min at 25°C and stopped
by adding 1 ml of washing buffer (20 mM TrisHCl, pH 8.0, 25 mM MgCl2, and 0.1 M NaCl).
The reaction mixture was filtrated through a nitrocellulose membrane filter. The membrane was
washed twice with 1 ml of washing buffer and
dried. The filter-bound radioactivity was counted
by a liquid scintillation counter (Aloka, LSC-5100).
Protein Sequence Analysis
For determination of the partial amino acid sequences, the purified enzyme (30 mg, 1.8 nmol)
was incubated with 0.3 mg of lysyl endopeptidase
at 30°C for 16 h in 100 ml of 50 mM Tris-HCl (pH
9.0) containing 4 M urea. The mixture was subjected to reverse phase high performance liquid
chromatography using a mBondasphere C18 column equilibrated with 0.1% (w/v) trifluoroacetic
acid. Peptide fragments were separated with a linear-gradient of acetonitrile, from 0 to 60% (v/v).
The amino acid sequences were determined by automated Edman degradation using a Shimadu
PPSQ-10 protein sequencer.
NDP Kinase Assay
Nucleotide diphosphate kinase assay was determined as follows. The standard assay (40 ml) contained 5 mM [g-32P] ATP (10 mCi), 1.5 mM GDP, 5
mM MgCl2 and 50 mM MES-NaOH (pH 6.0). Reaction was started by adding 10 ng of purified protein, carried out at 30°C for 2.5 min, and stopped
by adding 40 ml of 50 mM GTP. Ten-microlitter
aliquots of the samples were spotted on polyethyleneimine-cellulose sheets and then developed
in 0.75 M potassium phosphate buffer (pH 3.4)
for 90 min at room temperature. Reaction products
were located under an ultraviolet lamp. The radioactivities were counted using an imaging analysis,
BAS 1000 Man (Fuji Film Co., Tokyo, Japan). One
unit was defined as the amount of enzyme that
catalyze the production of 1 mM GTP min–1.
Protein Kinase Assay
The mixture with a final volume of 20 ml contains 20 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 1
mM EDTA, 1 mM DTT, 10 mM [g-32P] ATP (0.1 mCi),
and 1 mg of the protein. The phosphorylation was
started by adding 5 ml (10 ng) of NDP kinase to
20 ml of the reaction mixture. After 60 min incubation at 25°C, the reaction was terminated by adding 4 ml of SDS-sample buffer. The samples were
subjected to SDS-PAGE, and dried. 32P-labelled
Archives of Insect Biochemistry and Physiology
Purification of NDP Kinase
bands were visualized by exposing the gel to Kodak
X-Omat films.
Gel Filltration
The purified protein (7.8 mg, 40 ml) was applied
to a Superdex 75 PC 3.2/30 column (Smart system, Pharmacia) equilibrated with 62.5 mM TrisHCl (pH 6.7) and 0.1 M NaCl at a flow rate of 40
ml/min. The standard proteins are as follows; myoglobin (17.6 kDa), chymotrypsinogen A (25.6
kDa), ovalbumin (45 kDa), and BSA (67 kDa).
Activity of Dephosphorylated and Autophosphorylated
NDP Kinase
Purified enzyme (10 ng) was dephosphorylated
at 30°C for 10 min in 10 ml of reaction mixture A
[20 mM Tris-HCl (pH 8.0), 5 mM MgCl2, and 6
milli units of bacterial alkaline phosphatase]. The
NDP kinase reaction was started by adding 25 ml
of reaction mixture B [50 mM MES-NaOH (pH
6.0), 10 mM Na3VO4, 5 mM MgCl2, 5 mM [g-32P]
ATP and 1.5 mM GDP]. Reactions were incubated
at 30°C for the indicated period of time. The reaction was stopped with the addition of 50 mM GTP.
The reaction products were analyzed as described
previously. The NDP kinase reaction mixture of
autophosphorylated NDP kinase was made by adding 1 mM ATP in place of alkaline phosphatase in
reaction mixture A. As control, NDP kinase was
incubated in reaction mixture A omitting bacterial
alkaline phosphatase and reaction mixture B.
RESULTS AND DISCUSSION
Purification of NDP Kinase
The method to assay NDP kinase activity using
thin layer chromatography is time-consuming and
difficult. The GTPgS [Guanosine 5¢-O-(3-thiotri)
phosphate], which is GTP analog, filter binding assay was the easy and conventional method to detect NDP kinase indirectly (Inoue et al., 1996).
NDP kinase binds [35S] GTPgS, transfers the [35S]
-thiol phosphate to NDP kinase, and [35S] -thiol
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149
phosphate-bound NDP kinase is trapped on nitrocellulose membrane (Inoue et al., 1996). So, we
used [35S] GTPgS filter binding assay to purify NDP
kinase from the brain of Bombyx mori and identified the purified protein as NDP kinase by protein
sequencing. All procedures were carried out at 4°C.
Brains of pupae (11.84 g) were homogenized in
buffer A [20 mM Tris-HCl (pH 8.0), 0.25 M sucrose, 1 mM EDTA, 1 mM Diisopropyl flurophosphate, 1 mM DTT, and 2 mg/ml pepstatin A].
The homogenate was centrifuged at 20,000g for 30
min. To the supernatant, solid ammonium sulfate
was added to 70% saturation, followed by centrifugation. The precipitate was dissolved in 12 ml of
buffer A and dialyzed against buffer A for three
days. After centrifugation, the supernatant was applied to a DEAE-Cellulofine A-500 (f1.5 ´ 24 cm)
equilibrated with buffer A. After washing with
buffer A, proteins were eluted with a step-wise increase in NaCl (Fig. 1). GTPgS binding activity was
mainly recovered in 0.03 M NaCl and 0.2 M NaCl
eluates. The 0.03 M NaCl eluate was dialyzed
against buffer B [10 mM potassium phosphate (pH
7.2), 1 mM DTT, and 0.1 mM EDTA]. After centrifugation, the supernatant was applied to a hydroxyapatite column (f1.0 ´ 10 cm) equilibrated
with buffer B. After washing with buffer B, proteins were eluted with a step-wise increase in potassium phosphate. GTPgS binding activity was
recovered in 0.04 M potassium phosphate. The eluate was dialyzed against buffer C [20 mM sodium
acetate (pH 5.5)]. After centrifugation, the supernatant was applied to a Mono-S HR5/5 column
equilibrated with buffer C. After washing with
buffer C, the adsorbed proteins were eluted with a
linear gradient of 0-0.5 M NaCl in buffer C (Fig.
2A). GTPgS binding activity was detected between
0.04 to 0.07 M NaCl on the linear gradient. The
active fractions were pooled and dialyzed against
buffer D [20 mM Tris-HCl (pH 8.3)].
After centrifugation, the supernatant was applied to a Mono-Q HR5/5 column equilibrated
with buffer D. After washing with buffer D, the
adsorbed proteins were eluted with a linear gradient of 0–0.5 M NaCl in buffer D (Fig. 2B). GTPgS
binding activity was detected between 0.05 to 0.09
150
Uno et al.
Fig. 1. DEAE-Cellulofine column chromatography. The
crude enzyme preparation was applied to a column of
DEAE-Cellulofine (f1.5 ´ 24 cm), washed, and then eluted
stepwise with NaCl. The flow rate was 1.5 ml/min. Solid
line, absorbance profile at 280 nm. Dotted line, GTPgS
binding activity.
M NaCl on the linear gradient . A typical purification procedure is summarized in Table 1. Finally,
55 mg of GTPgS binding protein with a binding
constant of 0.36 nmol was purified from 11.8 g of
the brain of Bombyx mori. The sequential steps gave
an approximately 136-fold augmentation in specific content with a yield of 3.6%. As shown in
Figure 3A, the purified protein migrated as a single
band on SDS-PAGE. The estimated molecular mass
on SDS-PAGE was 18 kDa.
tain whether the purified protein was NDP kinase
or not, the NDP kinase activity was measured. The
purified protein showed NDP kinase activity. Optimal activity was observed at pH 6.0.
Protein Sequence of NDP Kinase
The 18-kDa protein was digested with lysyl endopeptidase, and the amino acid sequences of resulting peptides were determined. The amino acid
sequences were compared with those present in the
GenBank nucleotide database (by using the BlastN algorithm). The B. mori protein showed high
similarity to the deduced amino acid sequence of
the Drosophila NDP kinase (Fig. 4). Next, to ascer-
Enzymatic Properties of NDP Kinase
NDP kinase of Drosophila was found to function as a protein kinase (Inoue et al., 1996). So,
various exogenous protein substrates were tested
as possible substrates for phosphorylation in vitro.
When myelin basic protein (MBP) was incubated
with purified NDP kinase and [g-32P] ATP, incorporation of radioactivity into the protein was detected (Fig. 3B, lane 3). NDP kinase was also faintly
autophosphorylated (Fig. 3B, lane 1). BSA, BRab
(Uno et al, 1998) and histone were not phosphorylated (data not shown). MBP is a protein constitutent of myelin, nerve sheath, and used as a
marker protein of oligodendroglial cells (Shepard,
1994). NDP kinase in the brain of Bombyx mori
Archives of Insect Biochemistry and Physiology
Purification of NDP Kinase
Fig. 2. Mono-S column chromatography (A) and MonoQ column chromatography (B). The fractions were applied
to a column of Mono-S HR5/5 (f1.6 ´ 50 mm) and MonoQ HR5/5 (f1.6 ´ 50 mm), respectively, washed, and then
eluted with a 3-ml gradient of 0 to 0.5 M NaCl and with
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151
a 2-ml gradient of 0 to 0.3 M NaCl, respectively. Solid
line, dotted line, and dashed line, absorbance profile at
280 nm, GTPgS binding activity, and elution gradient of
NaCl, respectively.
152
Uno et al.
TABLE 1. Summary of NDP Kinase Purification From the Brain of Bombyx mori
Step
Cruse extract
70% (NH4)2SO4
DEAE-cellulofine
Hydroxyapatite
Mono-S
Mono-Q
Total protein (mg)
203
194
23.1
9.05
0.098
0.055
Total GTP g S binding (pmol)
9,720
9,030
8,470
4,640
374
357
Specific binding (pmol/mg)
47.9
46.5
367
512
3,820
6,490
Yield (%)
100
92.8
87.1
48.3
3.85
3.67
Purification (fold)
1.00
0.971
7.66
10.6
79.7
136
may regulate the connection between the neuron
and the glial cell by phosphorylating MBP-like protein in insect brain.
All known NDP kinase are oligomeric and made
of small polypeptides of about 150 residues (molecular mass of about 17 kDa) with a high degree
of sequence similarity (Hemmerich and Pecht,
1992). On a gel filtration column, the purified
Fig. 3. SDS-PAGE analysis of NDP kinase purified from
the brain of Bombyx mori (A) and phosphorylated proteins
(B). A: Lane 1: molecular mass marker. Lane 2: purified
NDP kinase (1 mg). B: MBP (1 mg) was incubated (60 min,
25°C) with purified NDP kinase (10 ng) and 10 mM [g32
P] ATP (0.1 mCi) in a reaction buffer [20 mM Tris-HCl
(pH 8.0), 5 mM MgCl2, 1 mM EDTA and 1 mM DTT]
(Lane 3). NDP kinase was autophosphorylated with [g32
P] ATP in a reaction buffer (Lane 1). Then, the reaction
was terminated by adding SDS-sample buffer. The samples
were subjected to SDS-PAGE. As control, MBP was reacted
with [g-32P] ATP in a reaction buffer (Lane 2).
Archives of Insect Biochemistry and Physiology
Purification of NDP Kinase
153
Fig. 4. Comparison of the deduced amino acid sequences
of Bombyx mori NDP kinase peptides with the sequences
of Drosophila melanogaster NDP kinase. Identical amino acids are indicated by asterisks.
protein exhibited a molecular mass of 67 kDa.
From this result, it appears that NDP kinase in
the brain of Bombyx mori associate to form a tetrameric structure.
To examine the interaction among subunits, Hill
plots of NDP kinase were constructed. The values
for Km and n were calculated to be 31.6 mM ± 0.1
and 1.14 ± 0.03, respectively. The n value so close
to 1.0 indicates little or no cooperativity on interaction among subunits of NDP kinase from the
brain of Bombyx mori. The purified NDP kinase
bound GDP with a higher affinity, compared to
the other NDP kinase with reported Km values,
125 mM (Ulloa et al., 1995).
autophosphorylated NDP kinase is suggested not
to be a phosphorylatted ntermediate during NDP
kinase GDP-phosphorylating reaction. NDP kinase
activities of autophosphorylated- and dephosphorylated forms, respectively, were measured (Fig. 5).
The basal and the autophosphorylated NDP kinsae
showed no significant differences in activity (Fig.
5; solid squares and triangles). NDP kinase activity was increased by dephosphorylation (Fig. 5;
open squares). These results suggest that autophosphorylation of NDP kinase inhibits the NDP
kinase GDP-phosphorylating reaction and the
autophosphorylated form is not an intermediate
during the NDP kinase GDP-phosphorylating reaction.
Both NDP kinase GDP-phosphorylating activity and autophosphorylation activity were affected
by magnesium ions. The maximal activities of
autophosphorylation and GDP-kinase activity were
observed at 0.1 mM (n = 3) and 7 mM (n = 3)
MgCl2, respectively. These results support the proposal that autophosphorylation and GDP-kinase
activity are different reactions. In the brain of insects, some protein phosphatase may regulate the
function of the phosphorylated NDP kinase by dephosphorylation.
What role does NDP kinase have in insect
brain? The enzyme has been shown to regulate
various cellular functions, including tumor metastasis, morphogenesis, cell proliferation, differentia-
Effect of Phosphorylation on NDP Kinase Activity
NDP kinase catalyzes the transfer of the terminal phosphate from nucleotide 5¢-triphosphate to
nucleotide 5¢-diphosphate (Parks and Agarwal,
1973). The mechanism of the NDP kinase reaction involves the formation of a high-energy phosphoprotein of a histidine residue (Morera et al.,
1995). In the absence of NDP as acceptor, NDP
kinase undergoes autophosphorylation utilizing
adenosine triphosphate as phosphate donor (Hemmerrich and Pecht, 1992). Using point mutational
analysis, it was shown that mutants replacing
autophosphorylated amino acid residues retained
NDP kinase activity (Almaula et al., 1995). So,
July 2002
154
Uno et al.
and GTP binding protein is responsible for effective GTP introduction into the GTP binding protein. Possibly NDP kinase in the brain is related
to PTTH secretion via GTP binding protein. Nothing is known about the role of NDP kinase in the
brain of Bombyx mori. Further studies are progressing to determine the functional role of NDP kinase in the brain relating to the PTTH release.
ACKNOWLEDGMENT
This work was supported by a grant-in-aid for
Scientific Research (No. 14760033) from the Ministry of Education and JSPS Research for the Future Program.
Fig. 5. NDP kinase activity of dephosphorylated- and
autophosphorylated- protein. Purified enzyme (10 ng)
was dephosphorylated at 30°C for 10 min in 10 ml of
reaction mixture A [20 mM Tris-HCl (pH 8.0), 5 mM
MgCl2 and 6 milli units of bacterial alkaline phosphatase]
(open square). The NDP kinase reaction was started by
adding 25 ml of reaction mixture B [50 mM MES-NaOH
(pH 6.0), 10 mM Na3VO4, 5 mM MgCl2, 5 mM [g-32P]
ATP and 1.5 mM GDP]. Reactions were incubated at 30°C
for the indicated period of time. The reaction was stopped
with the addition 50 mM GTP. The NDP kinase reaction
mixture of autophosphorylated NDP kinase was made
by adding 1 mM ATP in place of alkaline phosphatase
in reaction mixture A (solid square). As control, NDP
kinase was incubated in reaction mixture A omitting bacterial alkaline phosphatase and reaction mixture B (triangle). Y axis shows the amount of GTP produced per
10 ng NDP kinase.
tion, and transcriptional regulation in mammals
(Lacombe et al., 1992; Rosengard et al., 1989; Biggs
et al., 1990; Keim et al., 1992; Lakso et al., 1993;
Okabe-Kado et al., 1988; Postel et al., 1993). PTTH,
a neuropeptide hormone producing morphological changes and ecdysis in insects, is secreted from
the brain into hemolymph (Nagasawa, 1993). GTP
binding proteins contribute to the release of PTTH
(Shirai et al., 1998). NDP kinase has been proposed to interact with GTP binding proteins
(Kimura et al., 1990). The complex of NDP kinase
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