From the Division of Enzyme Chemistry, Institute for Enzyme
Research, The University of Tokushima, Tokushima 770, Japan
Hsp70 is a multifunctional molecular chaperone
whose interactions with protein substrates are regulated by ATP
hydrolysis and ADP-ATP exchange. We show here that, in addition to
ATPase activity, purified Hsp70 free from nucleoside-diphosphate (NDP) kinase exhibits intrinsic ADP-ATP exchange activity. The rate constants
for ATP hydrolysis and ATP synthesis were in a similar range at the
optimum pH of 7.5-8.5 in the presence of 5 mM ATP and 0.5 mM ADP. Hsp70 exhibited a considerably strict
preference for ATP as a phosphate donor, and a biased substrate
specificity, unlike NDP kinase; ADP, UDP, CDP > dTDP, dCDP > GDP, dGDP. During the reaction, Hsp70 formed an acid-labile
autophosphorylated intermediate, and nucleoside diphosphate-dependent
dephosphorylation of the latter then occurred. These properties of
Hsp70 are not identical but similar to those of NDP kinase, but are not
similar to those of adenylate kinase and ATP synthase.
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INTRODUCTION |
The cytoplasmic 70-kDa heat shock protein (Hsp70) is thought to
act as a "molecular chaperone" in protein folding by, at least, binding to nascent or misfolded segments of polypeptides, thereby regulating protein homeostasis (protein translocation, assembly, disassembly, and degradation) (1-3). Hsp70 binds tightly to ATP and
ADP (4, 5) and exhibits very weak ATPase activity (6-8), somewhat
diverse peptide binding activity (9-11), and the ability to couple
them. The nucleotides function to regulate peptide binding activity.
With ADP bound, Hsp70 binds tightly to peptides or denatured proteins,
but with ATP bound, the peptides are released (12). For peptide
dissociation, ATP binding but not ATP hydrolysis is essential (13, 14).
Following the hydrolysis of ATP, a relatively stable ADP·Hsp70
complex is formed, and the ADP-ATP exchange reaction then occurs for
the polypeptide binding and release cycles (3, 14, 15).
Despite the wealth of knowledge of ATP hydrolysis, as catalyzed by
Hsp70, and its regulation by unfolded proteins (15) and accessory
proteins (16), little is known as to the mechanism of the nucleotide
exchange reaction of Hsp70 in the cytosol. In this paper, we first
report intrinsic ADP-ATP exchange activity as a novel function of
Hsp70. The characteristics of the enzyme activity are similar to those
of nucleoside-diphosphate
(NDP1) kinases, but not to
those of adenylate kinase and ATP synthase.
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EXPERIMENTAL PROCEDURES |
Materials--
Hsp70 from bovine brain, nucleoside-diphosphate
kinase (EC 2.7.4.6) from bovine liver, adenylate kinase (EC 2.7.4.3)
from chicken muscle, various ribo- and deoxyribonucleoside tri-, di-, and monophosphates, AMP-PNP, and ATP
S were purchased from Sigma. [8-14C]ADP, [2-14C]CDP,
[8-14C]ATP, and [
-32P]ATP were obtained
from NEN Life Science Products. The monoclonal antibody against the
human nm23-H1 protein (human NDP kinase-A) was from Novocastra
Laboratories Ltd., UK. All other reagents were commercial products of
the highest grade available.
Purification of Hsp70--
Because Hsp70 purchased from Sigma
exhibits greater than 95% purity but there is still a possibility of
contaminating proteins with affinity for ATP, such as NDP kinase (17),
the preparation of Hsp70 was further purified to homogeneity by HPLC on
a Mono Q anion-exchange column and then on a gel permeation TSK-Gel
G3000SW column. Briefly, commercially available Hsp70 (600 µg) in
Buffer A (25 mM Tris-HCl, pH 7.2, 0.1 mM EDTA,
and 0.5 mM dithiothreitol) was applied to a Mono Q column
and then eluted with a narrow salt gradient of 0-0.15 M
KCl in Buffer A as shown in Fig. 1, which is able to separate conventional NDP kinase from Hsp70 (17). Hsp70 was
eluted with approximately 0.1 M KCl and separated from contaminating proteins. Conventional NDP kinase, eluted with
approximately 0.04 and 0.07 M KCl, was not detected among
the contaminants in the commercial products of Hsp70 on that
chromatography. The fractions of Hsp70 were concentrated and then
subjected to HPLC on a double-linked TSK-Gel G3000 SW column (7.5 × 600 mm each) with 50 mM ammonium formate buffer, pH 5.5, 0.1 mM EDTA, and 0.5 mM dithiothreitol. The
final preparation exhibited a purity of greater than 99% and was
judged to be free from conventional NDP kinase on silver-stained SDS-PAGE.

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Fig. 1.
Mono Q anion-exchange column
chromatography. Hsp70 (100 µg) and NDP kinase (30 µg)
purchased from Sigma in Buffer A were applied separately to a Mono Q
column (0.5 × 5 cm), equilibrated with Buffer A, and then eluted
with a salt gradient of 0.05-0.15 M KCl in Buffer A. ------, protein A280; , activity of ATP hydrolysis of the commercial
product of Hsp70; - - -, protein A280 of NDP kinase with a
molecular mass of 16 kDa; - , KCl gradient.
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Assaying of ATP Hydrolysis and ATP Synthesis--
Although
almost all the data for Hsp70 ATPase reported were obtained by
measurement of inorganic phosphate liberated from ATP, we found that
Hsp70 exhibits the reverse activity, i.e. ADP-ATP exchange,
and thus the amount of inorganic phosphate in the reaction is not
reflected by the ATPase activity. In this regard, we analyzed the
ATPase activity by measuring the conversion of [14C]ATP
to [14C]ADP. The ATP synthesis activity was analyzed by
measuring the conversion of [14C]ADP to
[14C]ATP as described (18, 19). The reactions were
carried out at 37 °C for 30 min in 100 mM Hepes-KOH
buffer, pH 8, containing 5 mM ATP, 0.5 mM ADP,
and 6 mM MgCl2 with 0-20 pmol of Hsp70
(monomeric form), and 0.05 µCi of [8-14C]ATP and 0.02 µCi of [8-14C]ADP for the assaying of ATPase and ATP
synthesis activities, respectively, in a total volume of 10 µl. After
incubation, the reaction mixtures were immediately spotted onto a
polyethyleneimine-cellulose TLC plate (Macherey-Nagel). ADP and ATP
were separated by ascending chromatography with 1 M formic
acid containing 0.7 M LiCl, and the radioactivity in the
resolved spots was quantitated with a Bio Imaging Analyzer BAS 1500 (Fuji Photo Film Co., Ltd., Japan).
Nucleotide Specificity as a Phosphate Acceptor of Hsp70--
The
nucleotide specificity of Hsp70 was examined at 37 °C for 30 min in
100 mM Hepes-KOH buffer, pH 8, containing 1 µCi of [
-32P]ATP and 6 mM MgCl2 with
10 pmol of Hsp70 and 0.5 mM of various ribo- or
deoxyribonucleoside diphosphates as phosphate acceptors in a 10-µl
reaction mixture. After incubation, the nucleotides were resolved by
polyethyleneimine-cellulose TLC with 0.75 M
KH2PO4, pH 3.5, followed by quantification with
an imaging analyzer.
Detection of an Autophosphorylated Hsp70
Intermediate--
Autophosphorylation and CDP-dependent
dephosphorylation of Hsp70 were analyzed using 10 µg of Hsp70, 10 µCi of [
-32P]ATP (6000 Ci/mmol), 100 µM ATP, and 6 mM MgCl2 in 100 mM Hepes-KOH buffer, pH 8, in the absence and presence of 5 mM CDP, respectively, in a total volume of 10 µl. After
incubation at 37 °C for 2 h, both reactions were quenched by
the further addition of 10 mM EDTA, and half of each sample
was then treated with the traditional (pH 6.8) SDS sample buffer
without boiling and subjected to 15% SDS-PAGE. After electrophoresis,
the gel was dried without acid fixation and analyzed with an imaging
analyzer. The remaining samples were analyzed as to the acid and base
stability of the phosphorylated Hsp70 as described (20). These samples
were then analyzed with an imaging analyzer.
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RESULTS |
Identification of ADP-ATP Exchange Activity of Hsp70--
We found
the purified Hsp70 free from NDP kinase exhibited ADP-ATP exchange
activity, in addition to ATPase activity. As shown in Fig.
2A, Hsp70 exhibited weak
ATPase activity in a dose-dependent manner, but no activity
was observed for the control protein, BSA. Although ADP is a product
inhibitor of common ATPase, a limited amount of ADP, i.e.
concentrations between 0.1 and 1.0 mM, in the reaction
mixture stimulated the ATPase activity of Hsp70 by 2-3.7 fold, as
shown in Fig. 2A. Inhibition by ADP was then observed at
concentrations in excess of 1 mM (data not shown). AMP had no effect on this activity.

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Fig. 2.
The ATP hydrolysis and ATP synthesis
activities of Hsp70. A, the activities of ATP hydrolysis of
Hsp70 ( , , ) and BSA ( , ) were assayed under the
conditions given under "Experimental Procedures" using 0 to 20 pmol
of each protein (monomeric form), 5 mM ATP, 0.05 µCi of
[8-14C]ATP, and 6 mM MgCl2 in the
presence ( , ) or absence ( , ) of 0.5 mM ADP.
The activity in the presence of 0.5 mM AMP instead of 0.5 mM ADP ( ) was also analyzed. After a 30-min reaction at pH 8, samples were analyzed by TLC, and ADP formation was calculated with an imaging analyzer. B, the ATP synthesis activities of
Hsp70 ( , , , ×) and BSA ( , ) were assayed under the
conditions given under "Experimental Procedures" using 0.02 µCi
of [8-14C]ADP in the presence ( , ) or absence ( ,
) of 0.5 mM ADP. The ATP synthesis activities of Hsp70
were also examined at 37 °C for 30 min in the reaction mixture
containing 0.5 mM AMP instead of ADP (×) and 5 mM Pi instead of ATP ( ).
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The reverse reaction of ATP hydrolysis, i.e. ATP synthesis
activity, of Hsp70 was analyzed (Fig. 2B). Surprisingly,
Hsp70 catalyzed ATP synthesis at a slow but linear rate in the presence of substrate ADP but not in the presence of AMP. The ATP synthesis activity of Hsp70 gave saturation curves with ADP as a phosphate acceptor and with ATP as a phosphate donor. The reaction of ATP synthesis, as catalyzed by Hsp70, was entirely different from that of
adenylate kinase, which catalyzes the reversible
ATP-dependent synthesis of ADP from AMP, as shown in Fig.
3C. Furthermore, the reaction
was different from that of conventional ATP synthase, which catalyzes
ATP synthesis from ADP and Pi. When ATP was replaced with 5 mM Pi, Hsp70 failed to produce ATP from ADP
(Fig. 2B). The conversion of ADP to ATP, i.e.
ADP-ATP exchange, catalyzed by Hsp70 was similar to the reaction
catalyzed by NDP kinase, which catalyzes the nucleoside
triphosphate-dependent synthesis of ribo- and
deoxyribonucleoside triphosphates from the corresponding diphosphates
(21-23). No ATP synthesis activity was observed for the control
protein, BSA (Fig. 2B).

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Fig. 3.
Substrate specificities of Hsp70 and NDP
kinase (A and B), and the failure of Hsp70 to
function as an adenylate kinase (C). A and
B, the substrate specificities of NDP kinase and Hsp70 were
assayed under the conditions given under "Experimental Procedures"
using 10 pmol of each protein (monomeric form), 1 µCi of
[ -32P]ATP (6000 Ci/mmol), 6 mM
MgCl2, and 0.5 mM various ribo- (lanes 1-8) and deoxyribonucleoside diphosphates (lanes
9-16), in 100 mM Hepes-KOH buffer, pH 8, in a total
volume of 10 µl. After incubation at 37 °C for 30 min, samples
were analyzed by TLC, followed by autoradiography with an imaging
analyzer. C, the adenylate kinase activity of Hsp70
(lane 2) and the activity of adenylate kinase from chicken
muscle (lane 3) were assayed as described (17, 19) using 1 mM AMP and 1 µCi [ -32P]ATP. Lane
1, control with [ -32P]ATP plus AMP in the absence
of the enzyme. Adk, adenylate kinase.
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From the kinetic results for ATP hydrolysis and for ATP synthesis, the
Km value of ATP for the ATP hydrolysis in the
presence of 0.5 mM ADP and the Km value
of ADP for the ATP synthesis of Hsp70 were calculated to be 4.3 mM and 0.31 mM, respectively (data not shown).
The specific activities of Hsp70 for ATP synthesis at pH 7.4, measured
under the conditions in Fig. 2B, were 2.8-4.0 molecules of
ATP min
1 (Hsp70 monomer)
1 with the five
different purified Hsp70 preparations tested. These values are nearly
the same as for the activity of ATP hydrolysis of Hsp70 in the presence
of 0.5 mM ADP at pH 8. The Km value of
ATP and the specific activity for the ATPase activity of Hsp70
determined by measuring the conversion of [14C]ATP to
[14C]ADP in our experiments were greatly different from
the data previously reported (15, 24), which were determined by
measuring the release of inorganic phosphate from ATP. Since Hsp70
exhibits both the activities of ATP hydrolysis and ATP synthesis, the
kinetic constants for Hsp70 ATPase determined by measurement of
inorganic phosphate in the reaction are not correct. Furthermore, the
Km value of ATP for the ATPase activity of Hsp70 in
our experiments is much higher than the values reported in the range of
0.7-1.4 µM (15, 24) and is in a range similar to that
reported for NDP kinase (25), because the Km value
was determined with 0.5 mM ADP in our experiments. Since
both the Km values are in a range similar to the
concentrations of ATP (
5 mM) and ADP (
0.5
mM) in the cytosol, changes in substrate availability would
be expected to significantly affect the in vivo enzyme
reaction.
The pH optima for Hsp70 ATPase and ATP synthesis were determined using
100 mM Mes buffer (pH 5.5-6), Hepes buffer (pH 7-8), Ches
buffer (pH 9), and Chaps buffer (pH 10). Without ADP, the pH optimum
for ATPase was found to be 7, consistent with a previous report (26).
The optimum pH of the stimulated Hsp70 ATPase in the presence of 0.5 mM ADP, however, shifted to 7.5-9. The pH optimum for
Hsp70 ATP synthesis was 7-9, which overlapped that of the stimulated
ATPase. It is noteworthy that, at pH 7.5-8.5, the rate constants for
ATP hydrolysis and ATP synthesis of Hsp70 were similar in the presence
of 5 mM ATP and 0.5 mM ADP in the reaction
mixture. The ATP synthesis activities were slightly higher below pH 7.5 and lower above pH 8.5 than the ATP hydrolysis activities.
Hsp70 Functions as a NDP Kinase-like Enzyme--
NDP kinase is
known to use any nucleoside triphosphate as a phosphate donor with
similar efficiency. Hsp70, however, utilized ATP most efficiently, with
the other ribo- and deoxyribonucleoside triphosphates being utilized at
rates 16-33% of the rate of ATP utilization (data not shown). NDP
kinase transfers the terminal phosphate of a nucleoside triphosphate to
a nucleoside diphosphate. To determine whether this mechanism is the
same for Hsp70, ATP analogs, such as ATP
S and AMP-PNP, were added
together with ATP to the reaction mixture, and then the transfer of the
-phosphate from ATP to ADP was analyzed. This transfer by both NDP
kinase and Hsp70 was competitively inhibited by ~60-50% in the
presence of equal concentrations of ATP
S and ATP and was completely
inhibited by 5-fold excess concentrations of these analogs over those
of ATP (data not shown).
Next we examined the specificity of each nucleotide as an acceptor of
the transfer of the [
-32P]phosphate of ATP, as
catalyzed by Hsp70. As shown in Fig. 3, A and B,
NDP kinase converted all ribo- and deoxyribonucleoside diphosphates to
the corresponding nucleoside triphosphates with almost similar
efficiency, consistent with previous reports (25, 27, 28). Hsp70,
however, exhibited a biased substrate specificity, i.e. UDP,
CDP > dTDP, dCDP > GDP, dGDP. Under these assay conditions, the conversion of ADP and dADP to ATP and dATP, respectively, could not
be analyzed, because the newly formed products overlapped the phosphate
donor, [
-32P]ATP. Nevertheless, it was confirmed that
the rates of conversion by Hsp70 of [14C]ADP and
[14C]CDP to the corresponding nucleoside triphosphates
exhibited almost similar efficiency (data not shown).
Since NDP kinase requires Mg2+ or Mn2+ for its
activity, the effects of divalent cations on the ATP hydrolysis and ATP
synthesis activities of Hsp70 were analyzed. Both activities of Hsp70
are almost equally stimulated by 6 mM Mg2+,
Mn2+, Co2+, and Ni2+ (data not
shown).
Formation of an Autophosphorylated Hsp70 Intermediate and Its
CDP-dependent Dephosphorylation--
We examined whether
or not the intrinsic ADP-ATP exchange activity of Hsp70 is due to a
contaminant, since its activity is similar to that of NDP kinase. This
possibility is unlikely for the following reasons. Firstly, the
commercially available Hsp70 was further purified by HPLC on a Mono Q
anion-exchange and then a gel permeation column to remove possible
contaminants with affinity for ATP, such as NDP kinase. The purified
Hsp70 was >99% pure and was confirmed to be free from conventional
NDP kinase. This was also confirmed by the fact that no
immunoreactivity of the Hsp70 preparation against anti-NDP kinase-A
antibodies was observed (Fig.
4A). Secondly, the nucleotide
specificity for the activity of ATP synthesis by Hsp70 was different
from that reported for NDP kinase. Thirdly, an autophosphorylated
intermediate was observed in the protein band of Hsp70, as described
below, as in the case of the autophosphorylated intermediate of
conventional 16-kDa NDP kinase.

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Fig. 4.
SDS-PAGE and immunoblot analyses of Hsp70
(A), autophosphorylation of Hsp70 in an alkali-stable and
acid-labile manner, and CDP-dependent dephosphorylation
(B and C). A, silver-stained SDS-PAGE (4-20% gradient) of 1 µg of Hsp70 (lane 2) and
immunoblot analysis of 1 µg of Hsp70 (lane 3) and NDP
kinase (lane 4) using a 1:100 dilution of a monoclonal
antibody raised against human nm23-H1 protein (NDP kinase-A), followed
by ECL Western blotting detection reagent (Amersham). Lane
1, molecular weight markers. B, the autophosphorylation
and CDP-dependent dephosphorylation of Hsp70 (10 µg) were
analyzed as described under "Experimental Procedures." The
autophosphorylation of Hsp70 in the absence (lane 1) and
presence (lane 2) of 5 mM CDP for 2 h. Half
of each sample was treated with the traditional (pH 6.8) SDS sample
buffer without boiling, subjected to 15% SDS-PAGE, and then dried
without acid fixation. To determine the stability of the
autophosphorylated Hsp70 as a function of pH, phosphorylated Hsp70 was
treated with basic (pH 8.5) (lane 3) SDS sample buffer
without boiling and then electrophoresed, and the gel was dried without
acid fixation. For acid stability, phosphorylated Hsp70 was boiled in
the SDS sample buffer at pH 6.8 and then electrophoresed. The gel was fixed in 20% trichloroacetic acid, followed by Coomassie staining, destaining in methanol/acetic acid, and drying (lane 4). The
samples were then analyzed with an imaging analyzer. C, the
remaining halves of the samples in B with (lane
1) or without (lane 2) 5 mM CDP in the
reaction mixture were then analyzed by TLC, followed by imaging
analysis.
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NDP kinase autophosphorylates the active site histidine of the
intermediate in the process of the catalytic phosphate transfer reaction (21-23). Hsp70 gave a single autophosphorylated intermediate band corresponding to a protein with a molecular mass of 70 kDa in the
absence of the nucleoside diphosphate, CDP, in the reaction mixture,
which contained 10 µCi of [
-32P]ATP, 100 µM ATP, and 6 mM MgCl2 (Fig.
4B, lane 1), and no formation of CTP was observed
(Fig. 4C, lane 1). In the presence of 5 mM CDP in the reaction mixture, however, the radioactivity
of the phosphorylated intermediate decreased significantly with the
concomitant formation of 32P-labeled CTP from CDP (Fig.
4B, lane 2, and Fig. 4C, lane
2, respectively). Furthermore, a significant decrease in the
radioactivity of the autophosphorylated intermediate was also observed
on the addition of 5 mM CDP to the reaction mixture after
the first reaction of the formation of an autophosphorylated
intermediate (data not shown). These results indicate that Hsp70
catalyzes the transfer of the
-phosphate group from ATP to CDP, and
that this transfer involves a phosphoenzyme intermediate. These
properties are similar to those of the conventional NDP kinase
reported. To characterize the phosphorylation of Hsp70,
autophosphorylated Hsp70 was subjected to acid, neutral, and basic
treatments as described (20, 22), which allow the evaluation of a high
energy phosphate on a histidine residue in NDP kinase and histidine
protein kinase. The phosphorylated intermediate of Hsp70 was stable as
to alkaline treatment, but labile as to acid treatment, with a
significant decrease in the autophosphorylation level, as compared with
neutral treatment, as shown in Fig. 4B, lanes 1,
3, and 4, respectively. That phosphorylation at
serine, threonine, and tyrosine is stable as to acid treatment suggests
that the acid-labile and alkali-stable phosphorylated residue(s) in
Hsp70 may be a basic amino acid residue, although the phosphorylated
residue has not yet been identified.
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DISCUSSION |
When we analyzed the ATPase activity of Hsp70, we observed a
discrepancy in the ATPase activity determined with two different methods; the ATPase activity determined as the conversion of
[14C]ATP to [14C]ADP was higher than that
determined as the release of Pi from ATP. In turn, we
obtained Hsp70 which is free from contaminants with affinity to ATP,
such as conventional NDP kinase, and measured the activities of ATP
hydrolysis as well as ATP synthesis of Hsp70 and found that Hsp70 also
exhibits the activity of ADP-ATP exchange.
A mechanism for Hsp70-protein/peptide interaction and ATP hydrolysis
has been proposed (3, 14). The first step in the mechanism involves the
binding of a substrate protein/peptide to an Hsp70·ADP complex,
resulting in a conformational change which causes acceleration of
ADP-ATP exchange in the presence of ATP (step 2). The binding of ATP
causes a conformational change which triggers substrate release from
the complex (step 3). Finally, ATP is hydrolyzed to ADP to afford an
Hsp70·ADP complex that can then participate in a new cycle of binding
(step 4). The mechanisms underlying ADP-ATP exchange (step 2) and ATP
hydrolysis (step 4) are best understood for the bacterial Hsp70
homolog, DnaK, with its cofactors, DnaJ and GrpE, which accelerate the
ATP-dependent cycle of substrate binding and release (3,
17, 29). Interaction with DnaJ stimulates the hydrolysis of ATP by
DnaK, resulting in the formation of a stable ternary complex of an
unfolded substrate polypeptide/protein, DnaJ and DnaK. The ADP-ATP
exchange factor, GrpE, promotes the release of ADP from DnaK, which is
rate-limiting in the cycle, following dissociation of DnaJ and
subsequent ATP binding and substrate dissociation from DnaK (13, 30).
Although the prokaryotic and eukaryotic Hsp70 systems have similar
functional properties, a GrpE-like nucleotide exchange factor has not
been found in the eukaryotic cytosol (13). On the basis of our present data, we propose that the intrinsic ADP-ATP exchange activity of Hsp70
accelerates the ATP-dependent reaction cycle in
protein/peptide folding. To our knowledge, this is the first report of
intrinsic ADP-ATP exchange activity of Hsp70. Determination of the
precise role of the activity in the function of Hsp70 awaits further
biochemical and genetic analysis.
We thank Dr. Niwa for the helpful
discussion.