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J Biol Chem, Vol. 273, Issue 10, 5435-5438, March 6, 1998
From the Division of Enzyme Chemistry, Institute for Enzyme Research, The University of Tokushima, Tokushima 770, Japan
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ABSTRACT |
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Abstract
Introduction
Procedures
Results
Discussion
References
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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 |
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Abstract
Introduction
Procedures
Results
Discussion
References
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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 |
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Abstract
Introduction
Procedures
Results
Discussion
References
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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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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 |
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Abstract
Introduction
Procedures
Results
Discussion
References
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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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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 ATPS 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 ATPS and ATP and was completely
inhibited by 5-fold excess concentrations of these analogs over those
of ATP (data not shown).
-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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-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 |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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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.
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ACKNOWLEDGEMENT |
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We thank Dr. Niwa for the helpful discussion.
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FOOTNOTES |
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* This work was supported in part by Grant-in-Aid 09276220 for Scientific Research from the Ministry of Education, Science and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 81-886-33-7423;
Fax: 81-886-33-7425.
1
The abbreviations used are: NDP, nucleoside
diphosphate; AMP-PNP, 5'-adenylyl-
,-imidodiphosphate; ATPS,
adenosine 5'-3-O-(thio)triphosphate; BSA, bovine serum
albumin; PAGE, polyacrylamide gel electrophoresis; HPLC, high
performance liquid chromatography; Mes, 4-morpholineethanesulfonic acid; Ches, 2-(cyclohexylamino)ethanesulfonic acid; Chaps,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
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REFERENCES |
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Abstract
Introduction
Procedures
Results
Discussion
References
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, activity of ATP hydrolysis of the commercial
product of Hsp70; - - -, protein A280 of NDP kinase with a
molecular mass of 16 kDa; 
- 
, 
, 
) and BSA (
, 
