Báo cáo khoa học: The duality of LysU, a catalyst for both Ap4A and Ap3A formation pdf

The duality of LysU, a catalyst for both Ap4A and Ap3A
formation
Michael Wright, Nonlawat Boonyalai, Julian A. Tanner, Alison D. Hindley and Andrew D. Miller
Imperial College Genetic Therapies Centre, Department of Chemistry, Imperial College London, London, UK

Keywords
Ap4A; Ap3A; dinucleoside polyphosphates;
heat shock response; LysU
Correspondence
A. D. Miller, Imperial College Genetic
Therapies Centre, Department of Chemistry,
Flowers Building, Armstrong Road, Imperial
College London, London, SW7 2AZ, UK
Fax: +44 20 75945803
Tel: +44 20 75945773
E-mail:
(Received 21 March 2006, revised 31 May,
accepted 6 June 2006)
doi:10.1111/j.1742-4658.2006.05361.x

Heat shock inducible lysyl-tRNA synthetase of Escherichia coli (LysU) is
known to be a highly efficient diadenosine 5¢,5¢¢¢-P1,P4-tetraphosphate
(Ap4A) synthase. However, we use an ion-exchange HPLC technique to
demonstrate that active LysU mixtures actually have a dual catalytic activity, initially producing Ap4A from ATP, before converting that tetraphosphate to a triphosphate. LysU appears to be an effective diadenosine
5¢,5¢¢¢-P1,P3-triphosphate (Ap3A) synthase. Mechanistic investigations
reveal that Ap3A formation requires: (a) that the second step of Ap4A
formation is slightly reversible, thereby leading to a modest reappearance
of adenylate intermediate; and (b) that phosphate is present to trap the
intermediate (either as inorganic phosphate, as added ADP, or as ADP
generated in situ from inorganic phosphate). Ap3A forms readily from
Ap4A in the presence of such phosphate-based adenylate traps (via a

‘reverse-trap’ mechanism). LysU is also clearly demonstrated to exist in a
phosphorylated state that is more physically robust as a catalyst of Ap4A
formation than the nonphosphorylated state. However, phosphorylated
LysU shows only marginally improved catalytic efficiency. We note that
Ap3A effects have barely been studied in prokaryotic organisms. By contrast, there is a body of literature that describes Ap3A and Ap4A having
substantially different functions in eukaryotic cells. Our data suggest that
Ap3A and Ap4A biosynthesis could be linked together through a single
prokaryotic dual ‘synthase’ enzyme. Therefore, in our view there is a need
for new research into the effects and impact of Ap3A alone and the intracellular [Ap3A] ⁄ [Ap4A] ratio on prokaryotic organisms.

Aminoacyl-tRNA synthetases (aaRSs) are a heterogeneous family of around 20 distinct enzymes involved in
maintaining the fidelity of protein synthesis through
the specific esterification of an amino acid to the 2¢- or
3¢-hydroxyl group of the 3¢-terminal adenosine of the
cognate tRNA(s) during the translation process [1].
Most prokaryotic aaRSs are usually coded for by single, unique genes (unlike eukaryotes), but Escherichia
coli lysyl-tRNA synthetase (LysRS) is an exception in
that it exists as two distinct synthetase isoforms, LysS

and LysU. These two isoforms share a high degree of
sequence identity (88%) but appear to have evolved
for different purposes. LysS is constitutively expressed
under normal growth conditions and is responsible for
the normal tRNA charging activity, whereas LysU
is the product of a normally silent gene, but can be
induced to a high-level expression under selected
physiological conditions, including heat shock, oxidative stress, and anaerobiosis [2,3]. LysU is a
highly efficient diadenosine 5¢,5¢¢¢-P1,P4-tetraphosphate

Abbreviations

aaRS, aminoacyl-tRNA synthetase; ACPR, 2-amino-6-chloropurine ribonucleoside; AMMPR, 2-amino-6-mercapto-7-methylpurine
ribonucleoside; AMPCP, adenosine 5¢-(a,b-methylene) diphosphate; AMPPCP, adenosine 5¢-(b,c-methylene) triphosphate; Ap3A, diadenosine
5¢,5¢¢¢-P1,P3-triphosphate; Ap4A, diadenosine 5¢,5¢¢¢-P1,P4-tetraphosphate; AppCH2ppA, methylene-substituted Ap4A analogue;
HA, hydroxyapatite; MMMP, 2-methyl-6-mercapto-7-methylpurine.

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FEBS Journal 273 (2006) 3534–3544 ª 2006 The Authors Journal compilation ª 2006 FEBS


LysU catalysis of Ap4A and Ap3A synthesis

M. Wright et al.

NH2
HO

O
N
N

O
P

O

N

N

(d)

OH

O

O

O

O

O

P P
O O O
O

N

N
N

OH
OH

N
NH2
Zn2+

Step 3

ADP
NH2
HO

OH

(b)
O

N
N

N

O

O
P

O

O

HO

O
P

O

O

O

Step 1

P
O
O

+

NH2

N

O

H2N
NH2

O

HO

ATP
Step 2

N

O

O

N
H2N

NH2

N

O

N

(c)
O
P
O

O

O
P

O

O

O

NH2

O

P P
O O O
O

O

N

N
N

OH
OH

N

N
(a)

OH
O

Zn2+

O
P

O

N

N

PPi, H2O
OH

NH2
O

3.Mg2+

N

OH

+ L-lysine

inorganic
pyrophosphatase

Pi
2.Pi

Step 4

Zn2+

HO

OH
O

N
N

N
N

O

O
P

O

O

O
P

O

+ L-lysine

O

(e)

NH2

Scheme 1. LysU-catalysed Ap4A and Ap3A synthesis. Both synthetic mechanisms are catalysed in the presence of Mg2+, Zn2+ and inorganic
pyrophosphatase. The pathways share a common step 1 in the formation of a lysyl-adenylate intermediate (a) from ATP (b) and L-lysine with
release of pyrophosphate (cleaved to phosphate by inorganic pyrophosphatase). Steps 2 and 3 are the combination of this intermediate and
either ATP to form Ap4A (c), or ADP (if present) to form Ap3A (d), with the concurrent release of L-lysine. Once the extraneous ATP (b) has
been exhausted, partial reversal of step 2 results in the cleavage of Ap4A (c) back to lysyl-adenylate (a) and ATP (which in turn forms further
intermediate). In this situation, step 4 allows the produced lysyl-adenylate (a) to slowly react with inorganic phosphate to give ADP (e), which
then allows the formation of Ap3A (d). The overall reaction is thus: 2ATP fi Ap4A + 2Pi fi Ap3A + 3Pi.

(Ap4A) synthase, and the production of Ap4A under
conditions of cellular stress appears to be a primary
function [4–7].
The synthesis of Ap4A catalysed by LysU occurs in
two steps (Scheme 1). The first (step 1) involves the
formation of a lysyl-adenylate intermediate in which
the amino acid is activated through combination with
the a-phosphate of the first nucleotide substrate ATP,
in a process involving the simultaneous displacement
of pyrophosphate. In step 2, the c-phosphate of a second nucleotide substrate ATP combines with enzymebound lysyl-adenylate, thereby generating Ap4A and
liberating free l-lysine [8]. Step 1 is highly specific and
conservative; ATP can only be replaced by deoxy-ATP
nucleotide substrates. Step 2 is much more catholic,
and the second nucleotide substrate ATP can be
replaced by a variety of diphosphate, triphosphate or

tetraphosphate nucleotide substrates. This flexibility in
step 2 means that LysU can be an efficient platform
catalyst for the synthesis of a wide variety of natural
and artificial polyphosphates [9–12].
Most previous studies of LysU catalysis have focused
on Ap4A synthase activity, but there have also been

several reports concerning the unexpected coisolation of
diadenosine 5¢,5¢¢¢-P1,P3-triphosphate (Ap3A) from
LysU catalysis mixtures [8,9,13,14]. Since ADP is not a
first nucleotide substrate for LysU (that is, LysU cannot
catalyse the formation of lysyl-adenylate directly from
ADP and l-lysine), this Ap3A has been assumed to be a
minor side product emanating from the combination of
the lysyl-adenylate intermediate with residual ADP present in ATP. Therefore, we decided to determine whether Ap3A was indeed a minor side product of LysU
catalysis or in fact a genuine second main product
alongside Ap4A. At the same time, we intended to
investigate the behaviour of LysU with respect to phosphorylation, looking for potential linkages between
LysU-mediated catalysis and phosphorylation state.
The results of our investigations are reported here.

Results and Discussion
Ap4A/Ap3A synthase duality
Typically, when a standard LysU catalysis mixture
is prepared (comprising LysU, excess l-lysine and

FEBS Journal 273 (2006) 3534–3544 ª 2006 The Authors Journal compilation ª 2006 FEBS

3535

LysU catalysis of Ap4A and Ap3A synthesis

M. Wright et al.

B

A

5

90 min

63 min

[fraction] / mM

4

3

2

36 min

1
9 min

0
3.5

4.0

4.5

5.0

5.5

6.0

0

t / min

20

40

60

80

100

t / min

Fig. 1. (A) Ion exchange HPLC measurement of mononucleotide and dinucleotide levels observed in a LysU catalysis mixture containing
5 mM ATP over 2 h. Traces show A260 during each separation. (B) Integration and normalization of the traces to give relative concentrations
for each component. Compounds are identified as: n, ATP; s, Ap4A; d, ADP; ., Ap3A; h, AMP. Errors are ± 0.05 mM.

inorganic pyrophosphatase in a buffer containing
Mg2+ and Zn2+), added ATP (> 99%, purified by
ion exchange chromatography) will be converted into
Ap4A after 20–30 min at 37 °C. Thereafter, significant
quantities of Ap3A will be formed at the expense of
Ap4A, such that triphosphate may easily become the
major product after 1 h (depending upon the concentrations of LysU and substrate involved). This phenomenon was clearly observed using an assay based
on a SOURCE 15Q ion exchange column attached to
an HPLC system set up to take repeated aliquots from
enzyme incubation mixtures every 9 min, thereby
allowing for the quantitative measurement of individual nucleotide substrate and diadenosine polyphosphate concentrations as a function of time. The results
obtained from the incubation of ATP (5 mm) with
LysU in a typical catalysis mixture are shown in
Fig. 1. From multiple data sets of this kind, we
observed that the fast initial Ap4A synthesis
(170 ± 40 min)1) converted the majority (perhaps all)
of the available ATP to tetraphosphate. This was followed by a slower process of Ap4A-to-Ap3A conversion (12 ± 3 min)1) with concurrent rise of ADP
levels to about 0.1 mm (initially negligible) over the
next 1.5 h. This dual catalytic phenomenon runs counter to the general expectation of LysU as a primary
Ap4A synthase and deserves an explanation. In the
absence of initially available ADP, it is apparent that
ADP is somehow generated in situ and then acts as an
alternative second nucleotide substrate in step 2, combining with lysyl-adenylate to form Ap3A (Scheme 1,
step 3). However, what is the origin of this in situ
ADP?
Initially, we considered two possibilities: option 1,
that LysU may act as an ATP-to-ADP hydrolase; or
3536

option 2 that LysU may be a symmetrical Ap4A
hydrolase. The rapidity of ATP consumption in the
first stage of polyphosphate formation with no apparent formation of ADP appeared to rule out option 1.
In fact, significant ADP was not seen in reaction mixtures until > 30 min from the start, by which time no
ATP remained (Fig. 1). Instead, the kinetics of ADP
appearance and Ap4A disappearance suggested that
Ap4A may be the source of ADP according to option
2. Consistent with option 2, when a mixture of
ATP and adenosine 5¢-(b,c-methylene) triphosphate
(AMPPCP) 1 : 1 (m ⁄ m) is combined with a LysU catalysis mixture, AppCH2ppA (a methylene-substituted
Ap4A analogue) forms without apparent formation of
Ap4A, and subsequently Ap3A is not isolated even
after 3 h at 37 °C (beyond which time, LysU begins to
denature) [10]. Even a single bisphosphonate methylene
linkage is known to confer resistance to hydrolysis
[15,16], and AMPPCP is unable to function as a first
nucleotide substrate for LysU (Scheme 1, step 1). It is,
however, an apparently more potent second nucleotide
substrate than ATP, able to trap the lysyl-adenylate
intermediate in a nonreversible equivalent to step 2
(Scheme 1), and leading to the exclusive formation of
AppCH2ppA [9]. The subsequent failure to generate
Ap3A could then be accounted for by the inability
of AppCH2ppA to undergo symmetrical hydrolysis to
form ADP, also owing to the presence of the bisphosphonate methylene linkage.
In order to obtain further evidence for a putative
‘symmetrical Ap4A hydrolase’ activity, Ap4A was
incubated with LysU in a standard catalysis mixture
(comprising Tris ⁄ HCl buffer) minus inorganic phosphate or any other nucleotide. Against expectations,
no ADP was generated over 3 h (Fig. 2A). However,

FEBS Journal 273 (2006) 3534–3544 ª 2006 The Authors Journal compilation ª 2006 FEBS


LysU catalysis of Ap4A and Ap3A synthesis

M. Wright et al.

A

B

4
[fraction] / mM

5

4
[fraction] / mM

5

3

2
1

3

2

1

0

0
0

20

40

60

80

0

20

40

0

100

20

40

t/min

C

80

60

80

100

D
5

4

4
[fraction] / mM

5

[fraction] / mM

60
t/min

3
2

3

2

1

1

0

0
0

20

40

60

80

100

t/min

100

t/min

Fig. 2. Evidence for LysU catalytic duality. (A) A LysU catalysis mixture containing 5 mM Ap4A shows no significant conversion to Ap3A nor

hydrolysis to ADP over 2 h. A constant AMP background is seen due to trace contaminants in the Ap4A stock, but no ADP or Ap3A is produced. (B) A catalysis mixture containing 5 mM Ap4A and 5 mM ADP shows rapid loss of ADP and Ap4A (fitting exponential decay curves),
and concurrent synthesis of Ap3A. The turnover of ADP is about twice that of Ap4A, which matches the Ap4A + 2.ADP fi 2.Ap3A + 2.Pi
stoichiometry. (C) A LysU catalysis mixture containing 5 mM Ap4A and made up in 50 mM potassium phosphate buffer, pH 7.8. Apparent
phosphate attack on lysyl-adenylate results in greatly increased turnover of Ap4A to ADP and Ap3A. Longer incubation times (> 2 h) show
that the conversion of Ap4A to Ap3A continues, while the concentration of ADP stabilizes at approximately 1 mM. (D) A LysU catalysis mixture containing 5 mM ATP and made up in 50 mM potassium phosphate buffer. The presence of inorganic phosphate disrupts the formation
of Ap4A, and the major product under these conditions is ADP. Compounds are identified as: n, ATP; s, Ap4A; d, ADP; ., Ap3A; h, AMP.
Errors are ± 0.05 mM.

when a mixture of ADP and Ap4A 1 : 1 (m ⁄ m) was
incubated with LysU in an identical catalysis mixture
(minus inorganic phosphate or any other nucleotide),
then Ap3A was generated rapidly with an estimated
turnover number for the conversion of Ap4A to
Ap3A of 90 ± 5 min)1 (10-fold higher than observed
previously) (Fig. 2B). The turnover rate was observed
to increase further as the ADP-to-Ap4A ratio was
increased. This latter result is consistent with a partial
reversibility of Ap4A formation (Scheme 1, step 2),
giving rise to an adenylate intermediate in situ that
can then be trapped by ADP to give Ap3A at the
expense of Ap4A (Scheme 1, step 3). The absence of
any observed accumulation of ATP formed from

reverse step 2 suggests that this is rapidly recombined
with lysine to form an adenylate intermediate
(Scheme 1, step 1) that would then be rapidly trapped
by ADP once again to give Ap3A. Evidence in support of this reverse-trap process was obtained by substituting for AMPPCP or adenosine 5¢-(a,bmethylene) diphosphate (AMPCP) for ADP. Trap
products AppCH2ppA and ApCH2ppA, respectively,
were generated exclusively, completely consistent with
our proposed reverse-trap mechanism. Hence, faced

with such evidence that LysU is clearly not an Ap4A
hydrolase (Fig. 2A), we needed to come up with
an alternative explanation for the source of ADP
generated in situ.

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LysU catalysis of Ap4A and Ap3A synthesis

M. Wright et al.

Returning to the original Ap4A synthesis scenario
(Fig. 1), we came to the realization that there could be
an alternative option (option 3). During the initial
synthesis of Ap4A, the main byproduct of step 1 is
inorganic phosphate formed through cleavage of pyrophosphate by the action of an inorganic pyrophosphatase enzyme (contained in the LysU catalysis mixture).
Therefore, we considered that this inorganic phosphate
may also be capable of trapping residual adenylate
intermediate arising from the partial reversibility of
Ap4A formation (Scheme 1, step 4), thereby generating
ADP in situ. An equivalent process to this (albeit much
slower) has been remarked upon in studies on E. coli
glycyl-tRNA synthetase [17]. Thereafter, ADP thus
formed in situ would be in a position either to trap
further adenylate deriving from the partial reversibility
of Ap4A formation or to combine with adenylate generated in the normal way from ATP, thereby forming
Ap3A in either case. All the proposed mechanistic

steps are summarized in Scheme 1. Given this, when
ATP is incubated in a standard LysU catalysis mixture
(Fig. 1), then the observed variations in concentrations
of mononucleotide and dinucleotide species with time
can be accounted for in the following way. Early ATP
consumption and Ap4A formation are rapid, consistent
with an initial process in which step 1 is committed
and step 2 is kinetically very favourable. Thereafter,
the partial reversibility of step 2 allows the reverse-trap
mechanism to give ADP (from inorganic phosphate)
and then Ap3A (from ADP; Scheme 1). The appearance of the lysyl-adenylate intermediate would appear
to be rate-limiting (at least in part) for the formation
of both ADP and Ap3A, although the former
clearly must convert readily into the latter (Scheme 1,
step 3) at the expense of Ap4A, keeping the overall
solution concentration of ADP at a minimum while
the concentration of end-product Ap3A begins to rise
steadily.
Evidence in support of our proposed mechanism
and the adenylate-trapping function of inorganic phosphate was obtained in the following way. Ap4A was
incubated with LysU in a catalysis mixture containing
50 mm potassium phosphate but minus any nucleotides. In this instance, > 20% of Ap4A was observed
to convert to Ap3A over 2 h. This contrasts with the
previous situation where Ap4A alone incubated in a
LysU catalysis mixture (Tris ⁄ HCl buffer, no inorganic
phosphate) failed to convert into Ap3A over a 3-h period (compare Fig. 2C with Fig. 2A). Furthermore,
when ATP alone was incubated with LysU in a catalysis mixture comprising 50 mm phosphate buffer,
then Ap3A ⁄ Ap4A synthase activities as a whole were
adversely affected, with fully 30% of the initial ATP
3538

being converted to ADP via attack of inorganic phosphate on the adenylate intermediate (compare Fig. 2D
with Fig. 1B). Curiously, unlike ADP and inorganic
phosphate, AMP did not appear to be able to trap
adenylate. The reasons for this are unclear but are
likely to be due to LysU active site topographies and
unfavourable steric contacts [18]. Finally, the summarized mechanistic steps (Scheme 1) can be used to provide an alternative explanation for our original
observation that AppCH2ppA is the exclusive product
when ATP and AMPPCP 1 : 1 (m ⁄ m) are combined
with a LysU catalysis mixture. As stated above, ATP
is the only possible first nucleotide substrate of LysU,
whereas both ATP and AMPPCP could be second
nucleotide substrates [9,10]. Given the situation in
which the formation of AppCH2ppA is essentially
irreversible but the formation of Ap4A is partially
reversible (as described above), the final outcome is
consistent with the formation of only transient Ap4A,
since adenylate intermediate regeneration at its expense
would then be trapped by excess remaining AMPPCP.
Therefore, our data describing the exclusive formation
of AppCH2ppA can be seen as a simple variation of
our newly proposed reverse-trap mechanism for the
Ap3A synthase activity of LysU.
Phosphorylation state duality
Previous purification protocols for LysS and LysU
have described the use of hydroxyapatite (HA) medium columns to subfractionate samples of LysU and
LysS into different charged ‘isoforms’ [4,5,19]. HA
makes this possible, as it is a crystalline form of calcium phosphate that interacts differently with globular
proteins depending upon their charge [20]. In our
hands, LysU (previously purified by S300 and Q-Sepharose chromatography [14]) could be easily resolved

into two main subfractions by elution through an HA
column, the first subfraction eluting at 20% and the
second at 30% from a linear gradient of potassium
phosphate (10–300 mm) (Fig. 3). Both subfractions
were confirmed to be greater than 95% pure LysU by
SDS ⁄ PAGE. However, since HA resolves proteins by
charge, we concluded that these two different subfractions should contain LysU isoforms of different overall
charge at neutral pH.
Differences in protein phosphorylation state were
considered the most likely explanation for the emergence of these two different LysU isoforms. Therefore,
separate aliquots of each subfraction were analysed for
phosphate by means of the malachite green assay [21]
calibrated against a standard curve generated with
known concentrations of inorganic phosphate. The

FEBS Journal 273 (2006) 3534–3544 ª 2006 The Authors Journal compilation ª 2006 FEBS


LysU catalysis of Ap4A and Ap3A synthesis

M. Wright et al.

20%

0.8

40
30%
30

0.6

20

0.4

10

0.2

0

A280 / AU

Buffer B / %

forms of eukaryotic lysyl-tRNA from rat liver have
been characterized [22,23], as have phosphorylated
forms of other aminoacyl-tRNA synthases (multiply
phosphorylated on the serine amino acid residue) from
rabbit reticulocytes [24]. The role of these phosphorylation events has not been explored to any great
extent, although nonphosphorylated yeast LysRS is
known to be more specific for lysine than its threonine-phosphorylated equivalent [22]. Furthermore, with
regards to Ap4A synthesis, phosphorylation of rabbit
reticulocyte SerRS and ThrRS has been shown to
enhance the catalytic rates of Ap4A synthesis rates by
two-fold and six-fold, respectively [25]. Consequently,
we elected to characterize the 20% and 30% subfractions of LysU in order to determine if there were any
major differences. In particular, in view of the dual
catalytic behaviour of LysU described here, we were

curious to characterize the effects or otherwise of
phosphorylation upon the catalytic rates for Ap4A and
Ap3A synthesis. A variety of catalysis characterization
techniques were employed to identify differences. A
radioactive assay using [14C]ATP was used to accurately
measure total polyphosphate synthesis over a fixed period, allowing for the calculation of average turnover
under kcat conditions (moles Ap3 ⁄ 4A per min per mole
LysU at 37 °C). Typical results were 134 ± 10 min)1
(20% subfraction) and 150 ± 12 min)1 (30% subfraction), suggesting little difference between phosphorylated LysU and the nonphosphorylated isoform within
experimental error. On the other hand, phosphorylated
LysU was able to retain activity (70 ± 5% activity

1.0

50

0.0
0

50

100

150
200
Elution volume / ml

250

300

350

Fig. 3. Hydroxyapatite (HA) chromatography. The HA column was
loaded with LysU and then eluted with a stepwise gradient of
potassium phosphate buffer, pH 6.5 (10–300 mM); fractions were
analysed for absorbance at 280 nm. Illustration of the elution profile
monitored by absorbance showing two main subfractions of LysU
eluting at 20% and 30% gradient.

results clearly suggest that the 20% subfraction was
unphosphorylated whereas the 30% subfraction
contained LysU phosphorylated at the level of a single
phosphate per monomer (Fig. 4), in line with our
explanation. Furthermore, western blot analyses of the
two subfractions using a phosphothreonine antibody
confirmed that not only was the 30% subfraction
phosphorylated, but also was the position of phosphorylation on the hydroxyl group of a threonine
residue (Fig. 4). LysU has a number of surface-accessible threonine residues according to the X-ray crystal
structure [2]. Therefore, enzyme digestion and fragment MS is likely to be the most effective way to determine precisely which threonine residue is involved.
Phosphorylated forms of aminoacyl-tRNA synthetase enzymes are known. For example, phosphorylated

A

B

2.0

C

97.4 kDa

30%

A810 / AU

1.5

66.2 kDa

1.0

0.5
45.0 kDa

20%
0.0

21.5 kDa
0

50

100

150

200

250

300

3-

PO4 / µM
20%

30%

20%

30%

Fig. 4. LysU phosphorylation. (A) SDS ⁄ PAGE of 20% and 30% subfractions (see Fig. 3), both showing clean bands matching the expected
mass for LysU monomer. (B) Calibration of Malachite green assay. (C) Anti-phosphothreonine western blot of 20% and 30% subfractions,
with only the 30% subfraction showing positive results.

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3539


LysU catalysis of Ap4A and Ap3A synthesis

M. Wright et al.

after 7 days) for significantly longer after storage at
4 °C than unphosphorylated LysU (30 ± 5% activity
after 7 days), suggesting that phosphorylated LysU is

significantly more stable than nonphosphorylated
LysU.
A 1H-NMR spectroscopy assay was then used to
characterize the conversion of ATP to Ap4A by the 20%
and 30% subfractions of LysU, respectively. For each
individual NMR assay, conversion of ATP into Ap4A
can be observed with time in an NMR tube by monitoring the disappearance as a function of time of the H2
and H8 proton signals of adenine (of ATP) matched by
the appearance of the H2 and H8 proton signals of
adenine (of Ap4A). Ap4A adenine signals are shifted
slightly upfield due to the p-stacking of adenine rings in
Ap4A (not observed with ATP) [26]. Assuming steadystate conditions, initial rates of catalysis of Ap4A formation may be calculated from H2 and H8 proton peak
integrations, and used to determine the main kinetic
steady-state parameters for ATP and l-lysine [14,27].
The effects of [ATP] and [l-lysine] on Ap4A formation
rates were determined by taking one substrate in excess
and varying the concentration of the other (excess concentrations estimated from previous results as 10 mm
ATP and 2 mm lysine), keeping the concentrations of
20% or 30% subfractions of LysU constant (400 nm,
dimer concentration) (Table 1). Unsurprisingly the kcat
constants of the two LysU isoforms were found to be
essentially identical within experimental error, although
the specificity constant (kcat ⁄ KM) of phosphorylated
LysU for l-lysine appears to be 2–3-fold higher than
that of nonphosphorylated LysU. The reason appears
to be that phosphorylated LysU has both a marginally higher kcat for l-lysine and a lower value of KM,
indicative of a situation wherein LysU phosphorylation
has enabled l-lysine to be an improved substrate under
these given assay conditions.
Next, a colorimetric-coupled assay was used to characterize the formation of the lysyl-adenylate intermediate by the 20% and 30% subfractions of LysU,

Table 1. Kinetic constants derived from 1H-NMR Ap4A synthesis
assays of 20% and 30% subfractions.
kcat
(s)1)

kcat ⁄ KM
(s)1ỈmM)1)

± 4 mM
± 12 lM

2.7 ± 0.4
1.8 ± 0.1

0.4 ± 0.3
78 ± 40

± 3 mM
± 5 lM

2.0 ± 0.2
2.9 ± 0.2

0.3 ± 0.2
290 ± 120

KM
LysU 20% subfraction
For ATP

7
For lysine
23
LysU 30% subfraction
For ATP
6
For lysine
10

3540

respectively. In each individual assay, the formation
of inorganic phosphate (produced by the action of
inorganic pyrophosphatase following lysyl-adenylate
formation) was coupled with the release of a UV-active
dye 2-methyl-6-mercapto-7-methylpurine (MMMP)
[28,29] from a chromogenic substrate 2-amino-6mercapto-7-methylpurine ribonucleoside (AMMPR).
The coupling enzyme purine nucleoside phosphorylase
catalyzes the combination of inorganic phosphate with
AMMPR, leading to MMMP release. This coupled
reaction must occur faster than the formation of lysyladenylate. Accordingly, only a low fixed LysU concentration (20 nm dimer concentration) was used per
assay, together with a correspondingly high excess of
ATP or l-lysine and a low incubation temperature of
20 °C (which significantly slows the catalysis of Ap4A
synthesis). The concentrations of AMMPR, purine
nucleoside phosphorylase and inorganic pyrophosphatase required were determined by experiment, and the
assay was calibrated against known concentrations of
potassium phosphate. The kinetic parameters determined are shown (Table 2). Since this coupled assay
monitors specifically the formation of lysyl-adenylate
alone and not Ap4A synthesis, these parameters can be

attributed solely to the binding and chemical equilibrium of step 1 (Scheme 1) alone. Once again, values of
kcat were found to be essentially identical within
experimental error, but the value of the specificity constant (kcat ⁄ KM) for ATP of phosphorylated LysU was
between one- and two-fold higher than that of nonphosphorylated LysU. The reason for this is that phosphorylated LysU has a lower value of KM for ATP,
indicative of a situation wherein phosphorylation of
LysU has enabled ATP to be a mildly improved
substrate for LysU-mediated catalysis under these
assay conditions. Finally, a complete repetition of the
SOURCE 15Q ion exchange chromatography assays
(as described earlier) suggested that the rates of formation of Ap3A from Ap4A were unaffected by the state
of LysU phosphorylation (results not shown).

Table 2. Kinetic constants derived from lysyl-adenylate, enzymecoupled assays of 20% and 30% subfractions.
kcat (s)1)

kcat ⁄ KM

3 mM
0.14 lM

16.0 ± 2.5
8.6 ± 1.1

1.7 ± 0.8 s)1ỈmM)1
32 ± 20 s)1ỈlM)1

1.2 mM
0.11 lM

12.2 ± 1.5

10.1 ± 0.7

3.0 ± 1.3 s)1ỈmM)1
30 ± 11 s)1ỈlM)1

KM
LysU 20% subfraction
For ATP
9.6 ±
For lysine
0.27 ±
LysU 30% subfraction
For ATP
4.0 ±
For lysine
0.34 ±

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M. Wright et al.

Roles of dualities
We have demonstrated that dimeric LysU has dual
Ap4A and consecutive Ap3A synthase activities, and
that it exists in either a phosphorylated or nonphosphorylated state. The phosphorylated LysU appears to
be a more robust enzyme than nonphosphorylated
LysU, although in catalytic terms they do not appear
to have substantially different activities. This suggests
that modifying LysU-catalysed Ap4A or Ap3A formation is probably not the main function of phosphorylation. Nevertheless, in our view, the Ap4A ⁄ Ap3A

synthase and phosphorylation state dualities of LysU
do appear to be linked, albeit not strongly. The Ap3A
synthase activities of LysU appear to be the product
of a number of possible contributory mechanisms.
Both ADP and inorganic phosphate are present in
cells, so Ap3A could be formed directly from ATP and
ADP (where ADP replaces ATP as the second nucleotide substrate in Scheme 1, step 2), or from Ap4A by
the illustrated reverse-trap mechanism (Scheme 1,
reverse step 2, then steps 3 and 4). However, there is
insufficient information in the current literature to
make sense of this catalytic behaviour. In eukaryotic
cells, there seems to be a general tendency for Ap4A
and Ap3A to have antagonistic effects in vivo. In particular, the relative concentrations of these polyphosphates appear to be indicative of cellular status, with
human cultured cells showing high Ap4A ⁄ Ap3A ratios
when undergoing induced apoptosis and the reverse
during differentiation [30,31]. Ap4A 10 lm was also
shown to be sufficient to trigger apoptosis in a variety
of human and mouse cell lines, a concentration not
significantly higher than that seen in human adrenal
vein blood serum [32].
Similarly, Ap3A has been reported to act as a coinducer of cell differentiation when combined with protein
kinase C activators. This antagonistic behaviour has
also been seen in a number of other studies. For example, submicromolar concentrations of Ap3A have been
reported to induce platelet aggregation, in contrast to
Ap4A which causes disaggregation [33], and these compounds have been shown to have opposing effects on
rabbit interocular pressure as well [34]. Regrettably,
there has been no evidence produced to date to suggest
that Ap4A and Ap3A should have antagonistic effects
in vivo in prokaryotes as well as in eukaryotes. Therefore, although Ap4A may appear to act as an immediate
modulator of stress responses in prokaryotes [30,35,36],

analogous links between Ap3A and longer-term prokaryotic stress accommodation, or high concentrations of
Ap4A and failure of the stress response, must remains
hypothetical. Our data presented here suggest that

LysU catalysis of Ap4A and Ap3A synthesis

Ap3A and Ap4A biosynthesis are linked together
through a single prokaryotic dual ‘synthase’ enzyme.
Therefore, in our view, there is now an urgent need for
new research into the effects and impact of Ap3A alone
and the intracellular [Ap3A] ⁄ [Ap4A] ratio on prokaryotes in order to make sense of the dual enzymology of
LysU in a fuller, more complete biological context.

Conclusion
LysU has dual Ap3A ⁄ Ap4A synthase activities and
dual phosphorylation state behaviour. These dualities
are linked, but only weakly. There is insufficient knowledge about the role of Ap3A in biological prokaryotic
systems to understand the implications of these dualities. More research into the function of Ap3A in prokaryotes is necessary.

Experimental procedures
General
LysU enzyme was overexpressed and purified according to
previously published protocols [18]. All LysU concentrations are of the dimer. LysU concentration was determined
from A280 measurement using an extinction coefficient of
30 580 m)1Ỉcm)1 [37]. All compounds used were obtained
from Sigma Aldrich (St Louis, MO) unless otherwise stated.

Ion exchange HPLC assays
A custom 2 mL SOURCE 15Q packed ion exchange column (Amersham Biosciences HR 5 ⁄ 10; Piscataway, NJ)
was attached to an Agilent 1100 series HPLC column (Agilent, Palo Alto, CA), equipped with an autosampler, a thermostatted sample chamber (37 °C) and a UV absorbance

detector set at 260 nm. The column was loaded (injection
aliquots 10 lL) in 5 mm Tris ⁄ HCl buffer, pH 8.0, and eluted with a gradient of NaCl (0–45%) over 5 min. The standard LysU catalysis mixture (500 lL) comprised 50 mm
Tris ⁄ HCl, pH 7.8, 10 mm MgCl2, 160 lm ZnCl2, 5 mm nucleotides, 2 mm l-lysine, 5 units of inorganic pyrophosphatase (Roche, Laval, Canada) and 1 lm LysU. The contents
of fractions postelution were identified by their retention
time compared with standards (AMP 3.85 min, AMPPCP
4.39 min, ADP 4.43 min, ATP 4.72 min, Ap4A 5.13 min).
Mononucleotide and dinucleotide concentrations were
calculated from peak area, assuming 260 nm extinction
coefficients to be roughly equivalent for AMP through
ATP and double for Ap3A and Ap4A. Ap3A was synthesized by incubating 5 mm ATP in a standard LysU mixture
overnight, and then extracted from the mixture using a
SOURCE 15Q ion exchange column (50 mL) (Amersham
Biosciences), loaded in water and eluted with a gradient of

FEBS Journal 273 (2006) 3534–3544 ª 2006 The Authors Journal compilation ª 2006 FEBS

3541


LysU catalysis of Ap4A and Ap3A synthesis

M. Wright et al.

2 m triethylammonium hydrogencarbonate buffer (0–70%)
[17]. Appropriate fractions were combined and freeze dried.
Contents were characterized by ESI-MS (Bruker Esquire
3000, negative ionization; Billerica, MA), giving a PMI of
754.9 m ⁄ z, and ion exchange HPLC, giving a single peak
at 4.71 min retention time.

HA column
An HA biogel column (3 · 20 cm) was mounted on an
FPLC (Amersham Biosciences, Piscataway, NJ) system and
equilibrated with 10 mm potassium phosphate, pH 6.5,
containing 2 mm b-mercaptoethanol, at a flow rate of
1.5 mLỈmin)1. LysU (3 mg) was dialysed to phosphate buffer and loaded onto the column. The column was eluted at
1.5 mLỈmin)1 with a gradient (10–300 mm) of potassium
dihydrogen phosphate, holding at 10%, 20% and 30% to
collect fractions (detected by A280 absorbance). The first
10% peak was discarded and the remainder were concentrated separately using a stirred cell with a 30 kDa molecular weight cutoff filter, before being stored at 4 °C in 20%
glycerol.

Malachite green assay
The reagent was made up from one part 0.2% Malachite
green in 1 m HCl to five parts 1 m HCl, and two parts
10% ammonium molybdate in 3 m HCl. The reagent was
centrifuged to clear precipitate, and an aliquot (100 lL)
was added to an aliquot of LysU (300 lL in water). A810
was measured after 5 min. Equal amounts of protein solution and 10% Mg(NO3)2 in 95% ethanol were combined
and then vigorously dehydrated in a glass flask over a
strong flame. The ‘ash’ was redissolved in 0.5 m HCl prior
to assay [38].

Anti-threonine western blot
SDS ⁄ PAGE gels of the two LysU fractions (20% and
30%) were electroblotted onto nitrocellulose membranes at
25 V, 0.2 mA for 2 h [15], in 20 mm Tris ⁄ HCl, pH 8.0, containing 152 mm glycine and 20% methanol. Nonspecific
binding was blocked with 10% free BSA in TBS (20 mm
Tris ⁄ HCl, 137 mm NaCl), pH 8.2, 0.05% Tween-20 for 1 h
at room temperature. Primary antibodies (mouse monoclonal IgG2b anti-phosphothreonine, Sigma-Aldrich) were

added at 1 : 1000 dilution, and the whole mixture was incubated for 12 h at 4 °C with gentle rocking. The membrane
was then washed with TBS (20 mL), pH 8.2, Tween-20
0.05%; 5 · 10 min washes were performed at room temperature. Thereafter, the secondary antibody (goat antimouse IgG conjugated to horse radish peroxidase; Santa
Cruz Biotechnology, Santa Cruz, CA) was added at
1 : 5000 dilution and the whole mixture was incubated for

3542

1 h. Finally, the membrane was washed again and then
incubated with 2 · 5 mL chemiluminescence reagents
(Santa Cruz) for 1 min before wrapping in clear film and
exposing to photographic film.

Radioactive [14C]ATP turnover assay
Initially, a LysU reaction mixture of 20 mm Hepes, pH 7.8,
was prepared, comprising 150 mm KCl, 25 mm ATP,
25 mm MgCl2, 0.5 mm l-lysine, and 150 lm ZnCl2.
Reaction buffer (50 lL) was added to an Eppendorf tube
(1.5 mL), followed by [14C]ATP (2.5 lL; 0.125 lCi),
0.25 lg of inorganic pyrophosphatase and a concentrated
aliquot of LysU (5 lL). This LysU catalysis mixture was
then incubated at 37 °C for 40 min and then briefly boiled
to denature the enzymes. After this, 10 units of alkaline
phosphatase was added to degrade remaining nucleotides
and the mixture was incubated at 37 °C for a further 2 h.
An aliquot (40 lL) of the mixture was then washed through
three-ply DE-81 filters (ion exchange paper) with 5 · 1 mL
of fresh 25 mm ammonium bicarbonate. The filters were
then transferred to 5 mL of scintillation fluid [Brady’s fluid:
10% (v ⁄ v) methanol, 6% (w ⁄ v) naphthalene, 2% (v ⁄ v)

ethylene glycol, 0.4% (w ⁄ v) diphenyloxazole in 1,4-dioxane]
and shaken vigorously for 20 min. The average LysU turnover was calculated from the scintillation count (minus
background) as moles Ap4A per min per mole of LysU at
37 °C.
1

H-NMR assays

Individual NMR assays were preformed at 37 °C in a 5 mm
NMR tube using a 400 MHz Bruker NMR spectrometer set
up with spectra taken every 2 min over a period of 20 min.
Each spectrum was a compilation of 64 scans acquired over
a period of 1 min. Initially, LysU reaction mixtures of
20 mm Hepes, pH 7.8, 150 mm KCl, 5 mm MgCl2, 150 lm
ZnCl2, 1–50 mm ATP, 0.05–5 mm l-lysine, 0.03 mg inorganic pyrophosphatase and 20% D2O were prepared. For
each NMR assay, an aliquot (600 lL) of an appropriate
LysU reaction mixture was equilibrated at 37 °C in the
NMR tube, after which a LysU aliquot (400 nm) was introduced by needle and spectral acquisition was begun. ATP
1
H-NMR signals were seen initially at: 8.55 p.p.m. (H2) and
8.29 p.p.m. (H8), and this was followed by the emergence of
Ap4A signals with time at: 8.40 p.p.m. (H2) and 8.18 p.p.m.
(H8). The H8 signals were seen to be the most reliable indicator of relative concentrations; therefore, the initial rate of
conversion of ATP into Ap4A observed in each given NMR
assay was determined from H8 signal integrations. Initial
rate data were then processed by standard Michaelis–Menten data-fitting to give values of KM and kcat for ATP conversion into Ap4A.

FEBS Journal 273 (2006) 3534–3544 ª 2006 The Authors Journal compilation ª 2006 FEBS

M. Wright et al.

AMMPR-coupled assay
This assay was performed on a thermostatted Ultraspec III
(Amersham Biosciences) set to detect A360 at 20 °C from
a 0.7 mL quartz cuvette. This assay uses AMMPR, which
was prepared in a three-step, one-pot reaction from the
reagent 2-amino-6-chloropurine ribonucleoside (ACPR,
1 g). ACPR was dissolved in distilled dimethyl formamide
(4 mL) and the solution was then placed in a stirred flask
(50 mL) under nitrogen. Methyl iodide (2 mL) was added,
and the mixture was stirred for 20 h at room temperature
before thiourea (1 g) was added. The mixture was stirred
for a further 30 min, after which 2 m ammonia in methanol was added dropwise until the mixture reached neutral pH. Finally, the mixture was poured into stirred
acetone (200 mL), causing AMMPR to precipitate out of
solution. The pale yellow solid was filtered and dried
under nitrogen before storage at ) 20 °C. The yield was
64%, and spectral characterization matched the literature
[16].
Purine nucleotide phosphorylase enzyme (Sigma-Aldrich)
was repurified before use by means of a Mono-Q column
(1 · 10 cm) packed in 50 mm Tris ⁄ HCl, pH 7.6, and eluted
with a gradient of NaCl (0–1.5 m) at a flow rate of 3 mLỈ
min)1. Individual reactions were tested for activity by incubation with 5 mm potassium phosphate and 150 lm
AMMPR. Active fractions were pooled, concentrated using
3 kDa cutoff centricon concentrators and dialysed into
20 mm Hepes, pH 7.8, before storage as a precipitate in
3.2 m ammonium sulphate at 4 °C.
Initially for the AMMPR assay, aliquots (500 lL each)
of a LysU reaction mixture comprising 20 mm Hepes,

pH 7.8, 150 mm KCl, 25 mm MgCl2, 150 lm ZnCl2,
0.25 lg of inorganic pyrophosphatase and 0.03 mg of
purine nucleotide phosphatase, were prepared. Each
aliquot was then used in two sets of experiments, one set
with l-lysine (1–200 lm) and fixed ATP (25 mm), and the
other with ATP (0.1–20 mm) and fixed l-lysine (0.5 mm).
For each assay, the LysU reaction mixture with added
l-lysine and ATP was transferred to the cuvette and
equilibrated at 20 °C for 2 min, after which AMMPR
(400 lm final concentration) and LysU (20 nm final concentration) were added, the mixture was vigorously agitated, and A360 was followed as a function of time. After
background correction, these absorbance data were converted into initial rate data. These initial rate data were
then processed by standard Michaelis–Menten data-fitting
to give values of KM and kcat for ATP and lysine conversion into lysyl-adenylate.

Acknowledgements
MW would like to thank IC-Vec, and NB would like
to thank the Royal Thai Government for personal sup-

LysU catalysis of Ap4A and Ap3A synthesis

port. We would also like to thank IC-Vec and the Mitsubishi Chemical Corporation for their support of the
Imperial College Genetic Therapies Centre.

References
1 Cusack S, Hartlein M & Leberman R (1991) Sequence,
structural and evolutionary relationships between class 2
aminoacyl-tRNA synthetases. Nucleic Acids Res 19,
3489–3498.
2 Onesti S, Miller AD & Brick P (1995) The crystal structure of the lysyl-tRNA synthetase (LysU) from Escherichia coli. Structure 3, 163–176.
3 Ito K, Oshima T, Mizuno T & Nakamura Y (1994)

Regulation of lysyl-tRNA synthetase expression by histone-like protein H-NS of Escherichia coli. J Bacteriol
176, 7383–7386.
4 Brevet A, Chen J, Leveque F, Blanquet S & Plateau P
(1995) Comparison of the enzymatic properties of the
two Escherichia coli lysyl-tRNA synthetase species.
J Biol Chem 270, 14439–14444.
5 Charlier J & Sanchez R (1987) Lysyl-tRNA synthetase
from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product. Biochem J 248, 43–
51.
6 Brevet A, Chen J, Leveque F, Plateau P & Blanquet S
(1989) In vivo synthesis of adenylylated bis (5¢-nucleosidyl) tetraphosphates (Ap4N) by Escherichia coli aminoacyl-tRNA synthetases. Proc Natl Acad Sci USA 86,
8275–8279.
7 Theoclitou ME & Miller AD (1994) Enhancement of
molecular ion intensity of synthetic analogues of diadenosine 5¢,5¢¢-P1,P4-tetraphosphate (Ap4A) in negativeion mode fast atom bombardment mass spectrometry.
Anal Biochem 218, 235–237.
8 Zamecnik PC, Stephenson ML, Janeway CM & Randerath K (1966) Enzymatic synthesis of diadenosine tetraphosphate and diadenosine triphosphate with a
purified lysyl-sRNA synthetase. Biochem Biophys Res
Commun 24, 91–97.
9 Theoclitou ME, El-Thaher TSH & Miller AD (1994)
Enzymatic synthesis of diadenosine 5¢,5¢¢¢-P1,P4-tetraphosphate (Ap4A) analogues by stress protein LysU.
J Chem Soc Chem Commun 1994, 659–661.
10 Wright M, Tanner JA & Miller AD (2003) Quantitative
single-step purification of dinucleoside polyphosphates.
Anal Biochem 316, 123–138.
11 Wright M & Miller AD (2004) Synthesis of novel fluorescent-labelled dinucleoside polyphosphates. Bioorg Med
Chem Lett 14, 2813–2816.
12 Wright M & Miller AD (2006) Novel fluorescent
labelled affinity probes for diadenosine-5¢,5¢¢¢-P1,P4tetraphosphate (Ap4A) binding studies. Bioorg Med
Chem Lett 16, 943–948.

FEBS Journal 273 (2006) 3534–3544 ª 2006 The Authors Journal compilation ª 2006 FEBS

3543


LysU catalysis of Ap4A and Ap3A synthesis

M. Wright et al.

13 Randerath K, Janeway CM, Stephenson ML & Zamecnik PC (1966) Isolation and characterization of dinucleoside tetra- and tri-phosphates formed in the
presence of lysyl-sRNA synthetase. Biochem Biophys
Res Commun 24, 98–105.
14 Theoclitou ME, Wittung EPL, Hindley AD, El-Thaher
TSH & Miller AD (1996) Characterisation of stress protein LysU. Enzymic synthesis of diadenosine 5¢,5¢¢¢P1,P4-tetraphosphate (Ap4A) analogues by LysU.
J Chem Soc Perkin Trans 1, 2009–2019.
15 Plateau P, Mayaux JF & Blanquet S (1981) Zinc (II)dependent synthesis of diadenosine 5¢,5¢¢-P(1),P(4)-tetraphosphate by Escherichia coli and yeast phenylalanyl
transfer ribonucleic acid synthetases. Biochemistry 20,
4654–4662.
16 McLennan AG, Taylor GE, Prescott M & Blackburn
GM (1989) Recognition of beta beta¢-substituted and
alpha beta,alpha¢beta¢-disubstituted phosphonate analogues of bis(5¢-adenosyl) tetraphosphate by the bis(5¢nucleosidyl)-tetraphosphate pyrophosphohydrolases
from Artemia embryos and Escherichia coli. Biochemistry 28, 3868–3875.
17 Led JJ, Switon WK & Jensen KF (1983) Phosphorolytic
activity of Escherichia coli glycyl-tRNA synthetase
towards its cognate aminoacyl adenylate detected by
31P-NMR spectroscopy and thin-layer chromatography.
Eur J Biochem 136, 469–479.
18 Hughes SJ, Tanner JA, Hindley AD, Miller AD & Gould
IR (2003) Functional asymmetry in the lysyl-tRNA
synthetase explored by molecular dynamics, free energy

calculations and experiment. BMC Struct Biol 3, 5–25.
19 Leveque F, Plateau P, Dessen P & Blanquet S (1990)
Homology of lysS and lysU, the two Escherichia coli
genes encoding distinct lysyl-tRNA synthetase species.
Nucleic Acids Res 18, 305–312.
20 Gorbunoff MJ (1984) The interaction of proteins with
hydroxyapatite. I. Role of protein charge and structure.
Anal Biochem 136, 425–445.
21 Cogan EB, Birrell GB & Griffith OH (1999) A roboticsbased automated assay for inorganic and organic phosphates. Anal Biochem 271, 29–35.
22 Vinogradova RP, Kucherenko NE, Verkhogliad IN &
Mirutenko NV (1982) Phosphorylation of lysyl tRNA
synthetase from the liver of rats exposed to x-rays.
Radiobiologiia 22, 446–449.
23 Vinogradova RP & Mirutenko NV (1979) Phosphorylation of aminoacyl-tRNA synthetase preparations of rat
liver tissues in vivo and in vitro. Ukr Biokhim Zh 51,
596–599.
24 Pendergast AM, Venema RC & Traugh JA (1987)
Regulation of phosphorylation of aminoacyl-tRNA
synthetases in the high molecular weight core complex
in reticulocytes. J Biol Chem 262, 5939–5942.

3544

25 Dang CV & Traugh JA (1989) Phosphorylation of
threonyl- and seryl-tRNA synthetase by cAMP-dependent protein kinase. A possible role in the regulation of
P1,P4-bis (5¢-adenosyl)-tetraphosphate (Ap4A) synthesis.
J Biol Chem 264, 5861–5865.
26 Tanner JA, Abowath A & Miller AD (2002) Isothermal
titration calorimetry reveals a zinc ion as an atomic
switch in the diadenosine polyphosphates. J Biol Chem

277, 3073–3078.
27 Plateau P & Blanquet S (1982) Zinc-dependent synthesis
of various dinucleoside 5¢,5¢¢¢-P1,P3-Tri- or 5¢¢,5¢¢¢P1,P4-tetraphosphates by Escherichia coli lysyl–tRNA
synthetase. Biochemistry 21, 5273–5279.
28 Lloyd AJ, Thomann HU, Ibba M & Soll D (1995) A
broadly applicable continuous spectrophotometric assay
for measuring aminoacyl-tRNA synthetase activity.
Nucleic Acids Res 23, 2886–2892.
29 Webb MR (1992) A continuous spectrophotometric
assay for inorganic phosphate and for measuring phosphate release kinetics in biological systems. Proc Natl
Acad Sci USA 89, 4884–4887.
30 Vartanian A, Prudovsky I, Suzuki H, Dal Pra I & Kisselev L (1997) Opposite effects of cell differentiation
and apoptosis on Ap3A ⁄ Ap4A ratio in human cell cultures. FEBS Lett 415, 160–162.
31 Vartanian A, Alexandrov I, Prudowski I, McLennan A
& Kisselev L (1999) Ap4A induces apoptosis in human
cultured cells. FEBS Lett 456, 175–180.
32 Jankowski J, Jankowski V, Laufer U, van der Giet M,
Henning L, Tepel M, Zidek W & Schluter H (2003)
Identification and quantification of diadenosine polyphosphate concentrations in human plasma. Arterioscler
Thromb Vasc Biol 23, 1231–1238.
33 Luthje J, Baringer J & Ogilvie A (1985) Effects of diadenosine triphosphate (Ap3A) and diadenosine tetraphosphate (Ap4A) on platelet aggregation in
unfractionated human blood. Blut 51, 405–413.
34 Pintor J, Peral A, Pelaez T, Martin S & Hoyle CH
(2003) Presence of diadenosine polyphosphates in the
aqueous humor: their effect on intraocular pressure.
J Pharmacol Exp Ther 304, 342–348.
35 Kisselev LL, Justesen J, Wolfson AD & Frolova LY
(1998) Diadenosine oligophosphates (Ap (n) A), a
novel class of signalling molecules? FEBS Lett 427, 157–
163.

36 Ogilvie A & Jakob P (1983) Diadenosine 5¢,5¢¢-P1,P3-triphosphate in eukaryotic cells: identification and quantitation. Anal Biochem 134, 382–392.
37 Gill SC & von Hippel PH (1985) Calculation of protein
extinction coefficients from amino acid sequence data.
Anal Biochem 182, 316–326.
38 Buss JE & Stull JT (1983) Measurement of chemical
phosphate in proteins. Methods Enzymol 99, 7–14.

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