Methylenebisphosphonate and triphosphate derivatives of the mevalonate pathway are substrates of yeast UTP:glucose-1-phosphate uridylyltransferase

Article abstract of DOI:10.1016/j.biochi.2012.04.022

UTP:glucose-1-phospate uridylyltransferase (EC 2.7.7.9) from Saccharomyces cerevisiae transfers the uridylyl moiety of UDP-glucose onto methylenebisphosphonate (pCH₂p) yielding uridine 5′-(β, γ-methylenetriphosphate) (UppCH₂p). The f

Full text of DOI:10.1016/j.biochi.2012.04.022

Biochimie 94 (2012) 1871e1875
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Research paper
Methylenebisphosphonate and triphosphate derivatives of the mevalonate
pathway are substrates of yeast UTP:glucose-1-phosphate uridylyltransferase
María Antonia Günther Sillero, Anabel de Diego, Antonio Sillero*
Departamento de Bioquímica, Instituto de Investigaciones Biomédicas Alberto Sols, UAM/CSIC, Facultad de Medicina, 28029 Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
UTP:glucose-1-phospate uridylyltransferase (EC 2.7.7.9) from Saccharomyces cerevisiae transfers the
Received 26 March 2012
Accepted 26 April 2012
Available online 2 May 2012
0
uridylyl moiety of UDP-glucose onto methylenebisphosphonate (pCH
ylenetriphosphate) (UppCH
2
p) yielding uridine 5 -(
p). The following bisphosphonates were not acceptors of UMP: alendronate,
b
,
g
-meth-
2
pamidronate, clodronate and etidronate. UDP-glucose serves as uridylyl donor to triphosphate deriva-
tives of the mevalonate pathway: farnesyl (far-PPP), geranyl (ger-PPP) and isopentenyl (iso-PPP), with
formation of farnesyl-tetraphosphouridine (far-ppppU); geranyl-tetraphosphouridine (ger-ppppU) and
Keywords:
Bisphosphonates
Ligases
Transferases
Mevalonate pathway
Osteoporosis
isopentenyl-tetraphosphouridine (iso-ppppU). The K
mined for these substrates were: 0.32 ꢀ 0.07 and 4.9 ꢀ 0.6; 0.21 ꢀ 0.06 and 5.7 ꢀ 0.8; 0.51 ꢀ 0.14 and
.0 ꢀ 0.2, respectively. The K and Vmax values for methylenebisphosphonate were 1.1 ꢀ 0.2 mM and
4055 ꢀ 96 mU/mg protein, respectively.
m
(mM) and Vmax (mU/mg protein) values deter-
2
m
Ó 2012 Elsevier Masson SAS. All rights reserved.
1
. Introduction
uridylyltransferase or as UDP-glucose pyrophosphorylase (in the
reverse reaction) [9,10], was able to transfer the UMP moiety of
UDP-glucose to a variety of nucleoside tri and polyphosphates with
formation of dinucleoside polyphosphates containing uridine
(Table 1, reaction 11) [11].
In this report, two selective, and so far not reported reactions are
described in which the enzyme transfers UMP from UDP-glucose to
methylenebisphosphonate, out of several bisphosphonates tested
(Table 1, reaction 12) and to compounds of the mevalonate pathway
with a terminal triphosphate, but not with a terminal pyrophos-
phate (Table 1, reaction 13).
It is well known that AMP-forming ligases may transfer AMP
from complexes of the type E$X-AMP to a proper acceptor (Table 1,
reaction 2) [1e3]. For the last years we have analysed the property
of some of these enzymes to transfer AMP to a nucleoside
triphosphate (reactions 3, 4) [4], to nucleoside polyphosphates
(
reaction 5), to a polyphosphate (reaction 6) [5], or even to
a bisphosphonate (reaction 7) [6,7]. We have also analysed the
capacity of some ligases to transfer AMP to compounds of the
mevalonate pathway with two or three terminal phosphates
(
reactions 8, 9) [8]. A great variety of compounds were synthesised
through these reactions. The mechanism of action of bisphospho-
nates in the treatment of osteoporosis has been approached else-
where and in previous publications from this laboratory [6,7].
This property of ligases is also shared by some transferases. We
had previously reported that the enzyme UTP:glucose-1-phosphate
uridylyltransferase (EC 2.7.7.9), known also as glucose-1-phosphate
2
2
2
. Materials and methods
.1. Materials
.1.1. Enzymes
Yeast UTP:glucose-1-phosphate uridylyltransferase. Cat. No.
U8501 (Lots 117K7430 and 117K74301) was from Sigma. One unit of
enzyme forms 1
m
mol of glucose-1-P from UDP-glucose and PPi per
ꢁ
min at 30 C;shrimp alkaline phosphatase (EC 3.1.3.1)wasfromRoche
0
Abbreviations: AppCH
2
p, adenosine 5 -(
b
,
g-methylenetriphosphate); far-PPP,
(
Ref.1758250); phosphodiesterase I (EC 3.1.4.1) from Crotalus durissus
farnesyl triphosphate; far-ppppU, farnesyl-tetraphosphouridine; ger-PPP, geranyl
triphosphate; ger-ppppU, geranyl-tetraphosphouridine; iso-PPP, isopentenyl
triphosphate; iso-ppppU, isopentenyl-tetraphosphouridine; N-BP, a bisphospho-
was from Boehringer Mannheim (now Roche) and yeast inorganic
pyrophosphatase (EC 3.6.1.1) was from BioLabs (Cat. No. 2403).
nate containing nitrogen; non-N-BP, a bisphosphonate not containing nitrogen;
0
pCH
2
p,
methylenebisphosphonate;
UppCH
2
p,
uridine
5 -(
b
,
g-
2
.1.2. Other materials
Glucose-1-phosphate, tripolyphosphate (P ), UDP-glucose,
3
methylenetriphosphate).
*
Corresponding author. Tel.: þ34 91 4975420; fax: þ34 91 5854401.
E-mail address: antonio.sillero@uam.es (A. Sillero).
2
methylenebisphosphonate, pCH p, (Cat. No. 274291; etidronate,
0300-9084/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.biochi.2012.04.022

1872
M.A. Günther Sillero et al. / Biochimie 94 (2012) 1871e1875
Table 1
3. Results
Reaction models catalyzed by ligases and transferases.
1
2
3
4
5
6
7
8
9
) E þ ATP þ X 4 E$X-AMP þ PPi
3.1. Yeast uridylyltransferase (EC 2.7.7.9) catalyses the transfer of
UMP to methylenebisphosphonate
) E$X-AMP þ Y / XeY þ AMP þ E
) E$X-AMP þ ATP / Ap A þ X þ E
4
) E$X-AMP þ NTP / E þ X þ AppppN
) E$X-AMP þ (p) A / E þ X þ A(p)nþ1
) E$X-AMP þ (P) / E þ X þ (p)nþ1
This enzyme catalyses the synthesis of UDP-glucose from UTP
and glucose 1-P (Table 1, reaction 10). Since this reaction is
reversible we assayed the transfer of the UMP moiety from UDP-
glucose to two types of compounds, both analogues of pyrophos-
phate: bisphosphonates (Table 1, reaction 12) and mevalonate
derivatives, mainly triphosphates (Table 1, reaction 13).
The bisphosphonates (BPs) tested as potential UMP acceptors
were of two types: nitrogen containing bisphosphonates, or N-
BP (alendronate and pamidronate) and non-containing nitrogen,
n
A
n
A
) E$X-AMP þ P-C(R1)(R2)-P / E þ X þ AppC(R1)(R2)p
) E$X-AMP þ mev-PP / E þ X þ mev-pppA
) E$X-AMP þ mev-PPP / E þ X þ mev-ppppA
1
1
1
1
0) UTP þ glucose-1-P 4 UDP-glucose þ PPi
1) UDP-glucose þ NTP / glucose 1-P þ Np
4
U
2) UDP-glucose þ pC(R1)(R2)p / glucose 1-P þ UppC(R1)(R2)p
3) UDP-glucose þ mev-PPP / glucose 1-P þ mev-ppppU
0
n
A, adenosine 5 -
X, a co-substrate of a ligase; NTP, a nucleoside triphosphate; (p)
polyphosphate; (P) , a polyphosphate; pC(R1)(R2)p, a bisphosphonate; mev-PP and
mev-PPP, components of the mevalonate pathway with a terminal pyrophosphate
or triphosphate, respectively. This work deals exclusively with reactions 12 and 13.
n
or non-N-BP (methylenebisphosphonate (pCH
and etidronate). When the enzyme was incubated with [
2
p), clodronate
32
a
- P]
UDP-glucose as UMP donor and with any of the above referred
bisphosphonates, only methylenebisphosphonate was substrate
pC(OH)(CH
compounds:
3
)p, (Cat. No. P5248) and the mevalonate pathway
-dimethylallyl pyrophosphate, Cat. No. D4287;
0
of the reaction with formation of UppCH
2
p (uridine 5 -(
b
,
g
-
g,g
methylenetriphosphate)) (Fig. 1, lane 5). Two control reactions
with no added bisphosphonate, Fig. 1, lane 1) and with added
tripolyphosphate (P ) (Fig. 1, lane 2) were run in parallel. After
h incubation, the amount of [
farnesyl pyrophosphate, Cat. No. F6892; farnesyl triphosphate, Cat.
No F1928; geranyl pyrophosphate, Cat. No. G6772; geranyl
triphosphate, Cat. No. G2418; isopentenyl pyrophosphate, Cat. No.
I0503, and isopentenyl triphosphate, Cat. No. I1282, were from
(
3
32
3
a- P]UDP-glucose remained
practically unchanged in the absence of bisphosphonate (lane 1)
or in the presence of any of the bisphosphonates tested (lanes 3,
Sigma. Clodronate, pCCl
2
p, Cat. No. 233183); pamidronate,
)p, Cat. No. 506600 and alendronate,
eNH )p, Cat. No. 126855, were from Cal-
pC(OH)(CH
pC(OH)(CH
2
eCH
eCH
2
eNH
eCH
2
4
, 6, 7), except methylenebisphosphonate (pCH
the presence of pCH p, the formation of a new radioactive spot,
presumably the corresponding UMP derivative, UppCH p (lane
was observed. Methylenebisphosphonate resulted to be
a better acceptor of UMP than P , an already known acceptor of
2
p) (lane 5). In
2
2
2
2
2
3
2
biochem. [a- P]UTP (3000 Ci/mmol) was from Perkin Elmer. Thin
2
layer chromatography (TLC) silicaegel fluorescent plates were from
Merck. Radioactively labelled nucleotides were quantified with the
help of a Typhoon Trio from GE Healthcare. The Hypersil ODS
column (4.6 ꢂ 100 mm) was from Hewlett Packard.
5
)
3
UMP [11], rendering uridine tetraphosphate (p
lanes 2 and 5).
4
U) (compare
The synthesis of the presumptive UppCH
HPLC. The enzyme was incubated with UDP-glucose in the absence
Fig. 2, a) or in the presence of pCH p (Fig. 2, b). Treatment of the
2
p was also followed by
2
2
.2. Methods
(
2
.2.1. Synthesis of [a
-32P]UDP-glucose
The reaction mixture (0.02 ml) contained 50 mM Hepes/KOH
3
2
(
pH 8.0), 1 mM
a
-glucose-1-P, 0.36 mM [
a- P]UTP (5 mCi), 2 mM
MgCl
2
, 0.1 mM dithiothreitol, 0.1 U pyrophosphatase and 0.25 U of
uridylyltransferase (Table 1, reaction 10). The mixture was incu-
ꢁ
bated until consumption of UTP (usually 30 min at 30 C) and then
3
2
used directly as a source of [
a- P]UDP-glucose. This preparation is
3
2
normally contaminated with about 10% [ P]UMP.
2.2.2. Synthesis of uridine derivatives of methylenebisphosphonate
and of compounds of the mevalonate pathway catalysed by
UTP:glucose-1-phosphate uridylyltransferase
Standard reaction mixtures (0.02 ml) contained 50 mM Hepes/
3
2
KOH (pH 8.0), 0.03 or 0.1 mM [
a- P]UDP-glucose (0.5 or 1 mCi);
2
mM MgCl ; 0.1 mM dithiothreitol; 0.1 U pyrophosphatase;
2
enzyme and potential uridylyl acceptors as indicated (Table 1,
ꢁ
reactions 12, 13). After incubation at 30 C, aliquots (1.5
ml) were
spotted onto silicaegel plates, the chromatograms developed as
indicated and the radioactivity quantified. For characterisation of
the reaction products, the mixtures were treated with shrimp
alkaline phosphatase (50 U/ml) and phosphodiesterase (100
ml), for 1 h at 37 C.
mg/
ꢁ
For the analysis by high-pressure liquid chromatography
HPLC), the reaction mixtures contained 50 mM Hepes/KOH (pH
0
2
b,g-methylenetriphosphate) (UppCH p) from [a
-32P]
Fig. 1. Synthesis of uridine 5 -(
UDP-Glu and pCH p catalysed by UTP:glucose-1-P uridylyltransferase from yeast. The
reaction mixtures (0.02 ml) contained 0.03 mM [
MgCl mM mM (clodronate, etidronate or methylenebisphosphonate
pCH p)), 1 mM (pamidronate or alendronate), and 0.25 U enzyme. Other compo-
(
2
3
2
8
.0), 10 mM MgCl
2
, 0.1 mM dithiothreitol, UDP-glucose, methyl-
a- P]UDP-Glu (0.5 mCi), 10 mM
enebisphosphonate and yeast uridylyltransferase as indicated
2
,
4
3
P , 2
(
2
(
Table 1, reaction 12). After incubation, aliquots were diluted 10-
nents as specified in Section 2. A control reaction with no added bisphosphonate was
run in parallel (lane 1). After 3 h incubation, aliquots were spotted on TLC plates,
developed in dioxane:ammonium hydroxide:water (6:1:6) and subjected to
autoradiography.
ꢁ
fold, heated for 1.5 min at 95 C; precipitated protein was
removed by centrifugation and 0.05 ml aliquots injected into
a Hypersil ODS column and eluted as described [5].

M.A. Günther Sillero et al. / Biochimie 94 (2012) 1871e1875
1873
3
.2. Uridylyltransferase (EC 2.7.7.9) catalyses the transfer of UMP to
a
phosphorylated compounds of the mevalonate pathway
Preliminary assays showed that only the triphosphate deriva-
tives from farnesyl (far-PPP) (Fig. 3, A), geranyl (ger-PPP) (Fig. 3, B)
or isopentenyl (iso-PPP) (Fig. 3, C) were UMP acceptors from UDP-
glucose. The corresponding diphosphate compounds (far-PP, ger-
PP, iso-PP) and dimethylallyl pyrophosphate, did no react with
UDP-glucose in our experimental conditions, even after prolonged
times of incubation (result not shown). Each triphosphate deriva-
tive was assayed at three different incubation times (Fig. 3, AeC;
lanes 2, 4, 6); controls in the absence of acceptor were run in
parallel (Fig. 3, AeC; lanes 1, 3, 5). Characterisation of the corre-
sponding products of the reaction was performed by treatment
with alkaline phosphatase (AP), showing their insensitivity to this
b
treatment, (Fig. 3, AeC; lane 8). In our TLC working conditions the R
value corresponding to Pi is concentration-dependent: while at low
concentration (50 M) Pi migrates ahead from the origin, at 4 mM it
f
m
practically does not move from it. Therefore in those samples that
contained far-PPP, ger-PPP or iso-PPP, the Pi formed by treatment
with alkaline phosphatase, diminished the mobility of Pi (compare
lanes 7 and 8). After inactivation of the phosphatase, the mixtures
were treated with snake venom phosphodiesterase (SVP) yielding
UMP in all cases (Fig. 3, AeC, lanes 9 and 10). The relative amounts
c
(
nmoles/mg protein) of far-p
these experiments are represented in Fig. 3, D.
The Vmax, K , kcat and kcat/K values calculated for far-PPP, ger-
PPP or iso-PPP are compiled in Table 2. Vmax and K values were
determined in the presence of a fixed (0.1 mM) concentration of
4 4 4
U, ger-p U and iso-p U synthesised in
m
m
m
d
3
2
[a
-
P]UDP-glucose and variable (0.1e0.6 mM) concentrations of
substrate. The kcat/K values are similar to the corresponding
values calculated for T4 RNA and DNA ligases [6,7], whereas the
value calculated here for pCH p is about 1 and 3 order of
m
k
cat/K
m
2
magnitude higher than the corresponding values determined for T4
RNA and T4 DNA ligases, respectively [6,7].
4
. Discussion
0
Fig. 2. Synthesis and characterisation of uridine 5 -(
UppCH p) form UDP-glucose and methylenebisphosphonate (pCH
UTP:glucose-1-P uridylyltransferase from yeast. A reaction mixture (0.08 ml) con-
taining 1 mM UDP-glucose, 10 mM MgCl , 1 mM pCH p, and 4 U enzyme (other
components as specified in Section 2), were incubated for 30 min and an aliquot
b
,
g
-methylenetriphosphate)
(
2
2
p) catalysed by
As expected [12], the terminal phosphate in UppCH
2
p or in
0
AppCH
2
p
(adenosine 5 -(
b,
g
-methylenetriphosphate)), forming
2
2
a phosphonate bond (CeP) [6], was not hydrolyzed by alkaline
phosphatase. In addition to the phosphonates obtained by chemical
synthesis [13], the carbonephosphorus bond is widely distributed
in nature, mainly in microorganisms [14,15].
analysed by HPLC (b). The rest of the reaction mixture was treated with 4
phodiesterase (SVP) for 1 h at 37 C (c), and thereafter further treated with 1
alkaline phosphatase (AP) (d). A control reaction with no added enzyme is shown in
a).
m
g phos-
ꢁ
m
l (1 U)
(
Concerning the mevalonate triphosphate derivatives (mev-PPP),
cosubstrates of the compounds of general formula mev-ppppU
here synthesised, recent findings point to a new and interesting
role for them: i) protein histidine phosphorylation by histidine
kinases (generating phosphoamide bonds) is emerging as a prom-
ising avenue in cellular signal transduction (see Ref. [16] for
a review); one of these kinases belongs to the group of the classical
nucleoside diphosphate kinases (NDPK); members of this family of
enzymes, named as Nm23/nucleoside diphosphate kinases, can
last reaction mixture with snake venom phosphodiesterase (SVP)
degraded both the remaining UDP-glucose and the pCH p deriva-
tive (UppCH p) to UMP (Fig. 2, c); further treatment with alkaline
phosphatase (AP) rendered uridine (Fig. 2, d). When in a similar
experiment (not shown) the reaction mixture containing UppCH
Fig. 2, b) was treated firstly with alkaline phosphatase, the result
obtained confirmed the insensitivity of the terminal phosphate in
UppCH p to this treatment [6]. Altogether these results show that
the enzyme transfers the moiety of UMP from UDP-glucose to
methylenebisphosphonate, with synthesis of UppCH p.
With this type of assay (HPLC), a K
value of 1.1 ꢀ 0.2 mM was
determined for pCH p in the presence of fixed (4 mM) UDP-glucose
and variable (0.3e2 mM) pCH p concentrations. From the Vmax
2
2
2
p
(
Table 2
2
Kinetics constants of UDP:glucose-1-phosphate uridylyltransferase towards
substrates of the mevalonate pathway and methylenebisphosphonate.
2
ꢃ1
ꢃ1
ꢃ1
M )
Substrates
V
max (mU/mg)
K
m
[M]
k
cat (s
)
kcat/K
m
(s
m
ꢃ3
ꢃ3
ꢃ3
ꢃ3
ꢃ3
ꢃ3
ꢃ3
far-PPP
ger-PPP
iso-PPP
4.9 ꢀ 0.6
5.7 ꢀ 0.8
2.0 ꢀ 0.2
(0.32 ꢀ 0.07)ꢂ10
(0.21 ꢀ 0.06)ꢂ10
(0.51 ꢀ 0.14)ꢂ10
(1.1 ꢀ 0.2)ꢂ10
33 ꢂ 10
38 ꢂ 10
13 ꢂ 10
27
103
181
25
2
2
value (4055 ꢀ 96 mU/mg) obtained in these experiments and
pCH
2
p
4055 ꢀ 96
24,545
assuming a molecular mass of 400 kDa for the yeast transferase,
ꢃ1
The above values were determined by LineweavereBurk analysis and represent the
mean and standard deviation of at least three determinations, using the following
range of concentrations: far-PPP (0.1e0.5 mM); ger-PPP (0.1e0.7 mM); iso-PPP
a kcat of 27 s
2
was calculated for the synthesis of UppCH p
(Table 2), this value is around 1350 times higher than that calcu-
32
lated for the synthesis of p U from P [11].
4
3
(0.1e0.6); [a- P]UDP-glucose (0.03 mM, 0.5 mCi), as indicated in Section 2.2.2.

1874
M.A. Günther Sillero et al. / Biochimie 94 (2012) 1871e1875
Fig. 3. Synthesis of farnesyl-tetraphosphouridine (far-p
transferase. The reactions (0.02 ml) were carried out in the presence of 0.03 mM [
4
U), geranyl-tetraphosphouridine (ger-p
4
U) and isopentenyl-tetraphosphouridine (iso-p
4
U) catalysed by yeast uridylyl-
3
2
a
P]UDP-Glu (0.5 Ci), 2 mM MgCl , 0.25 U enzyme (other components as indicated in Section 2),
m
2
and 0.60 mM of the following substrates: farnesyl triphosphate (far-PPP), geranyl triphosphate (ger-PPP) or isopentenyl triphosphate (iso-PPP), Fig. 3 (lanes 2, 4 and 6). Control
reactions with no added substrate (lanes 1, 3 and 5) were run in parallel. Samples were taken at the times indicated and subjected to TLC. After the last incubation time, the reaction
ꢁ
mixtures were treated with 1
were treated with 2
m
l (1 U) of shrimp alkaline phosphatase (AP) (lanes 7, 8). Once the phosphatase was denatured by heating at 90 C for 6 min, the reaction mixtures
m
g snake venom phosphodiesterase (SVP) (lanes 9 and 10). Plates (A and C) were developed in dioxane:ammonium hydroxide:water (6:1:5) for 90 min; plate B
4 4 4
in dioxane:ammonium hydroxide:water (6:1:4) for 120 min. The relative amounts of far-p U ger-p U and iso-p U synthesised along incubation time are represented in panel D.
also phosphorylate geranyl and farnesyl diphosphates to the cor-
responding triphosphates [17]; ii) cells with elevated levels of
Nm23 proteins contain both more farnesyl triphosphate [17] and
more farnesylated proteins in the 24e46 kDa range; iii) the possi-
bility that farnesyl triphosphates could serve as an alternative
substrate for the farnesylation of proteins has been also raised [17].
The fact that methylenebisphosphonate was the only
bisphosphonate, out of the ones tested, able to compete with PPi in
the reverse reaction catalysed by UTP:glucose-1-phospate uridy-
[2] C.S. Francklyn, E.A. First, J.J. Perona, Y.-M. Hou, Methods for kinetic thermo-
dynamic analysis of aminoacyl-tRNA synthetases, Methods 44 (2008)
100e118.
[
[
[
3] A. Sillero, M.A. Günther Sillero, Synthesis of dinucleoside polyphosphates
catalyzed by firefly luciferase and several ligases, Pharmacol. Ther. 87 (2000)
91e102.
4] A. Guranowski, M.A. Günther Sillero, A. Sillero, Firefly luciferase synthesizes
1
4
0
P , P -bis(5 -adenosyl)tetraphosphate (Ap
4
A) and other dinucleoside poly-
phosphates, FEBS Lett. 271 (1990) 215e218.
0
5] B. Ortiz, A. Sillero, M.A. Günther Sillero, Specific synthesis of adenosine(5 )
0
0
0
tetraphospho(5 )nucleoside adenosine(5 )oligophospho(5 )adenosine (n > 4)
catalyzed by firefly luciferase, Eur. J. Biochem. 212 (1993) 263e270.
lyltransferase and synthesising UppCH
2
p
with
a
high rate
[6] M.A. Günther Sillero, A. de Diego, E. Silles, F. Pérez-Zúñiga, A. Sillero, Synthesis
of bisphosphonate derivatives of ATP by T4 RNA ligase, FEBS Lett. 580 (2006)
ꢃ1
(
k
cat ¼ 27 s ), may be related of being comparatively a smaller
5723e5727.
molecule [18]. The other substrate of the enzyme, UDP-glucose,
holds a central position in the synthesis of glycogen and in the
[
7] M.A. Günther Sillero, A. de Diego, F.J. Pérez-Zúñiga, A. Sillero, Synthesis of
bisphosphonate derivatives of ATP by T4 DNA ligase, ubiquitin activating
enzyme (E1) and other ligases, Biochem. Pharmacol. 75 (2008)
2
interchange of sugars [14,19,20]. The possibility that pCH p inter-
feres with the synthesis of glycogen and/or sugar metabolism
in vivo could be raised.
When administered to humans, bisphosphonates tend to be fixed
to bone tissues, where they are captured by osteoclasts. In these, and
other types of cells, they evoke noxious effects through the synthesis
of bisphosphonate derivatives of ATP and inhibition of key enzymes
of cellular metabolism [21]. Perhaps, and in part secondary to the
reactions here described, methylenebisphosphonate is not used in
humans for the treatment of osteoporosis.
1959e1965.
[
8] M.A. Günther Sillero, A. de Diego, J.E. Tavares, J.A. Silva, F.J. Pérez-Zúñiga,
A. Sillero, Synthesis of ATP derivatives of compounds of the mevalonate
pathway (isopentenyl di- triphosphate; geranyl di- triphosphate, farnesyl
di- triphosphate, dimethylallyl diphosphate) catalyzed by T4 RNA ligase,
T4 DNA ligase and other ligases potential relationship with the effect of
bisphosphonates on osteoclasts, Biochem. Pharmacol. 78 (2009) 335e343.
[9] L.F. Leloir, C.E. Cardini, UDPG-Glycogen transglycosylase, in: P.D. Boyer,
H. Lardy, K. Myrbäck (Eds.), The Enzymes, Academic Press, New York, 1962,
pp. 317e326.
10] R.L. Turnquist, R.G. Hansen, Uridine diphosphoryl glucose pyrophosphor-
ylase, in: P.D. Boyer (Ed.), The Enzymes, Academic Press, New York, 1973, pp.
[
[
5
1e70.
0
11] A. Guranowski, A. de Diego, A. Sillero, M.A. Günther Sillero, Uridine 5 -poly-
0
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Acknowledgements
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This work was supported by a grant from Dirección General de
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Jaime Renart for critical reading of the manuscript and Javier Perez
for able technical assistance.
[
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