Báo cáo khoa học: Hydrolysis of diadenosine polyphosphates by nucleotide pyrophosphatases/phosphodiesterases ppt

Hydrolysis of diadenosine polyphosphates by nucleotide
pyrophosphatases/phosphodiesterases
Petra Vollmayer
1
, Timothy Clair
2
, James W. Goding
3
, Kimihiko Sano
4
,Jo¨ rg Servos
1
and Herbert Zimmermann
1
1
AK Neurochemie, Biozentrum der J. W. Goethe-Universitaet, Frankfurt am Main, Germany;
2
Laboratory of Pathology, NCI,
National Institutes of Health, Bethesda, Maryland, USA;
3
Department of Pathology and Immunology, Monash University,
Alfred Hospital, Prahran, Victoria, Australia;
4
Department of Pediatrics, Kobe University School of Medicine, Kobe, Japan
Diadenosine polyphosphates (Ap
n
As) act as extracellular
signaling molecules in a broad variety of tissues. They were
shown to be hydrolyzed by surface-located enzymes in an
asymmetric manner, generating AMP and Ap
n-1

from
Ap
n
A. The molecular identity of the enzymes responsible
remains unclear. We analyzed the potential of NPP1, NPP2,
and NPP3, the three members of the ecto-nucleotide pyro-
phosphatase/phosphodiesterase family, to hydrolyze the
diadenosine polyphosphates diadenosine 5¢,5¢¢¢-P
1
,P
3
-
triphosphate (Ap
3
A), diadenosine 5¢,5¢¢¢-P
1
,P
4
-tetraphos-
phate (Ap
4
A), and diadenosine 5¢,5¢¢ ¢-P
1
,P
5
-pentaphosphate,
(Ap
5
A), and the diguanosine polyphosphate, diguanosine
5¢,5¢¢¢-P

1
,P
4
-tetraphosphate (Gp
4
G). Each of the three
enzymes hydrolyzed Ap
3
A, Ap
4
A, and Ap
5
A at comparable
rates. Gp
4
G was hydrolyzed by NPP1 and NPP2 at rates
similar to Ap
4
A,butonlyathalfthisratebyNPP3.
Hydrolysis was asymmetric, involving the a,b-pyrophos-
phate bond. Ap
n
A hydrolysis had a very alkaline pH opti-
mum and was inhibited by EDTA. Michaelis constant (K
m
)
values for Ap
3
Awere5.1l
M

,8.0l
M
, and 49.5 l
M
for
NPP1, NPP2, and NPP3, respectively. Our results suggest
that NPP1, NPP2, and NPP3 are major enzyme candidates
for the hydrolysis of extracellular diadenosine polyphos-
phates in vertebrate tissues.
Keywords: diadenosine polyphosphate; diguanosine poly-
phosphate; ectonucleotidase; nucleotide pyrophosphatase;
nucleotide phosphodiesterase.
Diadenosine polyphosphates [adenosine-(5¢)-oligophospho-
(5¢)-adenosines, Ap
n
As] comprise two adenosine residues
linked together by a polyphosphate chain through phos-
phoester bonds at their ribose 5¢ carbons. Ap
n
As are present
intracellularly in prokaryotic and eukaryotic cells [1].
Recently, this group of nucleotides has attracted consider-
able interest because its members act as extracellular
signaling molecules in a broad variety of tissues [2,3]. They
are involved, for example, in the modulation of synaptic
transmission and sensory nerve function [2–4], inhibition of
platelet aggregation [5], or in the control of vascular tone
[6–9]. Vasoactive effects were also observed with adenosine
polyphosphoguanosines (Ap
n

Gs) and diguanosine poly-
phosphates (Gp
n
Gs) [10].
The diadenosine polyphosphates diadenosine
5¢,5¢¢¢-P
1
,P
3
-triphosphate (Ap
3
A), diadenosine 5¢,5¢¢¢-P
1
,P
4
-
tetraphosphate (Ap
4
A), and diadenosine 5¢,5¢¢¢-P
1
,P
5
-pen-
taphosphate (Ap
5
A) are stored in chromaffin granules at
millimolar concentrations together with noradrenaline
and other nucleotides such as ATP and ADP [11,12]. In
cholinergic synaptic vesicles, Ap
4

AandAp
5
A were found to
be co-stored with acetylcholine [13]. They can be released
from secretory cells in a stimulus-dependent manner [2].
Besides the adrenal medulla, platelets are thought to
represent the main source of Ap
n
As in blood. Stimulated
platelets release, from their storage granules, a mixture of
Ap
n
As(uptoAp
7
A),aswellasAp
n
Gs and Gp
n
Gs, together
with ATP, ADP and serotonin [14,15]. Ap
n
As exert their
function via ionotropic (P2X) or metabotropic (P2Y)
receptors for mononucleotides, or also via P1 adenosine
receptors [3,16]. In addition, Ap
n
As may act on a separate
group of receptors, termed dinucleotide receptors or P4
receptors [2,3,17].
In vertebrate tissues, extracellular Ap

n
As were shown
to be metabolized and thus functionally inactivated by
enzymes associated with the cell surface or present in
body fluids [18]. Primary cleavage of Ap
n
As is asym-
metric, generating AMP and Ap
n-1
from Ap
n
A. Cellular
systems analyzed include blood [19], the adrenal medulla
Correspondence to H. Zimmermann, AK Neurochemie,
Biozentrum der J.W. Goethe-Universitaet, Marie-Curie-Str. 9,
D-60439 Frankfurt am Main, Germany.
Fax: + 49 69 79829606, Tel.: + 49 69 79829602,
E-mail:
Abbreviations:Ap
4
, adenosine tetraphosphate; Ap
3
A, diadenosine
5¢,5¢¢¢-P
1
,P
3
-triphosphate; Ap
4
A, diadenosine 5¢,5¢¢¢-P

1
,P
4
-tetraphos-
phate; Ap
5
A, diadenosine 5¢,5¢¢¢-P
1
,P
5
-pentaphosphate; Ap
n
A,
diadenosine polyphosphate (adenosine-(5¢)-oligophospho-(5¢)-adeno-
sine); CHO, Chinese hamster ovary; E-NPP, ecto-nucleotide
pyrophosphatase/phosphodiesterase; Gp
4
G, diguanosine 5¢,5¢¢¢-P
1
,P
4
-
tetraphosphate; pNP-TMP, p-nitrophenyl thymidine 5¢ monophos-
phate; PC-1, plasma-cell differentiation antigen-1; PPADS,
pyridoxal phosphate-6-azophenyl-2¢,4¢-disulfonic acid.
Enzyme: nucleotide pyrophosphatase/phosphodiesterase (EC 3.1.4.1
or EC 3.6.1.9)
(Received 20 March 2003, revised 29 April 2003,
accepted 15 May 2003)
Eur. J. Biochem. 270, 2971–2978 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03674.x

[20], vascular endothelial cells [21,22], synaptic membranes
[23,24], airway epithelia [25], and Xenopus oocytes [26].
At present, little is known concerning the molecular
identity of the enzymes involved. One group of enzymes
with the potential to hydrolyze Ap
n
As are NPP1, NPP2,
and NPP3, the three members of the ecto-nucleotide
pyrophosphatase/phosphodiesterase (E-NPP) family
(revised nomenclature) [27,28]. They reveal a surprisingly
broad substrate specificity and are capable of hydrolyzing
phosphodiester bonds of nucleotides and nucleic acids, and
pyrophosphate bonds of nucleotides and nucleotide sugars,
resulting in the release of nucleoside 5¢ monophosphates.
Both purine and pyrimidine nucleotides are hydrolyzed.
Substrates include ATP, NAD
+
,andp-nitrophenyl thymi-
dine 5¢ monophosphate (pNP-TMP) [29–31]. To date, three
related mammalian NPPs have been cloned and function-
ally characterized: NPP1 (plasma-cell differentiation anti-
gen-1, PC-1) [32]; NPP2 (autotaxin) [33] and its splice
variant, PD-Ia [34]; and NPP3 (gp130
RB13)6
, B10, PD-Ib)
[35–37]. NPP1 and NPP3 share 50% amino acid identity
and are more distantly related to NPP2 (39–41% identity).
They have a broad and partially overlapping tissue distri-
bution and may be co-localized within the same cell [37]. All
can exist as integral glycoproteins (of  120 kDa) of the

plasma membrane. They are disulfide-linked homodimers
representing type II membrane proteins [18,29,30]. In
addition, active soluble forms have been identified that
presumably result from specific proteolytic cleavage near the
transmembrane domain [38–40].
We analyzed the potential of heterologously expressed
NPP1, NPP2, and NPP3 to hydrolyze the diadenosine
polyphosphates Ap
3
A, Ap
4
AandAp
5
A, and the diguano-
sine polyphosphate Gp
4
G.InthecaseofNPP1andNPP3,
the membrane-bound form of the enzyme was analyzed,
whereas, in the case of NPP2 (autotaxin), which was shown
to promote cell motility in its soluble form [38], the soluble
form was employed.
Experimental procedures
Materials
Culture medium and genectin G-418 sulfate were obtained
from Invitrogen (Karlsruhe, Germany). All nucleotides,
penicillin, streptomycin, sodium butyrate, Cibacron Blue,
suramin, heparin, heparan sulfate, benzamidine, and
pyridoxal phosphate-6-azophenyl-2¢,4¢-disulphonic acid
(PPADS) were obtained from Sigma-Aldrich (Deisenhofen,
Germany). Leupeptin, pepstatin A, chymostatin, and anti-

pain were from Calbiochem (Schwalbach, Germany), and
aprotinin was from Roche (Mannheim, Germany).
Cell culture and cell transfection
Chinese hamster ovary (CHO) cells were cultured in HAM’s
F-12 medium containing 10% fetal bovine serum,
100 UÆmL
)1
penicillin and 100 lgÆmL
)1
streptomycin. They
were transfected by electroporation [41] with plasmid DNA
containing human NPP1 (NPP1pSVT7) [42] or rat NPP3
(GenBank
TM
accession number D30649; NPP3pcDNA3).
Transfection with the empty plasmid pSVT7 or pcDNA3
served as a control. Stable transfectants for NPP3 were
selected for neomycin resistance using 800 lgÆmL
)1
of
genectin G-418 sulfate (Invitrogen) and subcloned by
limiting dilution. Stably transfected cells were cultured in
the presence of 750 lgofgenectinG-418sulfatepermLof
culture medium. Sodium butyrate (6 m
M
)wasaddedtothe
culture medium of stably transfected cells 16 h before
preparation of membrane fractions or analysis of cell
surface-located enzyme activity.
Preparation of membrane fractions

Transiently or stably transfected CHO cells were cultured in
150 · 25 mm culture dishes and membrane fractions were
prepared 48 h after electroporation or 16 h after stimulation
with sodium butyrate, respectively. After removal of culture
medium, cells were washed twice with buffer A (140 m
M
NaCl, 5 m
M
KCl, 20 m
M
Hepes, pH 8.5), scraped off with
5 mL of ice-cold buffer A containing protease inhibitors
(chymostatin, 2 lgÆmL
)1
;pepstatinA,1lgÆmL
)1
;benz-
amidine, 1 m
M
; antipain, 2 lgÆmL
)1
; aprotinin, 2 lgÆmL
)1
;
leupeptin, 2 lgÆmL
)1
), and centrifuged (300 g for 5 min at
4 °C). Cells were homogenized in buffer A using a Potter–
Elvehjem homogenizer and sonicated. The homogenate was
then centrifuged (10 min at 825 g at 4 °C). The resulting

supernatant fraction was centrifuged (100 000 g for 45 min
at 4 °C) and the pellets were resuspended in protease
inhibitor-containing buffer A prior to storage at )20 °C.
Protein was determined according to the method of
Peterson [43].
Measurement of nucleotide hydrolysis
The hydrolysis of substrates Ap
3
A, Ap
4
A, Ap
5
Aand
Gp
4
G was analyzed using membrane fractions containing
heterologously expressed NPP1 and NPP3, or the soluble
form of recombinant human NPP2. NPP2 was prepared
and isolated from a vaccinia virus lysate of BS-C-1 cells,
as described previously, through the concanavalin
A–agarose step [44], and stored in a solution containing
50 m
M
Tris, pH 7.5, 100 m
M
NaCl, 10 m
M
CaCl
2
,and

20% (v/v) ethylene glycol. Membrane fractions of mock-
transfected cells (NPP1, NPP3) or storage solution
(NPP2) served as controls. Hydrolysis was determined at
37 °C in buffer A (NPP1, NPP3) or buffer B (NPP2,
25 m
M
Tris/25 m
M
glycine, pH 9.0, 140 m
M
NaCl, 5 m
M
KCl, containing 500 lgÆmL
)1
bovine serum albumin).
Unless stated otherwise, CaCl
2
and MgCl
2
were added to
the assay medium at a concentration of 1 m
M
.The
reaction was started by addition of substrate and termin-
ated by heat inactivation (95 °C for 4 min). Nucleotides
were separated and quantified by HPLC. Samples were
first centrifuged (20 min, 14 500 g,4°C) and then diluted
with ultrapure water or buffer A. An aliquot was injected
into a Sepsil C
18

reversed-phase column (Jasco, Grob-
Umstadt, Germany) and eluted at 1.0 mLÆmin
)1
with the
mobile phase consisting of 50 m
M
potassium-phosphate
buffer (pH 6.4), 5 m
M
tetrabutylammonium hydrogen
sulfate and 10–18% methanol, depending on the nucleo-
tide analyzed. The absorbance at 260 nm was continu-
ously monitored and the nucleotide concentrations were
determined from the area under the absorbance peaks.
Hydrolysis rates represent initial rates.
2972 P. Vollmayer et al. (Eur. J. Biochem. 270) Ó FEBS 2003
pH dependence, ion dependence, and inhibitors
Two different buffer systems were applied for analyzing pH
dependence of Ap
3
A hydrolysis. For analysis from pH 6.0
to 7.5, a buffer solution containing 25 m
M
Hepes, 25 m
M
glycine, 140 m
M
NaCl, and 5 m
M
KClwasused.For

analysis from pH 7.5–11.0, the buffer solution consisted of
25 m
M
Tris, 25 m
M
glycine, 140 m
M
NaCl, and 5 m
M
KCl.
In these experiments the substrate concentration was
200 l
M
Ap
3
A for NPP1 and NPP3, and 2 m
M
for NPP2.
The rate of the Ap
3
A hydrolysis in the presence of divalent
metal cations was analyzed in buffer A (NPP1, NPP3) or
buffer B (NPP2) containing 2 m
M
CaCl
2
,2m
M
MgCl
2

,
2m
M
CaCl
2
and 2 m
M
MgCl
2
, or 500 l
M
EDTA. Because
the storage solution of NPP2 contained CaCl
2
,150l
M
EGTA was added to the solution containing 2 m
M
MgCl
2
.
The substrate concentration was 200 l
M
Ap
3
AforNPP1,
NPP2 and NPP3
1
. The inhibitory effect on Ap
4

Ahydrolysis
of the following compounds was analyzed: PPADS; Ciba-
cron Blue; suramin (all 100 l
M
); NaF (500 l
M
); heparin
(0.1 mgÆmL
)1
), and heparan sulfate (0.1 mgÆmL
)1
).
Enzymes were preincubated with the inhibitor for 10 min
at 37 °C followed by addition of substrate at a final
concentration of 200 l
M
.
Analysis of cell surface-located catalytic activity
For analysis of surface-located enzyme activity, CHO cells
transiently transfected with NPP1 were seeded, 24 h after
electroporation, in six-well plates (5 · 10
5
cells per well,
9.08 cm
2
) and analyzed after an additional 24 h. Stably
transfected CHO cells (NPP3) were seeded at a density of
2.5 · 10
5
cells per well and analyzed 2 days after seeding.

Cells were washed twice with phosphate-free saline solution
(140 m
M
NaCl, 5 m
M
KCl, 10 m
M
glucose, 20 m
M
Hepes,
pH 7.4) and subsequently incubated for 60 min at 37 °Cin
1 mL of identical saline solution containing 200 l
M
of the
substrate nucleotide, 1 m
M
MgCl
2
and 1 m
M
CaCl
2
.The
physiological saline solution collected from the cultures was
heat inactivated (95 °C for 4 min), followed by centrifuga-
tion (14 500 g for 20 min). Aliquots of the supernatant were
subjected to nucleotide analysis as described above.
Results
General biochemical properties of Ap
n

A hydrolysis
by NPPs
As members of the NPP family have been reported to share a
strongly alkaline pH optimum [18,29], we first determined
the pH dependence, using Ap
3
A as a substrate. Membrane
fractions containing heterologously expressed NPP1 and
NPP3 and soluble NPP2 revealed an alkaline pH optimum
for the hydrolysis of Ap
3
A. The pH optimum was between
8.5 and 9.0 for NPP1 and NPP3 and pH 10 for NPP2
(n ¼ 2–3). At the physiological pH of 7.4, the hydrolysis
rates for NPP1, NPP2, and NPP3 were approximately 10%,
15% and 30%, respectively, of maximal activity. In the
following we analyzed the catalytic activity of NPP1 and
NPP3 at pH 8.5 and that of NPP2 at pH 9. No diadenosine
polyphosphate hydrolysis was detected in isolated membrane
fractions or at the surface of mock-transfected CHO cells.
Hydrolysis of Ap
3
A by NPP1- and NPP3-containing
membrane fractions and by soluble NPP2 was determined
in the presence of 2 m
M
CaCl
2
,2m
M

MgCl
2
,2m
M
CaCl
2
plus 2 m
M
MgCl
2
2
,or500l
M
EDTA. As shown in
2
Fig. 1,
the catalytic rates were similar in the presence of Ca
2+
or Mg
2+
. Activity was not further stimulated by the
simultaneous addition of Ca
2+
and Mg
2+
, while EDTA
greatly reduced the catalytic activity. In the following,
catalytic activity was measured in the presence of 1 m
M
CaCl

2
and 1 m
M
MgCl
2
. Michaelis constant (K
m
)values
were calculated from Lineweaver–Burk plots for Ap
3
Aas
a substrate. Substrate concentrations tested ranged from
1 to 400 l
M
.TheK
m
and maximal rate (V
max
)values
corresponded, respectively, to 5.1 ± 3.6 l
M
and
25.8 ± 11.3 nmolÆmin
)1
Æmg protein
)1
(n ¼ 3) for NPP1,
49.5 ± 17.7 l
M
and 153.7 ± 41.6 nmolÆmin

)1
Æmg pro-
tein
)1
(n ¼ 4) for NPP3, and 8.0 ± 0.5 l
M
and
8.6 ± 0.8 nmolÆmin
)1
ÆmL
)1
(n ¼ 2) for NPP2.
Substrate specificity and product pattern
We subsequently analyzed the ability of NPP1 to NPP3
to hydrolyze various dinucleoside polyphosphates and
determined the pattern of product formation. In order to
avoid potential product inhibition, the enzyme concen-
tration was adjusted to obtain low catalysis rates. Table 1
compares the hydrolysis rates of the three enzymes for
the dinucleotides investigated. For each of the enzymes,
catalyticrateswerenormalizedtothesubstrateAp
4
A. In
the case of NPP1 and NPP3, substrates were added to
the surface of transfected intact and viable cells, or to
membrane fractions obtained from transfected cells.
Ap
3
A, Ap
4

AandAp
5
A were hydrolyzed by each of
the three enzymes at similar rates, and similar results
were obtained with viable cells and membrane fractions.
To investigate whether hydrolysis was restricted to
diadenosine polyphosphates, we also analyzed the
Fig. 1. Effect of divalent cations on the hydrolysis of diadenosine 5¢,5¢¢¢-
P
1
,P
3
-triphosphate (Ap
3
A) by NPP1, NPP2, and NPP3. Membrane
fractions derived from CHO cells containing heterologously expressed
NPP1 or NPP3, and soluble NPP2, were analyzed in the presence of
2m
M
CaCl
2
,2m
M
MgCl
2
,2m
M
CaCl
2
plus 2 m

M
MgCl
2
,or500l
M
EDTA. In the case of NPP2, 150 l
M
EGTA was added to the 2 m
M
MgCl
2
-containing solution to chelate CaCl
2
contained in the storage
solution. Substrate concentrations were 200 l
M
. The 100% values
correspond to 9.3 ± 1.0 nmolÆmin
)1
Æmg protein
)1
(NPP1), 13.7 ±
0.2 nmolÆmin
)1
ÆmL
)1
(NPP2), and 26.6 ± 11.0 nmolÆmin
)1
Æmg pro-
tein

)1
(NPP3). Values represent means ± SD of three experiments
with duplicate determinations in each (NPP1, NPP3) or mean values
± range of two experiments (NPP2).
Ó FEBS 2003 Hydrolysis of diadenosine polyphosphates (Eur. J. Biochem. 270) 2973
hydrolysis of diguanosine polyphosphates, choosing
Gp
4
G as an example. Gp
4
G was hydrolyzed by NPP1
and NPP2 at rates similar to Ap
4
A, but only at half this
rate by NPP3.
Ap
3
A, Ap
4
AandAp
5
A were asymmetrically cleaved
by all three members of the E-NPP family. Figure 2
shows representative HPLC profiles obtained with sol-
uble NPP2, or with NPP1 or NPP3 following substrate
application to the surface of transfected or mock-
transfected cells. Mock-controls revealed no significant
substrate hydrolysis. Ap
3
A was hydrolyzed to ADP and

AMP, Ap
4
AtoATPandAMP,andAp
5
AtoAp
4
and
AMP. Similarly, Gp
4
G was hydrolyzed to GTP and
GMP (results not shown).
Differential effect of inhibitors
We further investigated the possibility that the enzymes
differ regarding their sensitivity to potential inhibitors that
have previously been reported to affect hydrolysis of
diadenosine polyphosphates [18]. Ap
4
A(200l
M
)was
chosen as the substrate (Fig. 3). Cibacron Blue (100 l
M
)
strongly inhibited the hydrolysis of Ap
4
Abyallthree
enzymes. PPADS, at 100 l
M
, inhibited the hydrolysis of
Table 1. Substrate preference of NPP1, NPP2 and NPP3. Activity was determined at the surface of viable and transfected CHO cells or using

membrane fractions derived from transfected CHO cells (NPP1 and NPP3), or using soluble NPP2. Experiments were performed in the presence of
1m
M
CaCl
2
and 1 m
M
MgCl
2
. Substrate concentrations were 200 l
M
. Values are expressed relative to Ap
4
A hydrolysis. Absolute hydrolysis rates
for Ap
4
A were 539 ± 278 pmolÆmin
)1
Æ10
6
cells and 16.8 ± 12.1 nmolÆmin
)1
Æmg protein
)1
(NPP1, surface activity and activity of membrane
fractions, respectively), 781.7 ± 193 pmolÆmin
)1
Æ10
6
cells and 33.5 ± 10.6 nmolÆmin

)1
Æmg protein
)1
(NPP3, surface activity and activity of
membrane fractions, respectively) and 15.6 ± 0.3 nmolÆmin
)1
ÆmL
)1
(NPP2). Values represent means ± SD of three experiments with duplicate
determinations in each (NPP1, NPP3) or mean values ± range of two experiments (NPP2). ND, not determined.
Substrate
NPP1 (relative rate) NPP2 (relative rate) NPP3 (relative rate)
Cell surface Membrane fraction Soluble Cell surface Membrane fraction
Ap
4
A1 1 1 1 1
Ap
3
A 1.21 ± 0.03 1.06 ± 0.05 1.02 ± 0.02 1.19 ± 0.03 1.31 ± 0.08
Ap
5
A 0.98 ± 0.07 1.20 ± 0.13 1.07 ± 0.01 1.05 ± 0.05 1.54 ± 0.17
Gp
4
G ND 1.20 ± 0.20 1.14 ± 0.01 ND 0.45 ± 0.01
Fig. 2. Hydrolysis of diadenosine polyphosphates and product formation by heterologously expressed NPP1, NPP2 and NPP3. NPP1 and NPP3 were
analyzed after expression in CHO cells. The substrates Ap
3
A, Ap
4

AandAp
5
A were applied for 60 min to the surface of either mock-transfected
cells (control) or to the surface of transfected cells. In the case of NPP2, either storage buffer alone (control) or storage buffer containing the soluble
enzyme were analyzed (120 min). Experiments were performed in the presence of substrate (200 l
M
), 1 m
M
CaCl
2
and 1 m
M
MgCl
2
. Substrate
hydrolysis and product formation were determined by HPLC. Absorbance was measured at 260 nm. AU, arbitrary units.
2974 P. Vollmayer et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Ap
4
A by NPP1 and NPP3 by 82–88%, but was much less
effective on NPP2 (42% inhibition). Whereas NPP1 and
NPP2 were inhibited by suramin (100 l
M
) by about 70%
and 77%, respectively, NPP3 was not affected. Heparin and
heparan sulfate inhibited NPP1 by 22%. No inhibition was
obtained with NaF (500 l
M
).
Discussion

A direct comparison of all three heterologously expressed
members of the E-NPP family revealed that they are capable
of hydrolyzing the diadenosine polyphosphates Ap
3
Ato
Ap
5
A. This was true both for the membrane-bound enzymes
NPP1 and NPP3 and for the soluble NPP2. In addition, all
three enzymes hydrolyzed Gp
4
G. This suggests that they
are generally capable of hydrolyzing physiologically released
dinucleoside polyphosphates, including the vasoactive
adenosine polyphosphoguanosines and diguanosine poly-
phosphates [10]. Hydrolysis was asymmetric and involved
the a,b-pyrophosphate bond, resulting in the production of
AMP and Ap
n-1
as the primary hydrolysis products.
Hydrolysis of Ap
n
As had a very alkaline pH optimum,
comparable to that of alkaline phosphatases [18]. Dinucleo-
tide hydrolysis was inhibited by the divalent metal cation
chelator, EDTA, as previously described for other substrates
of the enzymes [29,30]. K
m
values for NPP1, NPP2 and
NPP3 were in the low micromolar range for Ap

3
Aasa
substrate. NPP2 (autotaxin) appears to be the most versatile
member of the enzyme family. Two recent publications
reported that it also exerts lysophospholipase D activity
[45,46]. The K
m
value for lysophosphatidylcholine was
found to be  250 l
M
. This is 30 times higher than that of
NPP2 for Ap
3
A (present data). In contrast to NPP2, NPP1
and NPP3 do not display phospholipase D activity [47].
The general catalytic properties of the heterologously
expressed E-NPPs compare well with previous data
describing the hydrolysis of diadenosine polyphosphates in
the blood or in association with the surface of cells [18],
suggesting that members of the E-NPP family represent a
major group of Ap
n
A-hydrolyzing enzymes in mammalian
tissues. From the data available it is, however, difficult to
conclude which of the isoenzymes has previously been
characterized. The low K
m
value (8 l
M
,Ap

4
A) dinucleoside
tetraphosphatase isolated from rat liver [48] shares its
general functional properties with members of the E-NPP
family. As NPP1, NPP2, and NPP3 are all expressed in liver
[49] it is difficult to assign the functional properties of the
enzyme fraction identified to a specific member of the
E-NPP family. Ap
3
AandAp
4
A are hydrolyzed asymmet-
rically by human plasma, and an enzyme chromatographing
at 230 kDa, with properties very similar to those of NPP1,
has been purified from plasma [50]. In human serum, three
isoenzymes splitting Ap
4
AandAp
3
A have been identified,
with K
m
values for Ap
4
A in the range of 2–10 l
M
[51]. These
isoenzymes may relate to the isoforms of the E-NPP family
present in serum [18]. In contrast to ATP, Ap
n

As are not
hydrolyzed by intact blood cells [19].
Cultured bovine chromaffin cells degrade members of the
diadenosine polyphosphate family from Ap
3
AtoAp
6
A
[2,20]. The pH optimum is in the alkaline range (8.5–9.0),
and K
m
values are in the order of 2–4 l
M
. A partially
purified enzyme isolated from bovine adrenal medullary
plasma membranes co-chromatographed with a protein
immunoreactive for an anti-NPP1 immunoglobulin [52],
suggesting that NPP1 is mainly responsible for diadenosine
polyphosphate hydrolysis in these cells. The K
m
value for
Ap
4
A was determined as 2 l
M
and relates closely to the
value obtained for Ap
3
A and recombinant NPP1 in this
study. A partially purified alkaline phosphodiesterase from

rat C6 glioma cells was found to hydrolyze Ap
4
A, Gp
4
G
and NAD
+
, compatible with the catalytic properties of
NPP1 [53]. Human airway epithelia contain mRNA enco-
ding all three forms of NPPs [54]. Diadenosine polyphos-
phates applied to the apical epithelial surface were
hydrolyzed, as would be expected for NPPs, but catalytic
activities cannot be assigned to an individual enzyme.
Not all Ap
n
A-hydrolyzing enzymes described share the
catalytic properties of NPPs. Whereas the diadenosine
polyphosphate hydrolase activity in the blood [50], in
synaptic membranes from the Torpedo electric organ, or on
chromaffin cells are activated by Ca
2+
ions, the enzyme on
cultured endothelial cells is inhibited by Ca
2+
[21,22]. The
enzyme from adrenomedullary vascular endothelial cells
also has a considerably lower K
m
for Ap
n

As (0.4 l
M
)than
the NPPs and – in contrast to NPPs – is effectively inhibited
by fluoride [22]. This implies the occurrence of Ap
n
A
hydrolases unrelated to the presently identified members of
the E-NPP family. Also unexplained is the release of Ap
n
A-
hydrolyzing enzyme activity by phosphatidyl inositol-speci-
fic phospholipase C, suggesting membrane anchorage via a
glycosylphosphatidyl inositol-anchor [20,55–57].
Hydrolysis of Ap
n
As by the three expressed NPPs can be
affected differentially by inhibitors. The non-specific P2
receptor inhibitor, Cibacron Blue, inhibits all enzymes to a
similar extent. In contrast, PPADS, a frequently applied
inhibitor of P2 receptors [16], has a considerably stronger
inhibitory effect on NPP1 and NPP3 than on NPP2.
Suramin inhibits only NPP1 and NPP2. This suggests that
Fig. 3. Effect of inhibitors on hydrolysis of Ap
4
A by NPP1, NPP2, and
NPP3. PPADS, Cibacron Blue, suramin (all 100 l
M
), NaF (500 l
M

),
heparin, and heparan sulfate (both 0.1 mgÆmL
)1
) were applied at an
Ap
4
A concentration of 200 l
M
. The 100% values correspond to
64.6 ± 16.1 nmolÆmin
)1
Æmg protein
)1
(NPP1), 12.3 ± 0.3 nmolÆ
min
)1
ÆmL
)1
(NPP2), and 109.0 ± 24.4 nmolÆmin
)1
Æmg protein
)1
(NPP3). Values represent means ± SD of three experiments with
triplicatedeterminationsineach(NPP1)ormeanvalues±rangeof
two experiments with triplicate determinations in each (NPP2, NPP3).
Ó FEBS 2003 Hydrolysis of diadenosine polyphosphates (Eur. J. Biochem. 270) 2975
differential inhibition by PPADS and suramin can be used
to assign Ap
n
A hydrolysis in individual tissues to defined

members of the E-NPP family. It should also be noted that
application of these P2 receptor inhibitors will potentially
obscure pharmacological experiments in which the effect of
Ap
n
As on P2 receptors are investigated – because Ap
n
A
hydrolysis and P2 receptors are simultaneously blocked.
PPADS and, in particular, suramin, are effective inhibitors
of ecto-Ap
n
AhydrolasefromTorpedo synaptic membranes
[58], suggesting that this enzyme is related to NPP1.
Submilligram quantities of heparin or heparan sulfate
inhibit strongly the hydrolysis of pNP-TMP and, to a lesser
extent, ATP hydrolysis by NPP1 endogenously expressed
by Jurkat T cells [59]. We found only a small effect on Ap
4
A
hydrolysis by heterologously expressed NPP1, suggesting
that the hydrolysis of individual substrates may be differ-
entially affected by the glycosaminoglycans.
Taken together, all three functionally characterized
members of the E-NPP family are capable of hydrolyzing
dinucleoside polyphosphates. They comprise a versatile
group of enzymes with a broad substrate specificity. In this
sense they resemble intracellular dinucleoside polyphos-
phate-hydrolyzing enzymes (Nudix hydrolases), unrelated
to members of the E-NPP family, that reveal a comparably

broad substrate specificity, including dinucleoside poly-
phosphates, nucleoside triphosphates, nucleotide sugars,
NADH and coenzyme A [60]. Our results suggest that
NPP1, NPP2, and NPP3 are major enzyme candidates for
the hydrolysis of extracellular dinucleoside polyphosphates
released in vertebrate tissues.
Acknowledgements
This work was supported by grants from the Deutsche Forschungs-
gemeinschaft (SFB 269, A4), from the Foerderfonds der Chemischen
Industrie (to H. Zimmermann) and by the National Health and
Medical Research Council of Australia (to J. W. Goding).
References
1. McLennan, A.G. (1992) Ap
4
A and Other Dinucleoside Polyphos-
phates. CRC Press, Boca Raton.
2. Miras-Portugal, M.T., Gualix, J., Mateo, J., Dı
´
az-Herna
´
ndez, M.,
Go
´
mez-Villafuertes, R., Castro, E. & Pintor, J. (1999) Diadeno-
sine polyphosphates, extracellular function and catabolism. Prog.
Brain Res. 120, 397–409.
3. Hoyle, C.H.V., Hilderman, R.H., Pintor, J.J., Schlu
¨
ter, H. &
King, B.F. (2001) Diadenosine polyphosphates as extracellular

signal molecules. Drug Dev. Res. 52, 260–273.
4. Oaknin, S., Rodrı
´
guez-Ferrer,C.R.,Aguilar,J.S.,Ramos,A.&
Rotlla
´
n, P. (2001) Receptor binding properties of di (1,N
6
-
ethenoadenosine) 5¢,5¢¢¢ -P
1
,P
4
-tetraphosphate and its modulatory
effect on extracellular glutamate levels in rat striatum. Neurosci.
Lett. 309, 177–180.
5. Zamecnik, P.C., Kim, B., Gao, M.J., Taylor, G. & Blackburn,
G.M. (1992) Analogues of diadenosine 5¢,5¢¢¢-P
1
,P
4
-tetraphos-
phate (Ap
4
A) as potential anti-platelet-aggregation agents. Proc.
NatlAcad.Sci.USA89, 2370–2373.
6. Hilderman, R.H. & Christensen, E.F. (1998) P
1
,P
4

-diadenosine 5¢-
tetraphosphate induces nitric oxide release from bovine aortic
endothelial cells. FEBS Lett. 427, 320–324.
7. Steinmetz,M.,Janssen,A.K.,Pelster,F.,Rahn,K.H.&Schlatter,
E. (2002) Vasoactivity of diadenosine polyphosphates in human
small mesenteric resistance arteries. J. Pharmacol. Exp. Ther. 302,
787–794.
8. Van der Giet, M., Schmidt, S., Tolle, M., Jankowski, J., Schlu
¨
ter,
H., Zidek, W. & Tepel, M. (2002) Effects of dinucleoside poly-
phosphates on regulation of coronary vascular tone. Eur. J.
Pharmacol. 448, 207–213.
9. Gabriels, G., Rahn, K.H., Schlatter, E. & Steinmetz, M. (2002)
Mesenteric and renal vascular effects of diadenosine polyphos-
phates (Ap
n
A). Cardiovasc. Res. 56, 22–32.
10. Ralevic, V., Jankowski, J. & Schlu
¨
ter, H. (2001) Structure–activity
relationships of diadenosine polyphosphates (Ap
n
As), adenosine
polyphospho guanosines (Ap
n
Gs) and guanosine polyphospho
guanosines (Gp
n
Gs) at P2 receptors in the rat mesenteric arterial

bed. Br.J.Pharmacol.134, 1073–1083.
11. Rodriguez del Castillo, A., Torres, M., Delicado, E.G. &
Miras-Portugal, M.T. (1988) Subcellular distribution studies of
diadenosine polyphosphates Ap
4
AandAp
5
A in bovine adrenal
medulla: presence in chromaffin granules. J. Neurochem. 51,
1696–1703.
12. Zimmermann, H. (1994) Signalling via ATP in the nervous system.
Trends Neurosci. 17, 420–426.
13. Pintor, J., Kowalewski, H.J., Torres, M., Miras-Portugal, M.T. &
Zimmermann, H. (1992) Synaptic vesicle storage of diadenosine
polyphosphates in the Torpedo electric organ. Neurosci. Res.
Commun. 10, 9–15.
14. Schlu
¨
ter, H., Groß, I., Bachmann, J., Kaufmann, R., van der Giet,
M.,Tepel,M.,Nofer,J.R.,Assmann,G.,Karas,M.,Jankowski,
J. & Zidek, W. (1998) Adenosine (5¢) oligophospho-(5¢)guano-
sines and guanosine (5¢) oligophospho-(5¢) guanosines in human
platelets. J. Clin. Invest. 101, 682–688.
15. Jankowski, J., Tepel, M., van der Giet, M., Tente, I.M., Henning,
L.,Junker,R.,Zidek,W.&Schlu
¨
ter, H. (1999) Identification and
characterization of P
1
,P

7
-di (adenosine-5)-heptaphosphate from
human platelets. J. Biol. Chem. 274, 23926–23931.
16. Ralevic, V. & Burnstock, G. (1998) Receptors for purines and
pyrimidines. Pharmacol. Rev. 50, 413–492.
17. Dı
´
az-Herna
´
ndez,M.,Pintor,J.,Castro,E.&Miras-Portugal,
M.T. (2001) Independent receptors for diadenosine pentaphos-
phate and ATP in rat midbrain single synaptic terminals. Eur. J.
Neurosci. 14, 918–926.
18. Zimmermann, H. (2001) Ecto-nucleotidases. In Handbook of
Experimental Pharmacology. Purinergic and Pyrimidergic Signal-
ling. (Abbracchio, M.P. & Williams, M., eds), pp. 209–250.
Springer Verlag, Heidelberg.
19. Lu
¨
thje, J. & Ogilvie, A. (1988) Catabolism of Ap
4
AandAp
3
Ain
whole blood. The dinucleotides are long-lived signal molecules in
the blood ending up as intracellular ATP in the erythrocytes. Eur.
J. Biochem. 173, 241–245.
20. Ramos, A., Pintor, J., Miras-Portugal, M.T. & Rotlla
´
n, P. (1995)

Use of fluorogenic substrates for detection and investigation
of ectoenzymatic hydrolysis of diadenosine polyphosphates: a
fluorometric study on chromaffin cells. Anal. Biochem. 228,
74–82.
21. Ogilvie, A., Lu
¨
thje,J.,Pohl,U.&Busse,R.(1989)Identification
and partial charactertization of an adenosine (5¢) tetraphospho (5¢)
adenosine hydrolase on intact bovine aortic endothelial cells.
Biochem. J. 259, 97–103.
22. Mateo, J., Miras-Portugal, M.T. & Rotlla
´
n, P. (1997) Ecto-enzy-
matic hydrolysis of diadenosine polyphosphates by cultured
adrenomedullary vascular endothelial cells. Am.J.Physiol.42,
C918–C927.
23. Mateo, J., Rotlla
´
n, P., Martı
´
, E., De Aranda, I.G., Salsona, C. &
Miras-Portugal, M.T. (1997) Diadenosine polyphosphate hydro-
lase from presynaptic plasma membranes of Torpedo electric
organ. Biochem. J. 323, 677–684.
2976 P. Vollmayer et al. (Eur. J. Biochem. 270) Ó FEBS 2003
24. Emanuelli, T., Bonan, C.D., Sarkis, J.J.F. & Battastini, A.M.O.
(1998) Catabolism of Ap
4
AandAp
5

A by rat brain synaptosomes.
Braz.J.Med.Biol.Res.31, 1529–1532.
25. Picher, M. & Boucher, R.C. (2000) Biochemical evidence for an
ecto-alkaline phosphodiesterase I in human airways. Am. J.
Respir. Cell. Mol. Biol. 23, 255–261.
26. Aguilar, J.S., Reyes, R., Asensio, A.C., Oaknin, S., Rotlla
´
n, P. &
Miledi, R. (2001) Ectoenzymatic breakdown of diadenosine
polyphosphates by Xenopus laevis oocytes. Eur. J. Biochem. 268,
1289–1297.
27. Zimmermann, H., Beaudoin, A.R., Bollen, M., Goding, J.W.,
Guidotti, G., Kirley, T.L., Robson, S.C. & Sano, K. (2000) Pro-
posed nomenclature for two novel nucleotide hydrolyzing enzyme
families expressed on the cell surface. In Ecto-ATPases and
Related Ectonucleotidases (Van Duffel, L. & Lemmens, R., eds),
pp. 1–8. Shaker Publishing BV, Maastricht.
28. Zimmermann, H. (2001) Ectonucleotidases: some recent devel-
opments and a note on nomenclature. Drug Dev. Res. 52, 44–56.
29. Bollen,M.,Gijsbers,R.,Ceulemans,H.,Stalmans,W.&Stefan,
C. (2000) Nucleotide pyrophosphatases/phosphodiesterases on the
move. Crit.Rev.Biochem.Mol.Biol.35, 393–432.
30. Goding, J.W. (2000) Ecto-enzymes: physiology meets pathology.
J. Leukoc. Biol. 67, 285–311.
31. Zimmermann, H. (2000) Extracellular metabolism of ATP and
other nucleotides. Naunyn Schmiedeberg’s Arch. Pharmacol. 362,
299–309.
32. Rebbe, N.F., Tong, B.D., Finley, E.M. & Hickman, S. (1991)
Identification of nucleotide pyrophosphatase/alkaline phospho-
diesterase I activity associated with the mouse plasma cell

differentiation antigen PC-1. Proc. Natl Acad. Sci. USA 88,
5192–5196.
33. Murata,J.,Lee,H.J.,Clair,T.,Krutzsch,H.C.,Arestad,A.A.,
Sobel, M.E., Liotta, L.A. & Stracke, M.L. (1994) cDNA cloning
of the human motility-stimulating protein, autotaxin, reveals a
homology with phosphodiesterases. J. Biol. Chem. 269,
30479–30484.
34.Kawagoe,H.,Soma,O.,Goji,J.,Nishimura,N.,Narita,M.,
Inazawa, J., Nakamura, H. & Sano, K. (1995) Molecular cloning
and chromosomal assignment of the human brain-type phos-
phodiesterase I/nucleotide pyrophosphatase gene (PDNP2).
Genomics 30, 380–384.
35. Deissler, H., Lottspeich, F. & Rajewsky, M.F. (1995) Affinity
purification and cDNA cloning of rat neural differentiation and
tumor cell surface antigen gp130
RB13)6
reveals relationship to
human and murine PC-1. J. Biol. Chem. 270, 9849–9855.
36. Jin-Hua, P., Goding, J.W., Nakamura, H. & Sano, K. (1997)
Molecular cloning and chromosomal localization of PD-Ib
(PDNP3), a new member of the human phosphodiesterase I genes.
Genomics 45, 412–415.
37. Scott, L.J., Delautier, D., Meerson, N.R., Trugnan, G., Goding,
J.W. & Maurice, M. (1997) Biochemical and molecular identifi-
cation of distinct forms of alkaline phosphodiesterase I expressed
on the apical and basolateral plasma membrane surfaces of rat
hepatocytes. Hepatology 25, 995–1002.
38. Stracke, M.L., Krutzsch, H.C., Unsworth, E.J., Arestad, A.A.,
Cioce,V.,Schiffmann,E.&Liotta,L.A.(1992)Identification,
purification, and partial sequence analysis of autotaxin, a novel

motility-stimulating protein. J. Biol. Chem. 267, 2524–2529.
39. Belli, S.I., van Driel, I.R. & Goding, J.W. (1993) Identification and
characterization of a soluble form of the plasma cell membrane
glycoprotein PC-1 (5¢-nucleotide phosphodiesterase). Eur. J. Bio-
chem. 217, 421–428.
40. Meerson, N.R., Delautier, D., Durand-Schneider, A. M., Moreau,
A., Schilsky, M.L., Sternlieb, I., Feldmann, G. & Maurice, M.
(1997) Identification of B10, an alkaline phosphodiesterase of the
apical plasma membrane of hepatocytes and biliary cells, in rat
serum: increased levels following bile duct ligation and during the
development of cholangiocarcinoma. Hepatology 27, 563–568.
41. Kegel, B., Braun, N., Heine, P., Maliszewski, C.R. & Zimmer-
mann, H. (1997) An ecto-ATPase and an ecto-ATP diphos-
phohydrolase are expressed in rat brain. Neuropharmacology 36,
1189–1200.
42. Belli, S.I. & Goding, J.W. (1994) Biochemical characterization of
human PC-1, an enzyme possessing alkaline phosphodiesterase I
and nucleotide pyrophosphate activities. Eur. J. Biochem. 226,
433–443.
43. Peterson, G.L. (1977) A simplification of the protein assay method
of Lowry et al. which is more generally applicable. Anal. Biochem.
83, 346–356.
44. Clair,T.,Lee,H.Y.,Liotta,L.A.&Stracke,M.L.(1997)Auto-
taxin is an exoenzyme possessing 5¢-nucleotide phosphodiesterase/
ATP pyrophosphatase and ATPase activities. J. Biol. Chem. 272,
996–1001.
45. Umezu-Goto, M., Kishi, Y., Taira, A., Hama, K., Dohmae, N.,
Takio, K., Yamori, T., Mills, G.B., Inoue, K., Aoki, J. & Arai, H.
(2002) Autotaxin has lysophospholipase D activity leading to
tumor cell growth and motility by lysophosphatidic acid produc-

tion. J. Cell Biol. 158, 227–233.
46. Tokumura, A., Majima, E., Kariya, Y., Tominaga, K., Kogure,
K., Yasuda, K. & Fukuzawa, K. (2002) Identification of human
plasma lysophospholipase D, a lysophosphatidic acid-producing
enzyme, as autotaxin, a multifunctional phosphodiesterase. J. Biol.
Chem. 277, 39436–39442.
47. Gijsbers, R., Junken, A., Arai, H. & Bollen, M. (2003) The
hydrolysis of lysophospholipids and nucleotides by autotaxin
(NPP2) involves a single catalytic site. FEBS Lett. 538, 60–64.
48. Cameselle, J.C., Costas, M.J., Sillero, M.A.G. & Sillero, A. (1984)
Two low K
m
hydrolytic activities on dinucleoside 5¢,5¢¢¢-P
1
,P
4
-
tetraphosphates in rat liver. Characterization as the specific
dinucleoside tetraphosphatase and a phosphodiesterase I-like
enzyme. J. Biol. Chem. 259, 2879–2885.
49. Stefan, C., Gijsbers, R., Stalmans, W. & Bollen, M. (1999) Dif-
ferential regulation of the expression of nucleotide pyrophos-
phatases/phosphodiesterases in rat liver. Biochim. Biophys. Acta
1450, 45–52.
50. Lu
¨
thje, J. & Ogilvie, A. (1985) Catabolism of Ap
3
AandAp
4

Ain
human plasma. Purification and characterization of a glycoprotein
complex with 5¢-nucleotide phosphodiesterase activity. Eur. J.
Biochem. 149, 119–127.
51. Lu
¨
thje, J. & Ogilvie, A. (1987) Catabolism of Ap
4
AandAp
3
Ain
human serum. Identification of isoenzymes and their partial
characterization. Eur. J. Biochem. 169, 385–388.
52. Gasmi, L., Cartwright, J.L. & McLennan, A.G. (1998) The
hydrolytic activity of bovine adrenal medullary plasma mem-
branes towards diadenosine polyphosphates is due to alkaline
phosphodiesterase-I. Biochim. Biophys. Acta 1405, 121–127.
53. Grobben, B., Anciaux, K., Roymans, D., Stefan, C., Bollen, M.,
Esmans, E.L. & Slegers, H. (1999) An ecto-nucleotide pyro-
phosphatase is one of the main enzymes involved in the extra-
cellular metabolism of ATP in rat C6 glioma. J. Neurochem. 72,
826–834.
54. Picher, M. & Boucher, R.C. (2001) Metabolism of extracellular
nucleotides in human airways by a multienzyme system. Drug Dev.
Res. 52, 66–75.
55. Rodriguez-Pascual, F., Torres, M., Rotlla
´
n, P. & Miras-Portugal,
M.T. (1992) Extracellular hydrolysis of diadenosine polyphos-
phates, Ap

n
A, by bovine chromaffin cells in culture. Arch. Bio-
chem. Biophys. 297, 176–183.
56. Itami, C., Taguchi, R., Ikezawa, H. & Nakabayashi, T. (1997)
Release of ectoenzymes from small intestine brush border mem-
branes of mice by phospholipases. Biosci. Biotechnol. Biochem. 61,
336–340.
Ó FEBS 2003 Hydrolysis of diadenosine polyphosphates (Eur. J. Biochem. 270) 2977
57. Minelli, A., Allegrucci, C., Liguori, L. & Ronquist, G. (2002)
Ecto-diadenosine polyphosphate hydrolase activity on human
prostasomes. Prostate 51, 1–9.
58. Mateo, J., Rotlla
´
n, P. & Miras-Portugal, M.T. (1996) Suramin – a
powerful inhibitor of neural ecto-diadenosine polyphosphate
hydrolase. Br. J. Pharmacol. 119,1–2.
59.Hosoda,N.,Hoshino,S.,Kanda,Y.&Katada,T.(1999)
Inhibition of phosphodiesterase/pyrophosphatase activity of PC-1
by its association with glycosaminoglycans. Eur. J. Biochem. 265,
763–770.
60. Fisher, D.I., Safrany, S.T., McLennan, A.G. & Cartwright, J.L.
(2002) Nudix hydrolases that degrade dinucleoside and diphos-
phoinositol polyphosphates also have 5-phosphoribosyl 1-pyro-
phosphate (PRPP) pyrophosphatase activity that generates the
glycolytic activator ribose 1,5-bisphosphate. J. Biol. Chem. 277,
47313–47317.
2978 P. Vollmayer et al. (Eur. J. Biochem. 270) Ó FEBS 2003