Inorganic pyrophosphatase in the roundworm
Ascaris
and its role in the development and molting process
of the larval stage parasites
M. Khyrul Islam
1
, Takeharu Miyoshi
1
, Harue Kasuga-Aoki
1
, Takashi Isobe
1
, Takeshi Arakawa
2
,
Yasunobu Matsumoto
3
and Naotoshi Tsuji
1
1
Laboratory of Parasitic Diseases, National Institute of Animal Health, National Agricultural Research Organization, 3-1-5,
Kannondai, Tsukuba, Ibaraki, Japan;
2
Division of Molecular Microbiology, Center of Molecular Biosciences, University of the
Ryukyus, Senbaru, Okinawa, Japan;
3
Laboratory of Global Animal Resource Science, Graduate School of Agricultural and
Life Sciences, University of Tokyo, Yayoi, Bunkyo, Tokyo, Japan
Inorganic pyrophosphatase (PPase) is an important
enzyme that catalyzes the hydrolysis of inorganic pyro-
phosphate (PP

Native PPases were expressed in all developmental stages of
A. suum. A homolog was also detected in the most closely
related human and dog roundworms A. lumbricoides and
Toxocara canis, respectively. The enzyme was intensely
localized in the body wall, gut epithelium, ovary and uterus
of adult female worms. We observed that native PPase
activity together with development and molting in vitro of
A. suum L3 to L4 were efficiently inhibited in a dose-
dependent manner by imidodiphosphate and sodium
fluoride, which are potent inhibitor of both soluble- and
membrane-bound H
+
-PPases. The studies provide evi-
dence that the PPases are novel enzymes in the roundworm
Ascaris, and may have crucial role in the development and
molting process.
Keywords: roundworm; inorganic pyrophosphatase;
sodium fluoride; imidodiphosphate; molting.
Geohelminth parasites are among the commonest and
widespread of human infections, particularly in the regions
where public health hygiene and nutritional status are
poorly maintained. The most prevalent geohelminth is
Ascaris lumbricoides (originally described by Linnaeus in
1758), which colonizes the small intestine of children, and is
estimated to infect a quarter of the world’s population [1].
Ascaris suum (originally described by Goeze in 1782) of pigs
is a very closely related species to A. lumbricoides,whichcan
develop in human hosts, indicating its zoonotic significance
[2,3]. Childhood infections with Ascaris worms are reported
to be associated with stunting growth, malabsorption,

PPases have been further divided into three subfamilies,
Correspondence to N. Tsuji, Laboratory of Parasitic Diseases,
National Institute of Animal Health, National Agricultural Research
Organization, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan.
Fax: + 81 29 8387749, Tel.: + 81 29 8387749,
E-mail: tsujin@affrc.go.jp
Abbreviations: PPase, inorganic pyrophosphatase; H
+
-PPase, proton-
translocating pyrophosphatase; AsPPase, Ascaris suum inorganic
pyrophosphatase; rAsPPase, recombinant A. suum inorganic pyro-
phosphatase; L3, third-stage infective larvae; L4, fourth-stage larvae;
ES, excretory and secretory; IDP, imidodiphosphate.
Enzyme: Soluble inorganic pyrophosphatase (EC 3.6.1.1).
Note: The nucleotide sequences reported in this paper has been
submitted to the DDBJ/EMBL/GenBank with accession number
AB091401.
(Received 4 March 2003, revised 7 May 2003, accepted 9 May 2003)
Eur. J. Biochem. 270, 2814–2826 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03658.x
prokaryotic, plant and animal/fungal PPases. Among the
subfamilies, plant PPases bear a closer similarity to prok-
aryotic than to animal/fungal PPases [12]. The H
+
-PPases,
which appear to work as a reversible H
+
-pump, are much
larger and do not have any sequence similarity to either of
the two families of soluble PPases [15–17]. All known soluble
PPases are homologous proteins, whose active site residues

to be a potent inhibitor of soluble PPases [23].
While PPases from diversified sources have been des-
cribed in some detail, no PPase has ever been studied in any
metazoan helminth parasite including the roundworm
Ascaris. To address this, we describe here the cloning,
sequencing and heterologous expression in E. coli of a gene
encoding PPase of A. suum. The amino acid sequence of
A. suum PPase (AsPPase) indicates that it is an authentic
member of the Family I soluble PPases. We also provide
information concerning the kinetics and properties of the
enzyme. More strikingly, we show a novel role of the PPase
enzyme in the development and molting process of A. suum
larvae in vitro.
Materials and methods
Parasites
Adult A. suum were obtained from infected pigs at a
slaughterhouse in Shimotsuma, Japan. Adult A. lumbric-
oides and T. canis were obtained from patients after
treatment with piperazine in Bac Gian, Vietnam and, from
an infected dog in Miyazaki, Japan, respectively. Unem-
bryonated and embryonated eggs were obtained essentially
as described elsewhere [24]. Third-stage infective larvae (L3)
from embryonated eggs and lung-stage L3 were obtained as
described previously [25,26]. Excretory and secretory (ES)
products from L3, lung-stage L3 and adult worms were
collected as described previously [27]. Animal studies were
performed in accordance with the approval of the National
Institute of Animal Health Animal Care and Use Commit-
tee (Approval no. 23).
RNA was isolated from embryonated eggs using an

proteins was revealed by incubation of the filters with
alkaline phosphate-conjugated goat anti-rabbit IgG (ICN)
for 1 h and developed with 5-bromo-4-chloro-3-indolyl-
phosphate/nitroblue tetrazolium (Gibco/BRL). Clones that
were reactive with the antibody were plaque-purified by
repeated cycles of immune selection. Plaque-purified clones
were converted using ExAssist
TM
helper phages and SOLR
E. coli (Stratagene) according to the in vivo excision
protocol described in the Stratagene ZAP-cDNA Synthesis
Kit (Stratagene). The nucleotide sequences of the cDNAs
were determined by the Sanger dideoxy chain termination
method using a PRISM
TM
Ready Dye Terminator Cycle
Sequencing Kit (PerkinElmer). DNA samples were ana-
lyzed using an automated sequencer (373A DNA sequencer,
Applied Biosystems).
BLASTX
(NCBI, National Institute of
Health) searches were performed to obtain cDNA clones
coding low similarity against mammalian proteins stored at
the current database. The
GENETYX
-
WIN
TM
DNA Sequence
Analysis Software System (Software Inc) and the

proteins
A full length cDNA (lacking signal peptides) was amplified
by PCR as previously described [29]. A sense primer
Ó FEBS 2003 Roundworm pyrophosphatase (Eur. J. Biochem. 270) 2815
(5¢-CCGAGCTCGAGACGTGAAGCGACAATCTCGC
AATCT-3¢) containing an XhoI (Promega) site upstream of
the start codon and an antisense primer (5¢-CAGCCAA
GCTTCTCACTCTTTGATGAAATGCATCT-3¢) con-
taining a HindIII (Promega) site just downstream of amino
acid residue were used. The PCR fragments digested with
XhoIandHindIII were ligated into plasmid expression
vector pTrcHisB
TM
(Invitrogen), which had also been
digested with the same enzymes according to the manufac-
turer’s instructions. The resultant plasmid was transformed
into E. coli strain TOP10F¢ (Invitrogen). The transformed
cellsweregrowntoaD
600
at 37 °CinSOBmedium
supplemented with 50 lgÆmL
)1
ampicillin. To induce pro-
tein expression, isopropyl thio-b-
D
-galactoside was then
added to a final concentration of 1 m
M
and the culture was
grown for an additional 4 h at 37 °C. The E. coli cells were

pH 7.5 and a decreasing concentration of NaCl in a Slide-
A-Lyzer Dialysis Cassette (Pierce). Fractions were collected
and the presence and purity of recombinant protein was
detected by 10% SDS/PAGE [30] and immunoblot [31]
using anti-T7 tag Ig (Invitrogen). Protein concentrations
were measured with the Micro BCA protein assay reagent
(Pierce).
Production of mouse polyclonal antibodies
BALB/c mice were immunized first with a subcutaneous
injection of 50 lg of recombinant AsPPase (rAsPPase)
emulsified with TiterMax Gold
TM
(CytRx), followed by
another injection 2 weeks later in the same adjuvant. The
mice were bled 2 weeks after the second injection. The
antiserafromthemiceweremixedandstoredat)20 °C
until used. Anti-(mouse rAsPPase) IgG from immune sera
and mouse preimmune IgG were affinity purified using
UltraLink
TM
immobilized protein G according to manu-
facturer’s instructions (Pierce) and used for evaluating the
native AsPPase-neutralizing activity.
Two-dimensional electrophoresis
Parasite extracts were treated with an equal volume of urea
mixture consisting of 9
M
urea, 4% Nonidet P-40, 0.8%
ampholine (pH 3.5–10; Pharmacia) and 2% 2-mercapto-
ethanol, and then subjected to 2D PAGE. Nonequilibrium

2
in NaCl/P
i
containing 10% ethanol to inacti-
vate endogenous peroxidases. For immunolocalization, the
slides were blocked in NaCl/P
i
containing 10% (v/v) goat
serum (Wako) for 30 min at room temperature. They were
then flooded with anti-(mouse rAsPPase) Ig diluted 1 : 100
in NaCl/P
i
/E. coli lysate, overnight at 4 °C. Afterwards, the
slides were rinsed thoroughly with NaCl/P
i
,andthe
antibody binding was resolved with a peroxidase-labeled
anti-mouse IgG and the substrate 3¢,3¢-diaminobenzidine
tetrahydrochloride (Sigma Fast
TM
tablets, Sigma). After
color development, the slides were dehydrated in a graded
series of alcohol and cleared in xylene. The slides were then
covered with cover slips and observed with a microscope
(Axiophot; Carl Zeiss).
Enzyme assay
The rAsPPase activity was determined spectrophotometri-
cally by measuring the rate of liberation of P
i
from PP

H
2
SO
4
)and125lL of 1% ascorbic acid (in 0.05%
KHSO
4
) were added to the mixture, and incubated for
30 min at 37 °C. Protein concentrations and reaction times
were chosen in order to obtain the linearity of the reactions.
As positive and negative controls, pure yeast-soluble PPase
from Sigma (1-1643) and an unrelated A. suum 14-kDa
recombinant protein (As14; [28]) were used, respectively.
The concentrations of individual components were varied as
indicated for the determination of Mg
2+
and pH dependent
rAsPPase activity. The amount of P
i
liberated from the
hydrolysis of PP
i
during the course of the reaction was
measured in comparison to a standard P
i
sample using a
2816 M. K. Islam et al. (Eur. J. Biochem. 270) Ó FEBS 2003
spectrophotometer (Model 600, Shimadzu) at an optical
density of 700 nm. The specific activity of rAsPPase was
defined as lmol P

20 m
M
Tris/HCl, pH 7.5) and the L3 ES products in the
culture fluids (dialyzed against 20 m
M
Tris/HCl, pH 7.5),
were assayed in the standard reaction mixture as described
above. Anti-(mouse rAsPPase) IgG were evaluated for
AsPPase-neutralizing activity. Recombinant AsPPase pro-
teins or A. suum L3 extracts were preincubated in the
standard reaction mixture containing 5 lgÆmL
)1
pre-
immune or anti-(mouse rAsPPase) IgG (15 min, 37 °C)
before adding PP
i
. The sensitivity of the native AsPPase to
inhibition by sodium fluoride (NaF, S-7920; Sigma) was
tested in the present study. The L3 extracts were assayed in
the standard reaction mixture for PP
i
hydrolysis, in the
presence of increasing concentrations of NaF. The PPase
activated PP
i
hydrolysis rate was then calculated.
Larval molting inhibition assay
To confirm whether the native PPase enzyme is involved in
the molting process, we examined the effects of two PPase
specific inhibitors, imidodiphosphate (IDP, 1-0631; Sigma)

penicillin/streptomycin were
cultured in 24-well flat-bottomed tissue culture plates
(CostarÒ). The cultures were incubated at 37 °Cina
humidified 5% CO
2
incubator in the absence (control) and
presence of increasing concentrations of inhibitors for
10 days, and the number of molting larvae was determined.
Molting was manifested by shedding of the L3 cuticle.
Numbers of molted larvae in a culture well were therefore
determined by counting the L3 cuticles shed from the larvae.
Furthermore, molted larvae (that had already shed their
cuticles) exhibited an intense motility compared with
unmolted larvae (that had not shed their cuticle). Aliquots
of larvae were removed at different days of postcultures and
photographs were taken.
Results
Identification of cDNA encoding
A. suum
inorganic
pyrophosphatases
A clone designated AdR44 was isolated initially by immu-
noscreening an A. suum female worm cDNA library with
serum obtained from a rabbit immunized with A. suum
infective eggs. AdR44 was selected for further characteriza-
tion because of its sequence homology to the inorganic
pyrophosphatase family of proteins. Sequence analysis
showed that AdR44 was 1,375-bp long with an open
reading frame (ORF) coding for 360 amino acids. The ATG
initiation codon is predicted to be at nucleotides 79–81 and

protein but few are at both ends. Sequence analysis revealed
that all 13 functionally important active site residues
(AsPPase numbering: E-125, K-133, E-135, R-155, Y-170,
D-192, D-194, D-197, D-224, D-229, K-231, Y-269 and
K-270) (Fig. 1), which have been reported previously to be
evolutionarily well conserved in Family I soluble PPases
[12,13,18,34,35], are identical in AsPPase.
Phylogenetic analysis of available PPases
We have constructed a phylogenetic tree using Family I
soluble PPase sequences by the neighbor-joining method
Ó FEBS 2003 Roundworm pyrophosphatase (Eur. J. Biochem. 270) 2817
and the confidence of the branching order was verified by
making 1000 bootstrap replicates with the
CLUSTALW
program (Fig. 2). The neighbor-joined trees reveal that
animal and fungal PPases including AsPPase represent a
separate group from plant and prokaryotic PPases. Fur-
thermore, within the animal/fungal subgroup, AsPPase is
more closely clustered with PPases from the free-living
model nematode C. elegans and the insect D. melanogaster.
Characterization of recombinant AsPPase
The gene encoding the soluble PPase of A. suum was
amplified by PCR with A. suum female worm cDNA.
AsPPase was then overexpressed in E. coli strain TOP10F¢
using pTrcHisB
TM
vector, to test whether the clone indeed
has an inorganic pyrophosphatase activity. Recombinant
AsPPase was expressed in E. coli with a yield of 1 mgÆL
)1

that could be
abolished by Ca
2+
or removal of Mg
2+
(Table 1). This
activity could not be due to copurification of endogenous
E. coli PPase as, the rAsPPase contained a His-tag that was
used for purification and the recombinant protein was
determined to be pure by SDS/PAGE analysis.
Expression and immunohistochemical detection
of native AsPPase
We performed 2D immunoblot analysis to identify native
AsPPase in adult female A. suum. Anti-(mouse rAsPPase)
serum reacted strongly with a protein having a molecular
mass of 39 kDa with a pI of 7.1 (Fig. 3A) confirming that it
corresponded to the predicted size of the putative mature
protein (38.771 kDa) calculated from the AsPPase amino
acid sequence except for a signal peptide. In addition, a native
AsPPase was identified on silver-stained 2D gels on which
more than 200 visible protein spots appeared (data not
shown). To determine the N-terminal residues, we excised the
native AsPPase spots from 2D immunoblotted polyvinylid-
ene difluoride membranes and subjected them to analysis by
the automatic Edman degradation method. The sequence 1-
MALAASATIS-10 of native AsPPase was identical to that
of the putative mature protein. This confirmed that our clone
encoded a soluble PPase of A. suum. A spot reacting with the
anti-mouse rAsPPase was also seen in parasite extracts and
ES products from various developmental stages, including

(Michaelis Constant) value of 0.117 ± 0.006 m
M
for PP
i
from three independent experiments (Fig. 5A).
The K
m
value is significantly higher than the values of
Fig. 1. Sequence alignment of representative members of Family I sol-
uble PPases.
CLUSTALW
alignment of soluble PPases (GenBank
accession numbers are indicated in parentheses): A. suum (AB091401),
C. elegans (CAA93107), D. melanogaster (O77460), Bos taurus
(P37980), S. cerevisiae (2781300) and Schizosaccharomyces pombe
(P19117). Identical residues among PPases are marked with asterisks.
The 13 essential, active site residues that are conserved in all Family I
soluble PPase sequences currently available in the GenBank are further
emphasized by bold typeface. The signal peptides are underlined.
Dashes indicate gaps inserted to optimize the alignment. The num-
bering is for the sequence of A. suum (As) PPase. As, A. suum,Ce,
C. elegans,Dm,D. melanogaster,Bt,B. taurus,Sc,S. cerevisiae,Sp,
S. pombe.
2818 M. K. Islam et al. (Eur. J. Biochem. 270) Ó FEBS 2003
0.0009–0.00147 m
M
from bovine retinal PPase [23],
0.005 m
M
from rat liver PPase [36] and 0.026 m

(> 5 m
M
Mg
2+
) has not been investigated in the
present study. Although excess of Mg
2+
is known to inhibit
the PPases, however, the mechanism is yet unclear. Both
E. coli and yeast PPases have four (M1–M4) subsites for
binding metal ions for catalysis [38,39]. It has been urgued
that binding of Mg
2+
at three subsites is required for
Fig. 2. Phylogenetic tree based on alignment of available Family I soluble PPase sequences. The sequences shown are those from (GenBank accession
numbers are indicated in parentheses): A. suum (AB091401), S. cerevisiae (2781300), Kluyveromyces lactis (P13998), Pichia pastoris (O13505),
S. pombe (P19117), D. melanogaster (O77460), C. elegans (CAA93107), B. taurus (P37980), Homo sapiens,from([12]),S. cerevisiae mitochondria
(P28239), Hordeum vulgare (O23979), Zea mays (O48556), Solanum tuberosum (O43187), Arabidopsis thaliana (AAC33503), Oryza sativa
(AAC78101), Chlamydia pneumoniae (AAD19056), Chlamydia trachomatis (O84777), Mycoplasma pneumoniae (P75250), Mycoplasma genitalium
(P47593), Bacillus stearothermophilus (BAA19837), Synechocystis (PCC6803, P80507), Thermoplasma acidophilum (P37981), Methanobacterium
thermoautotrophicum (O26363), Thermococcus litoralis (P77992), Pyrococcus horikoshii (O59570), T. thermophilus (P38576), Mycobacterium leprae
(O69540), Mycobacterium tuberculosis (CAB08851), Haemophilus influenzae (1170585), Sulfolobus acidocaldarius (P50308), Aquifex aeolicus
(O67501), Helicobacter pylori (P56153), Gluconobacter suboxydans (O05545), Bartonella bacilliformis (P51064), Rickettsia prowazekii (CAA15034),
Legionella pneumophila (O34955) and E. coli (P17288). The bar indicates the numbers of substitutions per site. Unrooted neighbor-joining trees
were generated from homologies of soluble PPase sequences and the confidence of the branching order was verified by making 1000 bootstrap
replicates using the
CLUSTALW
program. The tree was viewed and converted to graphic format with
TREEVIEW
.

Æmg
)1
protein for PP
i
hydrolysis.
Anti-(mouse rAsPPase) IgG partially inhibited recombinant
AsPPase (6 ng) activity up to 22% in the presence of
5 lgÆmL
)1
anti-(mouse rAsPPase) IgG relative to AsPPase
activity determined in the presence of 5 lgÆmL
)1
mouse
preimmune IgG (data not shown). Also, native AsPPase
activity in L3 extracts was shown to be partially inhibited
(25%) by anti-(mouse rAsPPase) IgG (data not shown)
indicating that AsPPase is responsible for the hydrolyzing
activity of PP
i
in A. suum L3 extracts. NaF, an anion, is a
potent inhibitor of PPases and was able to inhibit native
AsPPase activity at micromolar concentrations in a dose-
dependent manner (Fig. 6A). This agent is also known to
inhibit the H
+
-PPases from plants [42], trypanosomatids
[43,44] and apicomplexan protozoa [45].
The L3 of A. suum develop and molt to L4 in the lungs of
their vertebrate hosts that can also occur during in vitro
cultivation. To determine whether this complex process is

NaF with
progressive damage of the body wall and intestine was seen
on day 5 postculture and onwards (data not shown). The
molted L3 developed well to L4 in control culture with
increased body length and width (data not shown), and
changes in the structure of the head and tail (Fig. 7A–C)
compared with unmolted L3 which achieved little or no
development, being inhibited by IDP/NaF (Fig. 7D,E).
Under light microscopy, it was however, not possible to
detect the formation and/or separation of new cuticles of
unmolted L3 exposed to inhibitors that might be carried out
by electron microscopy. The mean molting percentage in
control culture was recorded as, 52.59 ± 4.12.
Discussion
Although PPases are distributed widely among living cells,
most of the previous studies have focused on microbial and
plant enzymes, and very little is known about the enzyme
Fig. 3. Identification of A. suum native PPase in adult female worm.
(A) Fifty micrograms of female worm extract was separated by 2D
nonequilibrium pH-gradient gel electrophoresis, and the proteins were
then transferred to a nitrocellulose membrane. The native AsPPase
bound to the anti-(mouse rAsPPase) serum was found by alignment of
the stained gel and immunoblot membrane. (B) Expression of AsPPase
homologs in ascarid roundworms. Sixty or 80 mg of protein equiva-
lents of each parasite extract were electrophoresed on a 10% SDS/
PAGE and blotted onto a nitrocellulose membrane. The AsPPase
homologs bound to the anti-(mouse rAsPPase) serum were detected by
5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium. Lane 1,
A. lumbricoides;lane2,T. canis;lane3,A. suum.
Table 1. Recombinant A. suum PPase activity. Pyrophosphatase

Mg
2+
(positive control)
13232.36 ± 183.42
As14 + 5.0 m
M
Mg
2+
(negative control)
–
2820 M. K. Islam et al. (Eur. J. Biochem. 270) Ó FEBS 2003
from mammalian tissues. In contrast, we do not have any
evidence of PPases from parasitic helminths. We report here
the cloning, sequencing and biochemical and functional
characterization of a novel PPase from the important
pathogenic roundworm A. suum. The deduced amino acid
sequence of AsPPase shows significant similarity with
animal/fungal PPase sequences in Family I soluble PPases
(Fig. 1). All members of Family I soluble PPases currently
available in the database have been shown to contain 13
functionally important active site residues that are evolu-
tionarily well conserved, and were found to be identical in
AsPPase. Several highly conserved regions, the most
prominent of which is an eight residue long sequence
(224-DEGETDWK-231), are also seen in the AsPPase
sequence. It will be interesting to see the significance of this
highly conserved region in AsPPase structure and function-
ing. Over 37 Family I soluble PPases have been identified.
The prokaryotic PPases are hexamers of  20 kDa and
reported to contain 162–220 amino acid residues per

Æmin
)1
Æmg protein
)1
)byPP
i
hydrolysis assay
that was found to be closer to those of the highly purified
and crystallized E. coli (2000 lmol P
i
Æmin
)1
Æmg
)1
[50]),
yeast (655 lmol P
i
Æmin
)1
Æmg
)1
[51]), rat liver 600–
700 lmol P
i
Æmin
)1
Æmg
)1
[36]) and bovine retinal PPases
(> 8 8 5 lmol P

bovine retinal PPases [23,41] that demonstrated much lower
concentrations of Ca
2+
were needed for enzyme inhibition.
Prior studies have shown that free PP
i
is a potent inhibitor,
and free Mg
2+
activates the enzyme and binds with PP
i
to
form a true substrate, Mg
2+
PP
i
for soluble PPases [38,55].
Family II PPases are easily distinguishable from Family I
PPases in having a preference for Mn
2+
over Mg
2+
as the
activator, and are not inhibited by Ca
2+
,ratherCa
2+
Fig. 5. PP
i
(A), Mg

2+
. (C) pH dependency of the enzyme was examined as
described above using several buffers with increasing pH values. The
buffers used were (100 m
M
), sodium acetate (pH 5.0–5.5), Mops
(pH 6.0–6.5), Tris/HCl (pH 7.0–8.5) and glycine/NaOH (9.0–10.5).
Data represent mean ± SEM from three independent experiments.
Fig. 6. Inhibition of A. suum native PPase activity and A. suum L3
molting by IDP and NaF. (A) Aliquots of A. suum L3 soluble extracts,
17 mg proteinÆmL
)1
was run in the standard reaction mixture
for PPase assays at 55 °C, in the presence of increasing concentrations
of NaF. Percentage activity compared to the control in the absence
of NaF (100%). Control activities were 1.58 ± 0.01 lmol
P
i
Æmin
)1
Æmg protein
)1
for PP
i
hydrolysis. Data represent mean ±
SEM from three independent experiments. (B) Lung-stage A. suum L3
were cultured for molting inhibition assays, in the presence of
increasing concentrations of IDP and (C) NaF. Molting percentage
was determined on day 10. Percentage activities are relative to the
control in the absence of inhibitor (100%). Molting percentage of

ES products, respectively) strongly suggested the important
roles of the PPase enzyme throughout the developmental
cycle of Ascaris parasites. The results of neutralization
studies indicate that AsPPase-specific IgG may interfere
with the development and molting process of Ascaris larvae.
We showed that the native AsPPases are very sensitive to
inhibition by NaF in the micromolar range (Fig. 6A). This
value is much lower than previously reported data for NaF
against H
+
-PPases from parasitic protozoa [44]. These
results prompted us to investigate the role of PPase enzymes
in the development and molting process of A. suum larvae
and to test whether this could be targeted by inhibitors. We
used IDP, a nonhydrolyzable PP
i
analogue, and NaF, a well
known inhibitor of Family I and Family II soluble PPases
(NaF competes with the hydroxide ion for binding to Mg
2+
in the active site of the enzyme [35,53]), to block enzyme
activity. We demonstrated for the first time that NaF is
highly effective in inhibiting the development and molting of
A. suum L3 to L4, in a concentration-dependent manner,
whereas, IDP has shown only partial inhibitory effect
(Fig. 6B,C). However, a much higher concentration of NaF
(> 1 m
M
) is required to completely block development and
molting of L3 as against micromolar concentration is needed

Ó FEBS 2003 Roundworm pyrophosphatase (Eur. J. Biochem. 270) 2823
still poorly understood. Based on our results presented
above, it is assumed that the target molecules of IDP and
NaF might be PPase enzymes in the hypodermis of L3
(Fig. 4 indicates the abundance of native PPases in the
hypodermal cells of sectioned adult worms in immuno-
histochemical staining), and that the inhibition of PPase-
catalyzed PP
i
hydrolysis by these inhibitors, is likely to
prevent the synthesis of the new cuticle from the hypodermis
and/or ecdysis of the old cuticle. This assumption is
supported by the fact that PPase-activated PP
i
hydrolysis
is essential to maintain the forward direction of many
biosynthetic reactions like synthesis of DNA, RNA, proteins
and polypeptides [8]. In addition, several investigators have
demonstrated that both IDP and NaF selectively inhibited
the plant and protist H
+
-PPases [42–45] and other potential
PP
i
analogues (bisphosphonates) selectively inhibited the
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metabolic and defending enzymes of microbial origin and
can alter expression of protein essential for survival and
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