From the Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Calcutta 700 032, India
Received for publication, September 15, 2000, and in revised form, November 21, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Experiments from other laboratories conducted
with Leishmania donovani promastigote cells had earlier
indicated that the plasma membrane Mg2+-ATPase of the
parasite is an extrusion pump for H+. Taking advantage of
the pellicular microtubular structure of the plasma membrane of the
organism, we report procedures for obtaining sealed ghost and sealed
everted vesicle of defined polarity. Rapid influx of H+
into everted vesicles was found to be dependent on the simultaneous presence of ATP (1 mM) and Mg2+ (1 mM). Excellent correspondence between rate of
H+ entry and the enzyme activity clearly demonstrated the
Mg2+-ATPase to be a true H+ pump.
H+ entry into everted vesicle was strongly inhibited by
SCH28080 (IC50 = ~40 µM) and by omeprazole
(IC50 = ~50 µM), both of which are
characteristic inhibitors of mammalian gastric
H+,K+-ATPase. H+ influx was
completely insensitive to ouabain (250 µM), the typical inhibitor of Na+,K+-ATPase.
Mg2+-ATPase activity could be partially stimulated with
K+ (20 mM) that was inhibitable (>85%) with
SCH28080 (50 µM). ATP-dependent rapid efflux
of 86Rb+ from preloaded vesicles was completely
inhibited by preincubation with omeprazole (150 µM) and
by 5,5'-dithiobis-(2-nitrobenzoic acid) (1 mM), an
inhibitor of the enzyme. Assuming Rb+ to be a true
surrogate for K+, an ATP-dependent,
electroneutral stoichiometric exchange of H+ and
K+ (1:1) was established. Rapid and 10-fold active
accumulation of [U-14C]2-deoxyglucose in sealed ghosts
could be observed when an artificial pH gradient (interior alkaline)
was imposed. Rapid efflux of [U-14C]D-glucose
from preloaded everted vesicles could also be initiated by activating
the enzyme, with ATP. Taken together, the plasma membrane
Mg2+-ATPase has been identified as an electroneutral
H+/K+ antiporter with some properties
reminiscent of the gastric H+,K+-ATPase. This
enzyme is possibly involved in active accumulation of glucose via a
H+-glucose symport system and in K+ accumulation.
Kala-azar or visceral leishmaniasis is a major public health
problem in many parts of the tropical and subtropical world including eastern regions of India (1). Leishmania donovani is the
established etiological agent for Kala-azar (2). This protozoal
pathogen belongs to the kinetoplastida group of parasites. The organism has a digenic life cycle. The flagellated vector or promastigote form
normally resides and divides in the alimentary tract of phlebatomine sandflies. Following a bite by the sandfly, the parasite is rapidly internalized in human liver macrophages where it overcomes the host
defense mechanism and undergoes a facile morphogenetic transformation to oval, aflagellated amastigote form in the phagolysosomal complex of
the macrophages. Rapid proliferation and invasion of neighboring macrophages initiates pathogenesis (3). The commonly available laboratory cultural form corresponds to the vector or promastigote form
of the organism.
Ion translocation and energetics of metabolite accumulation across the
plasma membrane are important aspects in the physiology of any
organism. Although some work has been done in this area for the
kinetoplastida group of pathogens, a reasonably clear picture is still
not available. L. donovani is known to have a Ca2+-ATPase (4, 5) and a Mg2+-ATPase (6, 7) in
its plasma membrane. Catalytic sites of both these transmembrane
proteins are oriented toward the cytosol (5, 7). Using sealed, everted
vesicles we had earlier unambiguously demonstrated the
Ca2+-ATPase to be a true extrusion pump for cytosolic free
Ca2+ (8). The specific function of the
Mg2+-ATPase, however, remains to be elucidated. Using
vesicles of undefined and mixed polarity and employing acridine orange
as the probe, Zilberstein and Dwyer (7) had earlier suggested that the
Mg2+-ATPase may be a H+ translocator. More
recently, using BCECF-loaded1
L. donovani promastigote cells, Jiang et al. (9)
have indicated the presence of a H+/K+
exchanger system in the surface of the parasite that is closely linked
to the functioning of this Mg2+-ATPase. The functional
capability, if any, of the Mg2+-ATPase to act as an ion
translocator needs to be demonstrated clearly and unambiguously. This
is also necessary to analyze the related problem of energy coupling in
glucose transport across the plasma membrane (10). Somewhat
contradictory results are available in the literature. Based on the
effect of ionophores and protonophores on 2-deoxyglucose uptake in
whole cells and also of inward flow of H+ across the plasma
membrane in de-energized cells on addition of glucose, Zilberstein and
Dwyer (11) suggested a glucose/H+ symport mechanism for
glucose entry and accumulation. Experiments in chemostat with varying
concentrations of glucose, on the other hand, indicate operation of a
facilitated diffusion mechanism for glucose entry independent of proton
gradient in L. donovani (12). A promastigote stage-specific
glucose transporter showing significant homology with mammalian
facilitative glucose transporter has also been cloned (13) and
functionally expressed in Xenopus oocytes (14). Analysis of
2-deoxyglucose entry process showed it to be a carrier-mediated
process, but the requirement for H+ could not be clearly
established (14).
In general sealed plasma membrane ghosts or vesicles of defined
polarity have been extremely valuable tools for transport studies in
many systems. In this paper, using sealed ghosts and vesicles of
opposite polarity, we demonstrate that the plasma membrane
Mg2+-ATPase of Leishmania is indeed an electroneutral
H+/K+ antiporter. Further, we observe that
imposition of an artificial pH gradient can drive glucose entry and
accumulation in the sealed ghost. It is therefore likely that the major
physiological roles for this enzyme is in generation of a pH gradient
across the plasma membrane that in turn helps in active accumulation of
metabolites like glucose. The enzyme is also probably involved in
accumulation of K+ inside the cell. The second role is
particularly important in view of the uncertain status of
Na+,K+-ATPase in this organism, as will be
discussed later.
Reagents
All biochemicals were purchased from Sigma, unless otherwise
mentioned. Bis-oxanol and BCECF were from Molecular Probes.
[ Organism
The organism used is a clinical isolate from a confirmed
Kala-azar patient, and the strain is designated as MHOM/IN/1978/UR6 (15). The UR6 cells were grown and maintained on a solid blood-agar medium as described earlier (16). Only the highly motile promastigote cells were used for all experiments.
Preparation of sealed ghost and everted vesicles: L. donovani promastigote plasma membrane has a characteristic
pellicular microtubular structure that provides strength to the
membrane against hypotonic shock. Unsealed ghosts devoid of flagella
were first prepared by exposing the promastigotes to controlled
hypotonic shock (4, 8). These unsealed aflagellated ghosts were then used for preparation of both sealed ghosts and everted vesicles. The
methods are briefly as follows. 2 gm of washed cells, collected at
mid-log phase, were suspended in 100 ml of 5 mM Tris-HCl
buffer, pH 7.4, containing 0.5 mM phenylmethylsulfonyl
fluoride that were kept in four long glass tubes at 4 °C. The tubes
were mixed by mild vortexing in a cyclomixer 10 times for a total
period of 90 min with occasional stirring. The hypotonic shock followed by gentle vortexing resulted in detachment of flagella from the cell
body. Formation of unsealed ghosts was confirmed by total leakage of
the marker cytoplasmic enzymes (8). CaCl2 was added at this
stage to a final concentration of 2 mM. The suspension was
then centrifuged at 3300 × g for 20 min at 4 °C,
and the pellet was resuspended in 5% sucrose in 50 mM
Tris-HCl buffer, pH 7.4. 5 ml each of this suspension was layered on to
a 10-ml cushion of 20% sucrose in the same buffer in a corex tube.
These were centrifuged in a Sorvall HB4 rotor at 1500 × g for 30 min. The band at the interface of the two layers
was highly enriched in flagella, whereas the pellet formed consisted
largely of unsealed ghosts. The process was repeated twice, and the
purified unsealed ghosts were utilized for the preparation of both
sealed ghosts and everted vesicles.
For preparation of sealed ghosts, unsealed ghost pellets were
resuspended in 30 ml of a medium containing 20 mM
Hepes-Tris buffer, pH 7.5, 140 mM KCl, and 10 mM CaCl2 at 35 °C for 40 min with gentle but
continuous shaking. The suspension was then immediately cooled to
4 °C and was incubated with gentle shaking for another 30 min. The
sealed ghosts obtained at this stage were pelleted by centrifugation at
3500 × g and resuspended in appropriate buffer for a
particular experiment as described in the text. An electron microscopy picture of the sealed ghost from a typical preparation following this method is presented in Fig. 1A. Sealing could
also be achieved by replacing KCl (140 mM) with sucrose
(250 mM). Presence of Ca2+ (10 mM)
was, however, essential for proper sealing.
Inside-out or everted vesicles were also prepared from unsealed ghosts.
For this purpose, the unsealed ghosts were sheared into smaller
membrane fragments, purified on a discontinuous sucrose gradient,
washed, and finally sealed in presence of 25 mM Hepes-Tris buffer, pH 7.4, 140 mM KCl, and 4 mM
Ca2+ or Mg2+ chloride. Everted vesicles were
then pelleted, washed, and resuspended in appropriate buffer for
subsequent experiments. Details of this procedure for preparing everted
vesicles have been recently published (8). An electron
microscopy picture of the everted vesicle is provided in Fig.
1B. Whenever necessary, everted vesicle could also be
prepared in presence of sucrose (250 mM), Hepes-Tris
buffer, pH 7.4 (20 mM), and Ca2+ (4 mM). This has been mentioned in the appropriate places in the text. Sealing of ghosts and of everted vesicles was confirmed with
the help of fluorescent dye calcein by exploiting its self-quenching property at high concentrations (17) and also by
86Rb+ trapping (18).
Electron Microscopy
Samples of sealed ghosts and everted vesicles were fixed with
3% gluteraldehyde for 15-20 h at 4 °C. After a wash with the respective buffers, the samples were post-fixed with 1% osmium tetroxide in Kellenburger buffer for 16-20 h at room temperature. The
pellets were then washed again and dehydrated with a graded series of
ethanol solutions and finally embedded in SPURRT resin. Thin sections
were collected using an ultrathin microtome and picked up on copper
grids. Observations were made in a transmission electron microscope
(JEOL IN ex) at different magnifications.
Functional Measurements for Sealed Ghosts and Everted
Vesicles
Internal pH--
Both the unsealed ghost and the membrane
fragment were sealed as described above in presence of BCECF at a
concentration of 4 µg ml Transmembrane Potential--
The development of transmembrane
potential was measured fluorimetrically in the same instrument with
bis-oxanol at Internal Volume--
The average internal volumes of the sealed
ghost and everted vesicle were determined by loading either with
86RbCl or with [3H]inulin during the sealing
process. These were then washed and counted. Internal volumes were
calculated according to Rottenberg (18).
Enzyme Assays
Mg2+-ATPase activity was determined either
colorimetrically by measuring the
Mg2+-dependent release of inorganic phosphate
or by measuring the liberation of 32P from
[ Generation of pH Gradient and [14C]2-Deoxyglucose
Uptake
pH gradient was imposed by using "pH jumps" (27). All assays
were performed at 25 °C. To initiate the reaction, everted vesicles
or sealed ghosts at pH 8.0 (protein concentration about 15 mg
ml Characterization of Ghost and Vesicle
Preparations--
Determination of polarity or sidedness of sealed
vesicles or ghosts in the absence of a marker for plasma membrane often
poses some problems. All flagellated trypanosomatids, including
Leishmania have an unique cytoskeletal microtubular arrangement that
can serve as an excellent marker for this purpose. Extensive
cytological studies had earlier shown that these microtubular beads are
closely associated with the inner lamina of the plasma membrane of
intact cells (28). This arrangement provides scaffolding and rigidity for the membrane structure and can be very conveniently used for determining polarity of closed structures. This is evident from the
thin section electron photomicrograph of a typical sealed ghost that
has an array of microtubules at regular intervals in the inner side of
the ghost (Fig. 1A). For this
purpose, unsealed ghosts were sealed in presence of Ca2+
(10 mM). The presence of Ca2+ was found to be
an obligatory requirement, and it could not be replaced by other
divalent cations. Mild hypotonic shock in the absence of drastic
homogenization resulted in the preparation of deflagellated unsealed
ghosts that retain most of the microtubular structure but lose all
internal structures and cytoplasmic marker enzymes. In contrast to the
sealed ghost preparation, the everted vesicles that were prepared from
sheared plasma membrane were of smaller dimension and clearly displayed
the marker microtubules on the outside (Fig. 1B). The lesser
number of microtubules were probably due to the shearing during the
membrane preparation.
Sealing was initially confirmed by calcein loading. Both the ghost and
the everted vesicle are effectively impermeable within experimental
time to cations such as 45Ca2+ and
86Rb+. These ions could be rapidly released
only in presence of A23187 and valinomycin, respectively. The average
internal volume of sealed ghost was calculated to be 3.6 µl
mg H+ Entry and Accumulation in Everted Vesicles--
The
plasma membrane Mg2+-ATPase, which is known to have its
catalytic site on the cytoplasmic side, will display its catalytic site
to the outer mileau in the everted vesicle. Rapid entry of H+ in the everted vesicle could be demonstrated only when
both ATP and Mg2+ were added, indicating the
Mg2+-ATPase to be responsible for H+ uptake.
Addition of FCCP resulted in an immediate release of accumulated
H+. ATP hydrolysis was obligatory for H+ entry
as nonhydrolyzable ATP analogue AMP-PNP completely failed to replace
ATP (Fig. 2). No H+ uptake
was observed on addition of Ca2+ and ATP, thus excluding
any role for the plasma membrane Ca2+-ATPase in the process
(data not shown). In a separate control experiment, we observed that
unlike the everted vesicle, the sealed ghost with original polarity
failed to show any significant uptake of H+ on addition of
ATP and Mg2+ (data not shown). Clearly, ATP was not
available to the catalytic site of Mg2+-ATPase in this
case.
Correlation between Mg2+-ATPase Activity and
H+ Translocation--
Role of Mg2+-ATPase in
H+ entry could be further confirmed when an excellent
correlation between activity of the enzyme and translocation of
H+ could be demonstrated in the everted vesicle with the
help of a reversible thiol-modifying reagent. We found that the enzyme could be completely inactivated (>95%) by
p-chloromercaribenzoate (0.5 mM) in 20 min, DTNB
(1 mM) in 30 min, and N-ethylmealamide (1 mM) in 40 min. Subsequent incubation with excess
dithiothreitol (10 mM) could reactivate (>60%) both the
p-chloromercuribenzoic acid and DTNB-inactivated enzymes
(data not shown). Based on these observations, an experiment was set up
to monitor the possible correlation between enzyme activity and
capacity for H+ uptake. Fig.
3 shows that an excellent correlation
exist between Mg2+-ATPase activity and the initial rate of
H+ transport (first 2 min) in the everted vesicle, leaving
little doubt that the plasma membrane Mg2+-ATPase is
exclusively responsible for H+ translocation across the
plasma membrane.
Effect of Metabolites and Inhibitors on H+
Transport--
Table I summarizes the
results of an experiment in which H+ accumulation in the
everted vesicle at the end of 5 min was monitored in presence of some
nucleotide triphosphates and inhibitors. ATP could not be replaced to
any significant extent by any of the other triphosphates tried. Strong
inhibition by orthovanadate suggests the H+ translocator to
be a P-type ATPase. Inhibition of this enzyme activity by vanadate (29)
and formation of Stimulation of Mg2+-ATPase Activity with
K+--
The strong inhibition of H+
translocating capacity of Mg2+-ATPase by omeprazole and
SCH28080 prompted us to see whether as in the case of mammalian gastric
H+,K+-ATPase, the parasite enzyme can also be
activated by K+. For this purpose, we used unsealed ghost
preparation as our source of enzyme. This preparation provides free
access for ions and nucleotides to the cytosolic face of the enzyme.
Further, the membrane-bound enzyme represents physiologically a more
authentic situation than the enzyme solubilized with detergents (5). The ATP hydrolytic activity was found to be only moderately but systematically stimulated by K+ ion in all the preparations
tested so far. At the saturating concentration of free Mg2+
that was controlled by CDTA-Mg2+ buffering system (24),
activation by K+ (20 mM) was 50%. More
importantly, the stimulated activity could be abolished in presence of
SCH28080, a specific competitor for K+ binding site of
H+,K+-ATPase (31, 34) (Fig.
4). We also observed that (a)
further increase in K+ concentration did not result in any
additional stimulation, (b) Na+ (upto 100 mM) had no stimulatory or inhibitory effect, and
(c) ouabin had no inhibitory effect on the
K+-stimulated activity (data not shown).
ATP-dependent 86Rb+
Extrusion--
To ascertain whether the parasite plasma membrane
Mg2+-ATPase can translocate K+ as a counterion
to H+, everted vesicles were sealed with
86Rb+, and then the reaction was initiated by
adding ATP. For this experiment, vesicles were sealed in presence of 25 mM Hepes-Tris buffer, pH 7.4, containing 100 mM
RbCl, 40 mM NaCl, 4 mM MgCl2, and
86Rb+ (20 µCi ml Stoichiometry--
Ion-translocating ATPases can either be
electrogenic or electroneutral depending upon the stoichiometry of the
overall ion movement. Rapid accumulation of H+ inside the
everted vesicle and simultaneous release of
86Rb+ as a surrogate for K+ from
the vesicle provided us with an opportunity to calculate the
stoichiometry of ion movement during ATP hydrolysis. Table II summarizes results of such an
experiment. For this experiment, a calibration curve for the
internal pH of the everted vesicle was first drawn according to the
method of Negulescu and Machen (21) and as described under "Materials
and Methods." The internal volume was calculated following the method
of Rottenberg (18), and with these two values the total influx of
H+ into the everted vesicle was calculated by multiplying
buffering capacity with change in pH according to Leavy et
al. (22). For calculation of K+ extrusion, kinetics of
the outward flow of 86Rb+ and K+
were assumed to be identical (35). Average internal volume of the
everted vesicle had earlier been calculated to be 1.4 µl mg
Table II shows that both for the initial rate (1.5 min) and for the
average rate over 3 min when the rates of both H+ influx
and Rb+ extrusion considerably slowed down, hydrolysis of
one molecule of ATP can maximally be related to the movements of one
H+ and one Rb+ across the plasma membrane of
the everted vesicle. Extrapolation of the rates to zero time of ATP
addition are expected to give the true initial values for these ion
movements, although no experiment was set up for this purpose. Higher
values for ATP hydrolytic activity were probably due to the presence of
some unsealed vesicles and contaminating membrane fragments. More
importantly, at both stages of measurement the ratio between
H+ influx and Rb+ extrusion remains very close
to one, strongly indicating the process to be an electroneutral one. In
separate experiments, when such Rb+ or
K+-loaded everted vesicles were suspended in the same
medium that also contained bis-oxanol to record changes in membrane
potential, activation of Mg+-ATPase by addition of ATP
brought about no change in bis-oxanol fluorescence (data not shown),
confirming once again the electroneutral character of this
ion-translocating ATPase. Assuming Rb+ to be a faithful
replacement for K+ for this enzyme, the plasma membrane
Mg2+-ATPase should therefore be regarded as an
electroneutral H+/K+ antiporter.
H+-driven 2-Deoxyglucose Accumulation in Sealed
Ghosts--
Results presented so far indicate that the operation of
the plasma membrane Mg2+-ATPase in a respiring L. donovani cell should generate a H+ gradient across the
membrane with interior alkaline. Can this proton gradient be involved
in the active accumulation of important metabolites? Earlier,
contradictory results were reported for glucose transport with resting
whole cell (11) or with growing whole cells in a chemostat (12). When a
pH gradient of 2.5 was imposed across the plasma membrane of sealed
ghost of original polarity (pH 5.5 and 8.0 for outside and inside,
respectively), a rapid accumulation of 14C-2-deoxyglucose
could be demonstrated (Fig. 6). In
absence of an imposed proton gradient or when a powerful protonophore
like FCCP (10 µM) was present, no such accumulation could
be observed. More importantly, based on an approximate internal volume
of 3.6 µl mg Direct Correlation between Plasma Membrane Mg2+-ATPase
Activity and Glucose Movement across the Membrane--
Active
accumulation of glucose in sealed ghost on imposition of a pH gradient
does not necessarily signify the involvement of the plasma membrane
Mg2+-ATPase in the process. To find out whether
H+ movement initiated by activation of
Mg2+-ATPase can directly be correlated to a simultaneous
movement of glucose across the plasma membrane, the following
experiment was set up with the everted vesicle. In this case, although
the catalytic site of the enzyme is exposed outside, the binding sites of the putative H+-glucose symporter are presumed to be
oriented inside the vesicle. Everted vesicles containing Tris-HCl
buffer, pH 7.4 (50 mM), KCl (120 mM), NaCl (10 mM), and MgCl2 (4 mM) were sealed
in presence of [U-14C]D-glucose (20 µCi
ml Ghosts and vesicles of defined polarity have proven to be
extremely powerful tools in many studies involving bioenergetics, ion
translocation, and active accumulation of metabolites (31, 36). Taking
advantage of the comparative rigidity provided by the pellicular
microtubular structure to the plasma membrane of L. donovani, we could quite conveniently prepare sealed ghosts and
everted vesicles of defined polarity that are free from cytoplasmic marker enzymes and internal metabolites (Fig. 1 and Methods). Everted
vesicles provide free access to ATP and other charged molecules to the
catalytic site of the plasma membrane Mg2+-ATPase, which
now becomes exposed to the suspending medium.
Mg2+-dependent ATP hydrolysis led to rapid
accumulation of H+ in everted vesicles (Fig. 2) and to
almost equally rapid depletion of loaded 86Rb+
(Fig. 5) from these vesicles. Direct involvement of the
Mg2+-ATPase in these ion movements was further confirmed
when an excellent correspondence between the rate of H+
influx and enzyme activity could be demonstrated (Fig. 3) or when
Rb+ efflux could be completely prevented by inactivating
the enzyme with DTNB (Fig. 5). Thus, in terms of its ion-translocating
capacity this Mg2+-ATPase can be considered as a
H+,K+-ATPase. Assuming Rb+ to be a
true surrogate for K+, we also calculated the stoichiometry
between H+ and K+ exchange. This appears to be
an electroneutral process giving a stoichiometry of 1:1 (Table II and
text). The electroneutral character of the antiporter is very similar
to the gastric mucosal H+,K+-ATPase from
different mammalian sources (31, 37, 38).
Can the Leishmania plasma membrane Mg2+-ATPase be regarded
as identical to the gastric H+,K+-ATPase? In
absence of any significant biochemical, mechanistic, and molecular
data, this remains an open question at the moment. In our initial
efforts we have failed to purify and biochemically characterize this
enzyme because of its severe aggregation problems. However, complete
abolition of the K+- stimulated activity by SCH28080 (Fig.
4) and also the strong inhibition of H+ uptake in and
Rb+ efflux from the everted vesicle by inhibitors of
H+,K+-ATPase (Table I, text, and Fig. 5)
clearly indicate that this enzyme has some characteristic properties
that are very similar to those of the gastric
H+,K+ATPase. The K+ translocating
activity and sensitivity to inhibitors of gastric H+,K+-ATPase also set this enzyme apart from
the well characterized plasma membrane H+-ATPase of lower
eucaryotes such as yeast and other fungi (39, 40). Thus, we conclude
that in terms of its functional capability this plasma membrane enzyme
is very similar to gastric H+,K+-ATPase.
What are the possible physiological roles of this
Mg2+-ATPase that we have identified as a
H+,K+ antiporter? The electroneutral but
energy-dependent extrusion of H+ from the
cytosol and uptake of K+ into the cytosol will result in
setting up of a pH gradient across the plasma membrane with interior
alkaline and in accumulation of K+ in the cytosol. The pH
gradient generated by this enzyme is fully capable of driving active
accumulation of metabolites. This has been exemplified by the
demonstration of rapid uptake and accumulation of glucose in sealed
ghosts (Fig. 6) and by the presence of a H+-glucose
symporter in the plasma membrane of the parasite that is functionally
closely coupled to this ATPase (Fig. 7). This is the first clear
demonstration of a H+-glucose symport mechanism for glucose
accumulation operative in Leishmania parasites. It is important to note
that developmentally regulated glucose transporter genes having
significant homology with such genes of higher eucaryotes were earlier
cloned from Leishmania, although their relationship to energy coupling
remained uncertain (14, 41).
Membrane potential of Leishmania was determined earlier to be around
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (5000 Ci mmol
1),
45Ca2+ (39 mCi mg
1),
86Rb+ (10 mCi mg
1),
[3H]Inulin (1 Ci mmol
1),
2-deoxy-D-[U-14C]glucose, and
D-[U-14C]glucose (300 mCi
mmol
1) were obtained from Amersham Pharmacia Biotech.
Omeprazole and SCH28080 were kindly provided by Dr. Pratap Das (IICB),
who got these chemicals as gift from Astra Hassle AB and Schering
Corporation, respectively.
1. For fluorescence
measurements, the dye-loaded and washed everted vesicles (500 µg
protein ml
1) were placed in a 1-ml fluorescence cuvette.
A Hitachi spectrofluorimeter model F4010 was used to monitor the
readings of BCECF at 505 nm excitation and at 525 nm emission
wavelengths (19). No appreciable leakage of the dye was observed during
the period in which experiments were completed. Calibration of the
internal pH of the everted vesicle was done by incubating the
BCECF-loaded vesicle in a buffer containing 140 mM KCl, 4 mM CaCl2, and 25 mM Hepes-Tris
buffer with varying pH (6.5-7.6) in presence of valinomycin (1 µg
ml
1) and FCCP (5 µM). FCCP and valinomycin
equilibrate the intracellular and extracellular proton and
K+, respectively (20). The pHi of the vesicle
is assumed to be the same as the extracellular pH as the
pHi and pHo become equal in presence of FCCP. A
calibration curve was made by plotting the ratio of the fluorescence at
535 nm when the samples were excited at 503 and 440 nm, respectively,
at various pH buffers (21) and found to be linear between pH 6.5 and
7.6 (data not shown). For stoichiometric calculations, the
H+ influx in the everted vesicle was calculated according
to the method of Levy et al. (22). In this case,
H+ influx = Buffering capacity ×
pH × internal volume.
ex535 nm and at
em at 559 mm (23). For this purpose, a final concentration of 80 nM
dye was added to the cuvette containing vesicle suspension (400 µg
protein ml
1) in sealing buffer pH 7.4.
-32P]ATP. Whenever required, the concentration of
free Mg2+ was controlled by complexation with CDTA. For
colorimetric assay, the incubation medium contained in a total volume
of 1 ml, 100 mM Tris-HCl, pH 7.4, 500 µM CDTA
with or without MgCl2, and 0.5 mM ATP. The
reaction was initiated by addition of everted vesicle (200 µg protein
ml
1) as the source of enzyme. MgCl2
concentration was varied to give the required free Mg2+
concentration that was calculated according to Parshadsingh and McDonald (24). Incubation was for 30 min at 28 °C. The reaction was
terminated by addition of 50 µl of 20% trichloroacetic acid. After
removal of the precipitate, the released inorganic phosphate was
measured following the method of Lowry and Lopez (25). To determine
free Mg2+ concentration, Mg2+ from the vesicle
(10 µM) and from other reagents (4 µM) were included in the calculations. Mg2+-stimulated ATPase
activity was determined by subtracting the values with chelator alone
from the values with Mg2+ and chelator. For the radioactive
assay, the method of Bais (26) was followed with few modifications. In
this case, the final volume of the assay mixture was reduced to 0.1 ml.
The composition and concentration of all the ingredients of the assay
mixture remained the same as before. In this assay, 50 nmol of
[
-32P]ATP (0.1 µCi) was added. After 10 min of
incubation at 28 °C, the reaction was stopped by addition of 5 µl
of 20% trichloroacetic acid. To this 10 µl of 100 mM
KH2PO4 and 0.1 ml of suspension of 50%
activated charcoal in water were added consecutively. After mild
agitation for 10 min, charcoal was precipitated by centrifugation. The
process was repeated once more, and finally 100 µl of the supernatant
was transferred to a scintillation vial.
1) were diluted 50-fold into mixtures containing
sealing buffer of either pH 5.5 or 8 and 50 µM of
[14C]2-deoxyglucose (1 µCi ml
1) or other
additions as required. Each tube containing 500 µl of mixture was
filtered at different time intervals through millipore filters (pore
size, 0.8 µm) and washed with 10 ml of cold resuspension buffer, and
trapped radiolabeled count was measured as described earlier (8).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (33K):
[in a new window]
Fig. 1.
Thin section electron micrograph of a single
sealed ghost with right-side-out orientation (×22,000) A,
an everted plasma membrane vesicle (×45,000) B,
mt indicates microtubules.
1 protein (n = 4), and everted vesicle
was 1.4 µl mg
1 protein (n = 4) (see
"Materials and Methods").
View larger version (16K):
[in a new window]
Fig. 2.
Mg2+-ATPase is a proton
translocator in everted vesicles. The BCECF-loaded vesicles each
containing 250 µg of protein were placed in 1-ml cuvettes, and
fluorescence intensities were measured as described under "Materials
and Methods." Each of the reaction mixture contained in a total
volume of 500 µl, 20 mM Tris-Hepes buffer, pH 7.4, 125 mM KCl, 15 mM NaCl, 4 mM
MgCl2, and 5 mM CDTA. In tube A, ATP
(1 mM), MgCl2 (2 mM), and FCCP (10 µM) were added sequentially at indicated times. In tube
B, order of addition of ATP and Mg2+ was
reversed. In tube C, AMP-PNP (1 mM) replaced
ATP.
View larger version (20K):
[in a new window]
Fig. 3.
Correspondence between
Mg2+-ATPase activity and H+ uptake. Each
tube contained in a total volume of 500 µl, BCECF-loaded everted
vesicle (250 µg ml 1 protein), 20 mM
Tris-Hepes buffer, pH 7.4, 125 mM KCl, 15 mM
NaCl, 4 mM MgCl2, and DTNB (1 mM)
except in zero time control where DTNB was omitted. In all cases,
reactions were initiated with ATP (1 mM). For reversal of
enzyme activity and of H+ uptake capacity, dithiothreitol
(10 mM) was added to a few tubes as indicated (
). Enzyme
activity was measured by removing suitable aliquots. H+
influx was measured as percent fluorescence quench at the end of 5 min.
-aspartyl phosphate intermediate (30) were earlier
shown by other workers. Most importantly, omeprazole and SCH28080 both
specific inhibitors for gastric H+,K+-ATPase
(31) showed strong inhibitory influence on H+ accumulation.
Direct participation of Na+,K+-ATPase or of a
V-type ATPase in H+ pumping could be ruled out because
neither ouabain nor bafilomycin A1 showed any effect on
H+ accumulation (32, 33). To analyze more carefully the
extent of inhibition that can be achieved by omeprazole and by
SCH28080, the initial rate of H+ entry (first 2 min) was
followed with increasing concentrations of these two inhibitors and
vanadate. The experimental protocol was the same as in Table I. Both
the drugs inhibited the entry process (up to 95 and 75%, respectively)
in a dose-dependent manner. IC50 for omeprazole
and SCH28080 were 50 and 40 µM, respectively. Omeprazole
could effectively abolish H+ influx (>90%) at 150 µM, whereas SCH28080 at that concentration had a less
pronounced effect. The inhibitory effect of omeprazole compared
favorably well with that of vanadate (data not shown). These results
suggested that the parasite H+ translocating plasma
membrane Mg2+-ATPase may have some enzymological and
physiological properties that may be similar to the mammalian gastric
H+,K+-ATPase (34).
Effect of energy sources and potential inhibitors on H+
transport in everted vesicles
View larger version (21K):
[in a new window]
Fig. 4.
Stimulation of Mg2+-ATPase
activity with K+ and inhibition with SCH28080. The
rate of Mg2+-dependent ATP hydrolysis was
estimated as [32P]Pi release (see
"Materials and Methods"). An appropriate control was run in the
absence of Mg2+. Each assay mixture contained in a total
volume of 100 µl, 100 mM Tris-HCl, pH 7.4, 0.5 mM ATP (Tris-salt), 500 µM CDTA, and varying
amounts of MgCl2 with other additions as indicated. The
reaction was started by the addition of a requisite amount of unsealed
membrane ghost preparation (~5 µg of protein). After 10 min of
incubation at 30 °C, the reaction was terminated by the addition of
5 µl of 20% trichloroacetic acid, and liberated
32Pi from [ -32P]ATP was
measured as Mg2+-ATPase activity. Additions, without KCl
(open circle), 20 mM KCl (open
square), and 20 mM KCl + 50 µM SCH28080
(open triangle). Mean ± S.D. values are plotted from
three separate experiments.
1). Vesicles
(200 µg protein ml
1) were washed twice and finally
suspended in a medium containing 25 mM Hepes-Tris buffer,
pH 7.4, 250 mM sucrose, and 4 mM
MgCl2. On addition of ATP (1 mM), an immediate
and rapid extrusion of 86Rb+ could be observed
(Fig. 5). When ATP was replaced with
AMP-PNP, no outward movement of 86Rb+ could be
observed, clearly implicating Mg2+-dependent
ATP hydrolysis to be essential for 86Rb+
extrusion. Preincubation with omeprazole (100 µM) for 10 min and DTNB (1 mM) for 30 min resulted in total blocked of
86Rb+ extrusion, providing strong evidence that
the Mg2+-ATPase is indeed a H+/K+
antiporter. Valinomycin (5 µM)-dependent
86Rb+ release served as the control (Fig. 5).
As with omeprazole, preincubation with SCH28080 could also completely
inhibit the Rb+ extrusion process (data not shown). We
failed to detect the presence of any H+/K+
exchanger in the plasma membrane of the parasite. For this purpose, 86Rb+-loaded everted vesicle (internal pH 7.6)
was diluted 50-fold in a medium containing Hepes-Tris buffer, pH 6.5. No efflux of 86Rb+ above the control (dilution
in pH 7.6 of Hepes-Tris buffer) could be observed.
View larger version (22K):
[in a new window]
Fig. 5.
ATP-dependent
86Rb+ extrusion from preloaded everted
vesicles. Everted vesicles (200 µg of protein) preloaded with
86Rb+ were suspended in 1.0 ml of 25 mM Hepes-Tris buffer, pH 7.4, 250 mM sucrose,
and 4 mM MgCl2 at 30 °C. Extrusion of
86Rb+ was measured for each individual tube at
indicated time after following additions: control without ATP
(open circle), immediately after addition of 1 mM ATP (open triangle), immediately after
addition of 5 µM valinomycin (close triangle),
immediately after addition of 1 mM AMP-PNP (reverse
open triangle), preincubation with 150 µM omeprazole
for 15 min and then 1 mM ATP (close circle), and
preincubation with 1 mM DTNB for 30 min and then 1 mM ATP (open square).
1 protein. For accurate determination of the extent of
ATP hydrolysis, [
-32P]ATP was used as the substrate.
Detailed procedures are provided under "Materials and Methods."
Stoichiometry of ATP hydrolysis, H+ influx, and Rb+
extrusion in everted vesicle
1), 20 mM Tris-Hepes
buffer, pH 7.4, 250 mM sucrose, and 4 mM
MgCl2. For one set of tubes quenching of BCECF fluorescence
(
pH from standard pH calibration curve, see methods) was determined
at 1.5 and 3.0 min. For the second set of tubes 86Rb+
extrusion was determined at also 1.5 and 3.0 min. For ATP hydrolysis
data, vesicles were scaled under identical conditions but in presence
of cold Rb+. After addition of 1 mM
[
-32P]ATP (0.5 µCi), appropriate aliquots were withdrawn
for estimation of liberated radiolabeled phosphate as described under
"Materials and Methods." Details of calculation procedures have
been described in the text. All tubes were in triplicate, and the mean
average was taken for calculation.
1 protein (see above), the ghost
accumulates more than 0.5 mM of deoxyglucose, which is at
least 10 times more than its external concentration. We could also show
similar uptake and accumulation with isotopic glucose and competition
between the two sugars (data not shown). Clearly, a
H+-glucose symport system is operative in the plasma
membrane of Leishmania promastigote cells.
View larger version (16K):
[in a new window]
Fig. 6.
Imposed pH gradient drives 2-deoxyglucose
influx and accumulation in sealed ghost. Sealed ghost was prepared
as described under "Materials and Methods." In the sealing medium
20 mM Tris-HCl buffer, pH 7.5, was replaced by the same
buffer of pH 8.0. Pelleted sealed ghost was resuspended (~15 mg
ml 1) in a incubation medium containing 20 mM
Tris-Hepes buffer, pH 8.0, 250 mM sucrose, and 10 mM CaCl2 for 10 min at 30 °C. Appropriate
aliquots of sealed ghost (~150 µg of protein) were rapidly diluted
50 times into 0.5 ml of medium containing 250 mM sucrose,
10 mM CaCl2 50 µM
[U-14C]2-deoxyglucose (0.8 µCi) with other ingredients
as described below and were rapidly filtered at indicated time
intervals. Additions, 50 mM Hepes-Tris buffer, pH 8.0 (open triangle), 50 mM Mes-Tris buffer, pH 5.5 (close circle), and 50 mM Mes-Tris buffer, pH
5.5, and 10 µM FCCP (open circle).
1; 7.5 mM). Everted vesicles loaded with
labeled glucose were washed twice and then suspended in the same
Tris-HCl buffer containing sucrose (250 mM) and
MgCl2 (4 mM). Fig.
7 shows an immediate and rapid efflux of
glucose on addition of ATP in the incubation mixture. When ATP was
replaced with AMP-PNP or the Mg2+-ATPase was inactivated by
pretreatment with DTNB, no efflux of glucose could be detected.
Finally, preincubation with FCCP (10 µM) also resulted in
total abolition of the efflux. Clearly, influx of H+ on
activation of Mg2+-ATPase results in the parallel
activation of the H+-glucose symport system that initiates
the rapid efflux of glucose from the preloaded vesicles.
View larger version (14K):
[in a new window]
Fig. 7.
Mg2+-ATPase dependent
[U-14C]D-glucose extrusion from preloaded
everted vesicles. For this experiment, everted sealed ghosts were
prepared in presence of 25 mM Tris-Hepes buffer, pH 7.4, 140 mM KCl,10 mM CaCl2, and 7.5 mM [U-14C]D-glucose (20 µCi
ml 1). Washed and preloaded sealed ghosts (200 µg
protein ml
1) were suspended in separate tubes, each
containing in 500 µl, 25 mM Tris-Hepes buffer, pH 7.4, 250 mM sucrose, and 4 mM MgCl2.
Other additions, if made, are indicated below. Incubation time was for
5 min at 30 °C. Reactions were initiated by additions as indicated
below. Released counts in the incubation medium were recorded at
indicated times by rapid filtration as described under "Materials and
Methods." Open circle, control without any addition;
closed circle, with 1 mM ATP; open
square, preincubated with 10 µM FCCP and then 1 mM ATP; open triangle, with 1 mM
AMP-PNP.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
100mV by two independent methods (42, 43). The concentration of
cytosolic K+ also appears to be typical of eucaryotic cells
(9). How is the potential built up and maintained? Several laboratories
have failed to detect the presence of a plasma membrane
Na+,K+-ATPase in L. donovani (7, 42,
44), although its occurence has been claimed in a different species of
Leishmania by Felibertt et al. (45). Whatever be the status
of Na+,K+-ATPase in this parasite, it is clear
that operation of this H+,K+-ATPase will
significantly help in the accumulation of K+ in the
cytosol. In fact, this process may be critical if
Na+,K+-ATPase is truly absent in L. donovani. Electroneutral accumulation of K+, mediated
by this enzyme, is obviously incapable of generating the desired
membrane potential. Very little is known about other ion translocators,
anion conducting pathways, and channels that may be involved in
generating this potential. So far, only a Cl
conducting
pathway has been demonstrated in this parasite (43), and a putative
Ca2+-activated K+ channel gene has been
uncovered in Leishmania major as part of the genome
sequencing program (46). Needless to say, much more work needs to be
done in this area before a comprehensive picture emerges. Further work
on this putative H+,K+-ATPase is expected to
throw considerable light on the enzymology and function of this
important and interesting enzyme in the life cycle and physiology of
this dreaded protozoal pathogen. This enzyme may also be explored as a
potential target for drug development.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Sailen Dey for assistance with electron microscopic photography and Suprabhat Bhattacharyya for typing the manuscript. We also thank Dr. Pratap Das for stimulating discussions.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Council of Scientific and Industrial Research and Department of Biotechnology (India).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Member of the Infectious Diseases Group.
§ These authors contributed equally to this work.
¶ Present address: The Johns Hopkins University, School of Medicine, Dept. of Physiology, 725, North Wolfe St., Baltimore, MD 21205.
To whom correspondence should be addressed. E-mail:
anbhaduri@yahoo.com.
Published, JBC Papers in Press, November 21, 2000, DOI 10.1074/jbc.M008469200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
BCECF, 2',7-bis(carboxyethyl)-5(6)-carboxyfluorescein;
bis-oxanol, bis-(1,
3-diethylthiobarbituric. acid) trimethine oxanol;
calcein, 2',7'-{[bis(carboxymethyl) amino] methyl}-fluorescein;
FCCP, Carbonylcyanide p-(trifluoromethoxy) phenylhydrazone;
CDTA, transcyclohexane-1,2-diamine-N, N,N',N'-tetraacetic acid;
AMP-PNP, ,
-imidoadenosine 5'-triphosphate;
DTNB, 5,5'-dithiobis-(2-nitrobenzoic acid).
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Ashford, R. W., Desjeux, P., and DeRaadt, P. (1991) Parasitol. Today 8, 104-105 |
2. | Schnur, L. F., and Greenblatt, C. L. (1995) in Parasitic Protozoa (Kreier, J. P., ed), Vol. 10 , pp. 1-30, Academic Press, Orlando, FL |
3. | Chang, K.-P., Fong, D., and Bray, R. S. (1985) in Leishmaniasis (Chang, K.-P. , and Bray, R. S., eds) , pp. 1-30, Elsevier Biomedical Press, Amsterdam |
4. |
Ghosh, J.,
Ray, M.,
Sarkar, S.,
and Bhaduri, A.
(1990)
J. Biol. Chem.
265,
11345-11351 |
5. |
Mazumder, S.,
Mukherjee, T.,
Ghosh, J.,
Ray, M.,
and Bhaduri, A.
(1992)
J. Biol. Chem.
267,
18440-18446 |
6. | Dwyer, D. M., and Gottlieb, M. (1983) J. Cell. Biochem. 23, 35-45[Medline] [Order article via Infotrieve] |
7. | Zilberstein, D., and Dwyer, D. M. (1988) Biochem. J. 256, 13-21[Medline] [Order article via Infotrieve] |
8. | Mandal, D., Mukherjee, T., Sarkar, S., Mazumder, S., and Bhaduri, A. (1997) Biochem. J. 322, 251-257[Medline] [Order article via Infotrieve] |
9. | Jiang, S., Anderson, S. A., Winget, G. D., and Mukkada, A. J. (1994) J. Cell. Physiol. 159, 60-66[Medline] [Order article via Infotrieve] |
10. | Tetaud, E., Barrett, M. P., Bringaud, F., and Baltz, T. (1997) Biochem. J. 325, 569-580[Medline] [Order article via Infotrieve] |
11. | Zilberstein, D., and Dwyer, D. M. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1716-1720[Abstract] |
12. | terKuile, B. H., and Opperdoes, F. R. (1993) Mol. Biochem. Parasitol. 60, 313-322[Medline] [Order article via Infotrieve] |
13. | Cairns, B. R., Collard, M. W., and Landfear, S. M. (1989) Proc. Natl. Acad. Sci. U. S. A. 85, 2130-2134 |
14. |
Langford, L. K.,
Little, B. M.,
Kavanaugh, M. P.,
and Landfear, S. M.
(1994)
J. Biol. Chem.
269,
17939-17943 |
15. | Chakraborty, P., and Das, P. K. (1988) Mol. Biochem. Parasitol. 28, 55-62[Medline] [Order article via Infotrieve] |
16. | Saha, A. K., Mukherjee, T., and Bhaduri, A. (1986) Mol. Biochem. Parasitol. 19, 195-200[Medline] [Order article via Infotrieve] |
17. | Tsao, Y., and Huang, L. (1985) Biochemistry 24, 1092-1098[Medline] [Order article via Infotrieve] |
18. | Rottenberg, H. (1979) Methods Enzymol. 55, 547-569[Medline] [Order article via Infotrieve] |
19. |
Ruben, L.,
Hutchinson, A.,
and Mochlman, J.
(1991)
J. Biol. Chem.
266,
24351-24358 |
20. |
Hao, L.,
Rigard, J. L.,
and Inesi, G.
(1994)
J. Biol. Chem.
269,
14268-14275 |
21. | Negulescu, P. A., and Machen, T. E. (1990) Methods Enzymol. 192, 38-81[Medline] [Order article via Infotrieve] |
22. |
Levy, D.,
Seigneuret, M.,
Bluzat, A.,
and Rigaud, J. L.
(1990)
J. Biol. Chem.
265,
19524-19534 |
23. | Apell, H. J., and Bersch, B. (1987) Biochim. Biophys. Acta 903, 480-494[Medline] [Order article via Infotrieve] |
24. |
Parshadsingh, H. A.,
and McDonald, J. M.
(1980)
J. Biol. Chem.
255,
4087-4093 |
25. | Lowry, O. H., and Lopez, J. A. (1957) Methods Enzymol. 3, 845-850 |
26. | Bais, R. (1975) Anal. Biochem. 63, 271-273[Medline] [Order article via Infotrieve] |
27. |
Schumaker, K. S.,
and Sze, H.
(1986)
J. Biol. Chem.
261,
12172-12178 |
28. | Vickerman, K., and Pneston, T. M. (1976) in Biology of Kinetoplastida (Lumsden, W. H. R. , and Evans, D. A., eds) , pp. 35-130, Academic Press, New York |
29. | Anderson, S. A., and Mukkada, A. J. (1994) Biochim. Biophys. Acta 1195, 71-80[Medline] [Order article via Infotrieve] |
30. | Anderson, S. A., Jiang, S., and Mukkada, A. J. (1994) Biochim. Biophys. Acta 1195, 81-88[Medline] [Order article via Infotrieve] |
31. |
Hersey, S. J.,
and Sachs, G.
(1995)
Physiol. Rev.
75,
155-189 |
32. | Ross, A., and Boron, W. F. (1981) Intracellular pH Physiol. Rev. 61, 296-434 |
33. | Bowman, E. J., Siebers, A., and Altendrof, K. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 7972-7976[Abstract] |
34. | Rabon, E. C., and Ruben, M. A. (1990) Annu. Rev. Physiol. 52, 321-344[CrossRef][Medline] [Order article via Infotrieve] |
35. | Skrabanja, A. T. P., Vander Hijden, T. W. M., and DePont, H. H. M. (1987) Biochim. Biophys. Acta 903, 434-440[Medline] [Order article via Infotrieve] |
36. | Kaback, H. R. (1986) Annu. Rev. Biophys. Biophys. Chem. 15, 279-319[CrossRef][Medline] [Order article via Infotrieve] |
37. | Sachs, G., Chang, H. H., Rabon, E., Schackman, R., Lewin, M., and Saccomani, G. (1976) J. Biol. Chem. 251, 7690-7698[Abstract] |
38. | Smith, G. S., and Scholes, P. B. (1982) Biochim. Biophys. Acta 688, 803-807[Medline] [Order article via Infotrieve] |
39. | Perlin, D. S., Kasamo, K., Brooker, R. J., and Slayman, C. W. (1984) J. Biol. Chem. 259, 7784-7792 |
40. | Blatt, M. R., Rodriguez-Navarro, A., and Slayman, C. L. (1987) J. Membr. Biol. 98, 169-189[Medline] [Order article via Infotrieve] |
41. | Langford, C. K., Burchmore, R. J. H., Hart, D. T., Wagner, W., and Landfear, S. M. (1994) Parasitology 108, 73-83 |
42. | Glaser, T. A., Utz, G. L., and Mukkada, A. J. (1992) Mol. Biochem. Parasitol. 51, 9-16[CrossRef][Medline] [Order article via Infotrieve] |
43. |
Vieira, L.,
Slotki, I.,
and Cabantchik, Z. I.
(1995)
J. Biol. Chem.
269,
16254-16259 |
44. | Blum, J. J. (1992) J. Cell. Physiol. 152, 111-117[Medline] [Order article via Infotrieve] |
45. | Felibertt, P., Bermudez, R., Cervino, V., Dawidowicz, K., Dragger, F., Proverbio, T., Marin, R., and Benaim, G. (1995) Mol. Biochem. Parasitol. 74, 179-187[CrossRef][Medline] [Order article via Infotrieve] |
46. |
Myler, P. J.,
Audleman, L.,
Devos, T.,
Hixson, G.,
Kiser, P.,
Lemley, C.,
Magness, C.,
Rickel, E.,
Sisk, E.,
Sunkin, S.,
Swartzell, S.,
Westlake, T.,
Bastein, P.,
Fu, G.,
Ivens, A.,
and Stuart, K.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
2902-2906 |