Báo cáo Y học: Effects of ATP depletion and phosphate analogues on P-glycoprotein conformation in live cells docx

Effects of ATP depletion and phosphate analogues
on P-glycoprotein conformation in live cells
Katalin Goda
1
, Henrietta Nagy
1
, Eugene Mechetner
2
, Maurizio Cianfriglia
3
and Ga
´
bor Szabo
´
Jr
1
1
Department of Biophysics and Cell Biology, University of Debrecen, Hungary;
2
Chemicon International, Inc., Temecula, CA, USA;
3
Laboratorio di Immunologia, Istituto Superiore di Sanita
`
, Rome, Italy
P-glycoprotein (Pgp), a membrane pump often responsible
for the multidrug resistance of cancer cells, undergoes con-
formational changes in the presence of substrates/modula-
tors, or upon ATP depletion, reflected by its enhanced
reactivity with the UIC2 monoclonal antibody. When the
UIC2-shift was elicited by certain modulators (e.g. cyclo-
sporin A or vinblastine, but not with verapamil or

Tween 80), the subsequent binding of other monoclonal
anti-Pgp Ig sharing epitopes with UIC2 (e.g. MM12.10) was
abolished [Nagy, H., Goda, K., Arceci, R., Cianfriglia, M.,
Mechetner, E. & Szabo
´
Jr, G. (2001) Eur. J. Biochem. 268,
2416–2420]. To further study the relationship between
UIC2-shift and the suppression of MM12.10 binding, we
compared, on live cells, how ATP depletion and treatment of
cells with phosphate analogues (sodium orthovanadate,
beryllium fluoride and fluoro-aluminate) that trap nucleo-
tides at the catalytic site, affect the two phenomena. Similarly
to modulators or ATP depleting agents, all the phosphate
analogues increased daunorubicin accumulation in Pgp-
expressing cells. Prelabeling of ATP depleted cells with UIC2
completely abolished the subsequent binding of MM12.10,
in accordance with the enhanced binding of the first mAb.
Vanadate and beryllium fluoride, but not fluoro-aluminate,
reversed the effect of cyclosporin A, preventing UIC2
binding and allowing for labeling of cells with MM12.10.
Thus, changes in UIC2 reactivity are accompanied by
complementary changes in MM12.10 binding also in re-
sponse to direct modulation of the ATP-binding site, con-
firming that conformational changes intrinsic to the catalytic
cycle are reflected by both UIC2-related phenomena. These
data also fit a model where the UIC2 epitope is available for
antibody binding throughout the catalytic cycle including
the step of ATP binding, to become unavailable only in the
catalytic transition state.
Keywords: P-glycoprotein; multidrug resistance; UIC2;

MM12.10; conformation.
P-glycoprotein (Pgp) is an integral plasma membrane
protein that functions as an ATP-dependent efflux pump
for a broad range of lipophylic or amphiphylic compounds
[1–3]. Expression of this, and related pumps on cancer cells,
renders them resistant to a wide range of cytotoxic
compounds, causing multidrug resistance (mdr) [1–3]. Based
on the structure of its two ATP binding sites and the
mechanism of ATP hydrolysis, Pgp belongs to the ATP
Binding Cassette (ABC) family of transport ATPases [4,5].
The molecule is comprised of two homologous halves, each
containing an ATP binding site characterized by Walker A
and B sequence motifs, and six transmembrane segments.
The two halves of Pgp are connected by a linker peptide
( 75 amino acids) composed of charged amino-acid
residues with several phosphorylation sites [1,6]. As no
difference was found in resistance between cells transfected
with wild-type and phosphorylation defective forms of the
human Pgp, the role of phosphorylation sites is not clearly
understood [7].
Pgp interacts directly with its substrates, probably within
the cell membrane, and transports them out reducing their
intracellular concentration [1,8]. A number of compounds,
often referred to as modulators (reversing agents, chemo-
sensitizers), are capable of decreasing or eliminating mdr by
preventing Pgp-mediated substrate export [1,9].
The protein exhibits a substrate-stimulated ATPase
activity, suggesting that ATP hydrolysis and drug transport
are intimately linked [10]. Phosphate (P
i

) analogues, e.g.
vanadate (V
i
), beryllium fluoride (BeF
x
; the exact compo-
sition of the complex is unknown) and fluoro-aluminate
(AlF
4
) are potent inhibitors of Pgp ATPase activity [10–15].
The characteristics of their inhibitory effect are similar in
general, as it is due to trapping of nucleotides at the catalytic
site in a noncovalent but tenaciously bound form [10–15].
However, BeF
x
and V
i
trap only ADP, while AlF
4
traps
both ATP and ADP, when Pgp molecules are preincubated
with MgATP [11,12]. PP
i
protects effectively against BeF
x
inhibition of Pgp by competing with BeF
x
,whereasPP
i
had

no effect on inhibition by V
i
[12]. These data were
interpreted to suggest that P
i
analogues may trap Pgp at
different steps of the catalytic process [13].
The V
i
-trapped intermediate of Pgp (i.e. PgpÆMg–
ADPÆV
i
) is thought to be equivalent to the PgpÆMg–ADPÆP
i
complex, which represents the catalytic transition-state in
the normal reaction pathway [14,15]. The formation of
Correspondence to G. Szabo
´
Jr, Department of Biophysics
and Cell Biology, University of Debrecen, PO Box 39,
H-4012 Debrecen, Hungary.
Fax/Tel.: + 36 52 412 623,
E-mail:
Abbreviations: Pgp, P-glycoprotein; mdr, multidrug resistance;
ABC, ATP Binding Cassette; V
i
, vanadate; BeF
x
, beryllium
fluoride; AlF

4
, fluoro-aluminate; CsA, cyclosporin A, FITC,
fluorescein-5-isothiocyanate.
(Received 6 December 2001, revised 18 March 2002,
accepted 12 April 2002)
Eur. J. Biochem. 269, 2672–2677 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02929.x
Pgp Mg–ADP V
i
intermediate occurs randomly at one of
the two nucleotide-binding sites of Pgp, but not simulta-
neously [14–16]. It has been demonstrated in photoaffinity
labeling experiments that the affinity of Pgp to different
substrates is decreased after V
i
trapping, supporting the idea
that during ATP hydrolysis Pgp undergoes conformational
changes also affecting the drug binding site [17].
Conformational changes of Pgp upon its catalytic cycle
can be detected by measuring the binding of certain
monoclonal antibodies (mAbs; UIC2 [18], MC57 [19]). It
was previously shown that reactivity of the UIC2 mAb with
Pgp is increased in the presence of substrates/modulators or
ATP depleting agents, or when both nucleotide-binding
sites are inactivated by mutations [18]. The upshift of UIC2
binding in the presence of substrates/modulators is applied
as an indicator of the expression of functional Pgp
molecules in clinical tissue samples [20]. Changes in the
proteolysis profile of Pgp were also detected in the presence
of nucleotides alone or ATP with V
i

, indicative of
conformational changes propagating to the extracellular
domains of the pump upon its interaction with these
nucleotides [21].
We have recently shown [22] that drug transport-related
conformational changes of Pgp can also be detected via
mAb competition involving UIC2. Using the UIC2-
MM12.10 mAb pair, we have described that cyclosporin A
(CsA), vinblastine, and valinomycin (and several other
drugs; N. Nagy, K. Goda, F. Fenyvesi & G. Szabo
´
Jr,
unpublished data)
1
interactwithPgpinsuchamannerthat
preincubation of the cells with UIC2 completely suppresses
the subsequent binding of MM12.10. In contrast, UIC2
mAb added at saturating concentration decreased the extent
of MM12.10 labeling only mildly (up to  40%) without
drug treatments, or when the cells were preincubated with
verapamil or Tween-80 (and other drugs; N. Nagy, K.
Goda, F. Fenyvesi & G. Szabo
´
Jr, unpublished data)
2
.The
conformational state characterized by enhanced UIC2/
MM12.10 mAb competition (i.e. complete suppression of
MM12.10 labeling after UIC2 binding) may be intrinsic to
the normal catalytic cycle, or, alternatively, the CsA-type

drugs may induce a special conformation adopted by Pgp
only in the presence of these drugs. The first possibility
would be confirmed if the same conformational state could
be induced by nonsubstrate agents interfering with the
ATPase cycle. To study this question, we compared the
effects of P
i
analogues (V
i
,BeF
x
,AlF
4
), trapping nucleotides
at the catalytic site of Pgp, and of ATP depletion, on UIC2-
shift and UIC2/MM12.10 competition. Accurate molecular
interpretation of the changes in mAb binding are expected
to help to visualize the conformational steps during the
catalytic cycle.
MATERIALS AND METHODS
Cell lines
The human mdr1-transfected NIH 3T3 (NIH 3T3 MDR1
G185) mouse fibroblast cells [23] and the drug sensitive
human epidermoid carcinoma cell line KB-3-1 and its
multidrug resistant relative KB-V1 [24], obtained by
vinblastine selection, were used. The cell lines (obtained
from M. Gottesman, NIH, Bethesda, USA) were grown as
monolayer cultures at 37 °C in an incubator containing 5%
CO
2

and maintained by regular passage in Dulbecco’s
minimal essential medium (supplemented with 10% heat-
inactivated fetal bovine serum, 2 m
ML
-glutamine and
25 lgÆmL
)1
gentamycin). KB-V1 and NIH 3T3 MDR1
cells were cultured in the presence of 180 n
M
vinblastine or
670 n
M
doxorubicin, respectively. Cells were trypsinized
2–3 days prior to the experiments and maintained without
vinblastine or doxorubicin until use. The cells were occa-
sionally checked for mycoplasma
3
by the mycoplasma tissue
culture rapid detection system with a
3
H-labeled DNA
probe from General-Probe Inc. and were found to be
negative.
Chemicals
The cells were treated with the modulators or other agents at
the following concentrations: 100 l
M
verapamil, 100–
500 l

M
Na-orthovanadate (from a 13.5-m
M
translucent
stock solution freshly prepared in distilled water [25]),
fluoro-aluminate (AlF
4
–
; prepared from 1 m
M
AlCl
3
and
5m
M
NaF), beryllium fluoride (BeF
x
; prepared from 2 m
M
BeSO
4
plus 10 m
M
NaF) [11], 10 l
M
cyclosporin A, 50–
100 l
M
vinblastine or 10 l
M

valinomycin. Intracellular
ATP was depleted by 5 l
M
oligomycin or 5 m
M
sodium
azide applied together with 5 m
M
2-deoxy-D-glucose. The
optimal concentration of P
i
analogues (V
i
,BeF
x
and AlF
4
)
and ATP depleting agents were pretitrated in daunorubicin
accumulation experiments. The concentrations of the
chemicals applied increased steady-state daunorubicin
accumulation to the approximate level reached in Pgp
–
parental cells and did not significantly increase the ratio of
dead cells, as assessed by propidium iodide exclusion. All of
the above agents were from Sigma–Aldrich (Budapest). Cell
culture media and supplements were also from Sigma.
Fluorescein isothiocyanate (FITC) was purchased from
Molecular Probes (Eugene, OR, USA). All the other
chemicals used in the experiments were of analytical grade,

from Sigma. The MRK16 (Cancer Chemotherapy Center,
Tokyo, Japan), UIC2, 4E3, MC57, MM12.10 and MM8.15
anti-Pgp mAb preparations were > 97% pure by SDS/
PAGE. The FITC conjugates of MM12.10 and UIC2 were
prepared as described previously [26,27].
Flow cytometric assays
Nearly confluent monolayers of cells were harvested by
2–3 min trypsin treatment [0.05% trypsin and 0.02%
EDTA in NaCl/P
i
(pH 7.4)] and washed twice with
NaCl/P
i
before antibody labeling. The mAbs MRK16,
MC57, MM8.15, MM12.10 (8 lgÆmL
)1
)and10lgÆmL
)1
of UIC2 and 4E3 were applied. The antibody competi-
tion test was performed as follows: 10
6
cells in 1 mL
NaCl/P
i
supplemented with 8 m
M
glucose were preincu-
bated in the absence or presence of different drugs/
modulators at 37 °C for 20 min, then the first mAb,
UIC2 was added, without washing the cells. After further

30 min incubation at 37 °C, the FITC-conjugated secon-
dary mAb MM12.10 was added (again without washing
the cells) and incubation followed at 37 °C for another
30 min. The extent of competition between mAbs UIC2
and FITC-MM12.10 were expressed as R
competition
,the
difference of mean fluorescence intensities of cell-bound
FITC-MM12.10, in the absence and in the presence of
Ó FEBS 2002 Catalytic cycle of P-glycoprotein (Eur. J. Biochem. 269) 2673
UIC2, divided by the fluorescence intensity obtained in
the absence of UIC2.
In the case of the ÔUIC2 shift assayÕ [18], the cells were
pretreated with drugs, labeled first with UIC2 then with the
secondary antibody [FITC-conjugated goat anti-(mouse
IgG2a) Ig, F/P ¼ 4.3, from Sigma) on ice in 100 lLof
NaCl/P
i
for 45 min. The samples were washed twice after
staining and resuspended in 500 lLofNaCl/P
i
for flow
cytometric analysis.
Daunorubicin and calcein accumulation was measured as
described previously [28,29]. Briefly, the cells were preincu-
bated with modulators for 10 min or with P
i
analogues (V
i
,

BeF
x
or AlF
4
) for 20 min and then with daunorubicin or
calcein-AM for further 40 min, at 1 and 0.5 l
M
final
concentration, respectively. All the incubations were carried
out at 37 °C. The cells were washed and the samples stored
on ice until their measurement. The mean cellular fluores-
cence in each sample was determined using a modified
Becton Dickinson FACStar Plus flow cytometer (Mountain
View, CA, USA) equipped with an argon ion laser (Spectra-
Physics Inc. Mountain View, CA, USA). Dead cells stained
with propidium iodide were excluded from the analysis.
Fluorescence signals were collected in logarithmic mode and
the cytofluorimetric data were analyzed by the
FLOWIN
software (written by M. Emri & L. Balkay, University of
Debrecen, Positron Emission Tomography Center,
Hungary).
RESULTS
As ATP depletion is known to increase UIC2 reactivity
(UIC2-shift assay, [18]), we investigated if treatment of
cells with oligomycin, or sodium azide together with
2-deoxy-
D
-glucose, elicit a similar effect on UIC2/
MM12.10 competition. R

competition
(i.e. the relative decrease
of FITC-MM12.10 labeling after UIC2 pretreatment, see
Materials and methods) was used to characterize the effect
of the treatments. In ATP-depleted cells, UIC2 completely
abolished MM12.10 labeling similarly to CsA treated cells,
as shown by the R
competition
 1valuesinFig. 1.Thus,ATP
depletion affects UIC2 binding and UIC2/MM12.10 com-
petition in a parallel manner.
P
i
analogues are often used for blocking Pgp ATPase
activity in plasma membrane vesicles [10], and it was
recently shown that V
i
also affects UIC2 binding in living
cells [30]. We compared the effects of CsA and P
i
analogues
(V
i
,AlF
4
,BeF
x
) on Pgp function, measuring the cellular
accumulation of different Pgp substrates. As demonstrated
in Fig. 2, treatments with P

i
analogues restored steady-state
daunorubicin levels in NIH 3T3 MDR1 cells, similarly to
the effect of CsA applied at a concentration that completely
inhibits the pump. V
i
treatment also increased calcein
accumulation of Pgp
+
cells, albeit to a lesser degree
( twofold, as opposed to the 30-fold increase in intracel-
lular calcein levels observed after CsA treatment; data not
shown). As the addition of P
i
analogues did not influence
significantly either the daunorubicin or calcein uptake of the
parental cells (NIH 3T3 cells; Fig. 2B), their effect must be
Pgp-specific.
The consequences of V
i
,BeF
x
and AlF
4
treatments on
UIC2 binding were also examined. It was shown previously
by Druley et al. [30] that V
i
prevents UIC2 binding even on
vinblastine-treated cells. We have found that BeF

x
pretreat-
ment also decreased the reactivity of UIC2 with cell surface
Pgp molecules (data not shown), in contrast with CsA
treatment or ATP depletion that increased UIC2 binding in
accordance with what was previously shown by Mechetner
et al.[18].V
i
and BeF
x
also suppressed the effect of
substrates/modulators (verapamil, CsA, vinblastine) on
UIC2 binding when the cells were incubated in the
simultaneous presence of V
i
or BeF
x
, and any of the above
agents. The order of treatments with substrates/modulators
vs. V
i
or BeF
x
seemed to be indifferent as it is shown in the
case of CsA and P
i
analogues in Fig. 3. In contrast to V
i
and
BeF

x
,AlF
4
did not affect the binding of UIC2 despite its
inhibitory effect on Pgp function (compare Figs 2 and 3).
Fig. 1. Effect of ATP depletion on UIC2-MM12.10 mAb competition in
NIH 3T3 MDR1 cells. Cellular ATP production was inhibited by
30 min pretreatment of cells with 5 l
M
oligomycin or 5 m
M
sodium
azide applied together with 5 m
M
2-deoxy-
D
-glucose. Labeling with
mAbs was carried out and R
competition
was calculated as described in
Materials and methods. Means of three independent experiments are
shown (± SEM).
Fig. 2. Effect of phosphate analogues and cyclosporin A (CsA) on the
accumulation of daunorubicin into Pgp
+
(NIH 3T3 MDRl) and Pgp
–
(NIH 3T3) cells. The cells were preincubated in the presence of phos-
phate analogues (V
i

,BeF
x
,AlF
4
), or 10 lmCsAfor20 min,then1 lm
daunorubicin was added for 40 min. Solid line: daunorubicin only;
bold line: 10 l
M
CsA; empty triangles: 500 l
M
V
i
; black triangles:
BeF
x
; empty squares: AlF
4
treatment. Preparation of phosphate ana-
logues is described in Materials and methods.
2674 K. Goda et al. (Eur. J. Biochem. 269) Ó FEBS 2002
The reactivity of several other anti-Pgp mAbs (MRK16,
4E3, MM.12.10, MC57, MM.8.17) was not affected by
either V
i
or substrate/modulator treatment (data not
shown).
The effects of P
i
analogue treatments on UIC2/MM12.10
competition are shown in Fig. 4. The degree of the UIC2/

MM12.10 competition (i.e. the suppression of MM12.10
binding after UIC2) correlates with the level of UIC2
binding upon the same treatment (compare Figs 3 and 4).
DISCUSSION
The UIC2-shift [18] and the mAb competition phenomenon
involving UIC2 and MM12.10 mAbs [22] were affected by
pharmacological modulation of the ATPase cycle in a
parallel manner. Near the catalytic transition state stabilized
by V
i
or BeF
x
, Pgp apparently undergoes a global
conformational change involving the extracellular loops,
manifest in the drastic decrease of its affinity to UIC2. ATP
depletion increased UIC2 reactivity as expected [18],
preventing subsequent MM12.10 binding. AlF
4
that prob-
ably freezes the catalytic cycle at an earlier phase, trapping
unhydrolysed ATP [11], inhibited transport function with-
out preventing UIC2 binding.
In our experiments, different P
i
analogues affected Pgp
function and conformation in live cells. As the nucleotide-
binding domains of Pgp do not seem to be accessible from
the extracellular surface of the cell [31], our results suggest
that P
i

analogues penetrate the cell membrane, despite of
their negative charge.
V
i
can increase phosphorylation of Pgp at its linker
peptide region [32] similar to phorbol esters or phosphatase
inhibitors (e.g. ocadaic acid). However, the latter agents do
not affect or decrease drug accumulation [33,34], while in
our experiments, V
i
significantly inhibited drug transport. In
addition, other P
i
analogues, e.g. BeF
x
and AlF
4
also
inhibited Pgp mediated drug transport. Thus, the V
i
effect
seems to be independent of the phosporylation state of the
pump and is best interpreted assuming that V
i
trapping of
Pgp also occurs in live cells.
Coupling of Pgp-mediated drug transport and ATP
hydrolysis are generally interpreted in terms of ligand-
induced ATPase activation and concomitant transitions
between substrate-binding and substrate-releasing conform-

ational states. The consecutive steps of the catalytic cycle
appear to be discriminated by the changing affinity of Pgp
to the UIC2 mAb [18,30]. Reactivity of Pgp with UIC2 is
Fig. 4. Effect of phosphate analogues on UIC2/MM12.10 mAb
competition in NIH 3T3 MDR1 cells. Cells were preincubated for
20 min in the presence or absence of phosphate analogues (V
i
,BeF
x
,
AlF
4
), followed by the addition or omission of 10 l
M
CsA for an
additional 30 min and finally labeled with UIC2 and MM12.10 mAbs
(upper panel). Incubations with CsA and phosphate analogues were
also carried out in the reverse order (lower panel). Preparation
of phosphate analogues, labeling with mAbs and calculation of
R
competition
values are as described in Materials and methods. Means of
three independent experiments are shown (± SEM).
Fig. 3. Flow-cytometric fluorescence intensity distribution histograms
demonstrating UIC2-shift assays performed on NIH 3T3 MDR1 cells.
Cells were preincubated for 20 min in the presence or absence of
phosphate analogues, then 10 l
M
CsA was added and the cells were
further incubated for 30 min and finally labeled with UIC2 mAb. In

parallel experiments, incubations with CsA and phosphate analogues
were carried out in the reverse order. Phosphate analogues and CsA
were continuously present till the end of labeling with UIC2. UIC2
binding was visualized by indirect immunofluorescence. Solid line: no
CsA and phosphate analogue treatment; bold line: 10 l
M
CsA; empty
triangles: 10 l
M
CsA + 500 l
M
V
i;
black triangles: 10 l
M
CsA +
BeF
x
; empty squares: 10 l
M
CsA + AlF
4
treatment.
Ó FEBS 2002 Catalytic cycle of P-glycoprotein (Eur. J. Biochem. 269) 2675
increased in the presence of substrates/modulators or ATP
depleting agents, and in double Walker A and B mutants
incapable of ATP binding and hydrolysis [18,35]. These
findings may be interpreted suggesting that changes in the
UIC2 reactivity of Pgp are brought about by nucleotide
binding and release [18,30], or assuming that the hydrolysis

of ATP is the key element in driving Pgp from UIC2-
reactive conformation to a nonreactive state. We favor the
latter interpretation considering the following data. Previ-
ous studies demonstrated that linker region mutants capable
of binding ATP but unable to hydrolyze it, are recognized
by UIC2 similarly to the functional molecules that have
been preincubated by Pgp substrates [36], suggesting that
changes in the availability of the UIC2 epitope cannot be
explained by the step of ATP binding itself [35]. This
conclusion was further confirmed by the following obser-
vations. Mutation restricted to the N-terminal Walker B
motif abolished ATP binding at both ATP sites, while
mutation of the C-terminal motif did not affect ATP
binding at the N-terminal site [35]. Interestingly, both of the
above mutants were recognized by UIC2, implying that
ATP binding per se does not affect the availability of the
UIC2 epitope. In our experiments, V
i
(in agreement with
[30]) and BeF
x
treatment induced a significant decrease of
UIC2 binding, despite the presence of substrates or
modulators, while AlF
4
,knowntotrapbothATPand
ADP produced upon hydrolysis [11], inhibited drug
pumping similarly to V
i
and BeF

x
but did not affect UIC2
binding (Figs 3 and 4). These data also support a model
where the UIC2 epitope is available for antibody binding
throughout the catalytic cycle including the step of ATP
binding, to become unavailable only in the catalytic
transition state. ATP hydrolysis could cover the energy
expenses of a global conformational change effecting a
decreased affinity for substrates, as shown in vanadate-
trapping experiments [17].
As both substrate/modulator category identified on the
basis of UIC2/MM1210 mAb binding include agents that
stimulate, and others that inhibit, ATPase activity [37–39],
the two classes of substrates/modulators may not be
distinguished based on the overall catalytic rate achieved
by Pgp in their presence. The investigated conformational
effect of CsA (or vinblastine) manifest at the externally
located UIC2-binding epitopes is apparently reproduced by
ATP depletion and can be thwarted
4
by P
i
analogues,
confirming that the state elicited by CsA-type substrates/
modulators is part of the normal catalytic cycle. This latter
conformational state must be present for periods long
enough to be recognized by UIC2 in a way that leads to
mAb competition. In the presence of verapamil-like agents,
this state may be by-passed or its duration may be
diminished.

As UIC2 binding and UIC2/MM12.10 competition
could be influenced by nonsubstrate agents in a parallel
manner, the two phenomena are, in the case of the CsA-type
modulators, negative replicas of each other. Thus, the
epitopes opening up upon UIC2-shift are eventually titrated
back by MM12.10, without modulator treatment. A
practical implication of this finding is that the often variable
functional modulation of Pgp by modulators (e.g. CsA) in
the UIC2-shift assay can be substituted by the consecutive
application of UIC2 and MM12.10 for the labeling of Pgp
+
cells, in the absence of modulators.
ACKNOWLEDGEMENTS
This work was financially supported by OTKA funding T 032563 and
the research grant of the Hungarian Academy of Sciences AKP 98-83
3,3. This publication was also sponsored by the research grant of the
Ministry of Public Health ETT T01/103 and the OMFB grant 02692/
2000. M. C. is in part supported by grant 502 from Istituto Superiore di
Sanita
`
, Rome, Italy. The technical assistance of Eniko
¨
Pa
´
sztor is
gratefully acknowledged.
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