nucleotide binding to the human multidrug resistance protein 3, mrp3
TRANSCRIPT
Nucleotide Binding to the Human Multidrug Resistance Protein 3,MRP3
Andrea D. Hoffman • Ina L. Urbatsch •
Pia D. Vogel
Published online: 19 June 2010
� Springer Science+Business Media, LLC 2010
Abstract We used a spin-labeled ATP analog, SL-ATP,
to study nucleotide binding to highly purified human
multidrug resistance protein 3, MRP3, which had been
expressed in the yeast Pichia pastoris. SL-ATP was shown
to be a good substrate analog and is hydrolyzed by MRP3
at about 10% of the Vmax for normal ATP. ESR titrations
showed that 2 mol of SL-ATP readily bound per mole of
MRP3 with a dissociation constant of about 100 lM in the
presence of Mg2? ions. The binding curve was easily fitted
for a hyperbolic binding relationship. SL-ATP also bound
readily to MRP3 in the absence of divalent ions and
presence of EDTA. The resulting binding curve, however,
could not be satisfactorily fitted using the equation for
hyperbola. Analysis showed that a good fit was only
obtained with the Hill equation using a Hill coefficient of 4
or close to 4. Lower Hill coefficients resulted in lower
goodness of the fit. Such cooperative binding may be
explained by a dimerization event triggered in the absence
of divalent ions and a close communication of nucleotide
binding sites of the interacting dimers. These findings may
be of great importance for the overall mechanism and
regulation of multidrug resistance proteins.
Keywords Drug resistance pumps � ESR �Nucleotide binding � Spin-label � SL-ATP
1 Introduction
Multidrug resistance protein 3, MRP3, a member of the
ABCC3 subfamily, was discovered over a decade ago as
one of the first proteins in the family of multidrug resis-
tance proteins [16]. MRP3 has been of particular interest in
cancer biology due to its implicated role in conferring
multi-drug resistance in several carcinoma cell lines. Since
its discovery, considerable effort has been made to char-
acterize the transport substrates and substrate specificities
of MRP3 [8, 13, 28–30], for reviews see [4, 17, 22, 24],
with the goal to define a physiological role for the enzyme.
Despite these efforts, the physiological function of the
proteins remains elusive.
Relatively little focus has been placed so far on the
energy-harvesting events during substrate transport, i.e.
nucleotide binding and hydrolysis, mainly because the
enzyme was not available in sufficient amounts and at
acceptable purity for biochemical and biophysical analyses.
Only recently, basal and drug stimulated ATPase activity
of human MRP3 were reported for the enzyme expressed in
Pichia pastoris [7].
In this present study, we employed a highly sensitive
biophysical technique, Electron Spin Resonance Spectros-
copy (ESR) with use of a ribose-modified spin labeled
ATP, 20,30-SL-ATP, (SL-ATP) [5, 6, 9, 10, 12, 19, 20, 21,
23] to investigate the nucleotide binding characteristics of
human MRP3 under equilibrium conditions for the first
time. This technique allows us to obtain not only data
regarding the binding stoichiometry and dissociation con-
stants, but may also give independent information about the
A. D. Hoffman � P. D. Vogel (&)
Department of Biological Sciences, Southern Methodist
University, Dallas, TX 75275, USA
e-mail: [email protected]
I. L. Urbatsch
Cell Biology and Biochemistry, Texas Tech University Health
Science Center, Lubbock, TX 79430, USA
Present Address:A. D. Hoffman
Department of Physiology, University of Texas Southwestern
Medical Center, Dallas, TX 75390, USA
123
Protein J (2010) 29:373–379
DOI 10.1007/s10930-010-9262-4
relative structure and structural dynamics within the
nucleotide binding sites of MRP3 similar to studies we
performed with the mouse homolog of the human P-gly-
coprotein, MDR1 [9].
The information gained from these titration experiments
addresses the issue as to whether the two most likely func-
tionally different nucleotide binding sites of MRP3 also
differ in their ability to bind nucleotide, as has been sug-
gested for several of the ABCC family members, i.e. MRP1
[11, 14, 15] and CFTR [1–3]. We also addressed the question
on how the presence or absence of divalent cations like Mg2?
affects the overall nucleotide binding to the protein.
2 Results
2.1 ATPase Activity of Purified MRP3
The purified, detergent-soluble MRP3 (see Fig. 1) was
active in ATP-hydrolysis assays and was stimulated by the
putative transport substrate 17b-estradiol-17b-D-glucuro-
nide (E217bG). No additional stimulation of ATP hydrolysis
was achieved, however, upon addition of methotrexate or
taurocholate as shown in Table 1. All ATPase assays were
carried out for 20 min at 37 �C with solubilized enzyme. The
putative transport substrates E217bG (150 lM), methotrex-
ate (800 lM) or taurocholate (150 lM–1 mM) were added
directly to the cocktail mixture prior to the addition of pro-
tein. The ATPase activities determined from two different
methods (Pi release as in [25] or coupled enzyme assay [26]
resulted in comparable values. The data presented in Table 1
represent average values from both methods and are the
results of 4 to 36 individual experiments and their standard
deviations of the mean.
2.2 ESR Determination of SL-ATP Binding Affinity
Under Equilibrium Conditions
We assessed the usefulness of SL-ATP as an ATP analog
for MRP3 by investigating the enzyme’s capability to
hydrolyze the analog. ATPase assays using [25] showed
that SL-ATP is indeed a good ATP analog to study MRP3,
as it supported basal ATPase activity to *10% of that
determined with the same concentration of normal ATP.
The specific activities were determined at a concentration
of 1 mM ATP or SL-ATP, which is the upper range of
SL-nucleotide where the signal amplitude of the ESR spectra
is linearly correlated to the concentration of the SL-nucleo-
tides in ESR experiments, i.e. the upper concentration of
SL-ATP used in our ESR experiments. A specific activity
of 33 (±1) nmol/min/mg was determined in the presence of
1 mM Mg2? ATP, compared to 3.2 (±0.6) nmol/min/mg
determined in the presence of 1 mM Mg2? SL-ATP. Con-
trols showed that the detected activity was significantly
above background values. These results are from three
independent assays, which all gave very similar values.
Titration experiments employing SL-ATP allowed us
for the first time to directly characterize the binding stoi-
chiometry and affinity of the ATP analog to MRP3 under
equilibrium conditions. Using this technique we were able
to show that SL-ATP readily bound to human MRP3.
Figure 2a shows that in the presence of 2 mM Mg2?, non-
linear, hyperbolic curve fit extrapolated to a maximum
binding of 2.3 (±0.2) mol SL-ATP bound per mol of
MRP3 with an apparent Kd of 103 lM (±17 lM).
Figure 2b shows the ESR spectra of SL-ATP in the
presence of MRP3. The ESR spectrum of sub-stoichiom-
etric amounts of SL-ATP, with respect to putative
1 2 3 4 5 6 7 8
250
130
95
72
55
kDa
Fig. 1 Steps of wt human mrp3 purification from P. pastoris cells,
along with representative samples of purified MRP3 from indepen-
dent purifications. MRP3 is a 150 kDalton protein when expressed in
P. pastoris. All samples were run on an 8% SDS-PAGE gel followed
by Coomassie blue staining. Lanes 2–6 represent steps associated
with the purification of MRP3, lanes 7 and 8 represent purified MRP3
from independent purifications. Lanes are as follows: (1) molecular
weight marker, (2) 10 lg solubilized supernatant, (3) 10 lg of
Ni-NTA column flow-through, (4) 10 lg of Ni-NTA column wash
with 10 mM imidazole, (5) 10 lg of Ni-NTA column wash with
50 mM imidazole, (6) 20 lg of the 300 mM Ni-NTA column eluate.
Lanes (7) and (8) contain 10 lg of Ni-NTA column eluate from
independent purifications
Table 1 ATPase activity of wild-type human mrp3 in nmols/min/mg
protein as measured with either a coupled-enzyme assay (Delannoy
et al. [9]; Vogel and Steinhart [26]) or Pi-release assay (Van Vel-
dhoven and Mannaerts [25])
Wild-type mrp3 ATPase activity in
nmol/min/mg
Soluble enzyme 83 ± 20
? E217bG 135 ± 18
? methotrexate 86 ± 1
? taurocholate 76 ± 5
374 A. D. Hoffman et al.
123
nucleotide binding sites, in the presence of MRP3 is shown
in full. The three sharp signals in the center correspond to
free, not protein-bound SL-nucleotides. The signal ampli-
tude of the high field signal of the free SL-ATP was used to
determine the overall free SL-nucleotide in the experiment,
see Sect. 4. The relatively broad signals in the low and high
field regions correlate to the enzyme-bound SL-nucleotide
population and were re-recorded at higher signal gain for
better visualization (arrows). The signals were normalized
for protein concentration for better comparability. The
binding of SL-ATP to MRP3 is concentration-dependent,
as the intensity of the protein-bound SL-ATP signal
increased with increasing SL-ATP concentrations. MgATP
efficiently displaced SL-ATP binding, demonstrating that
the observed binding is specific to ATP and is not directed
by the spin label moiety (data not shown). The outermost
distance of the signals of the bound SL-nucleotide (2Azz-
value) was estimated to be about 54 G, indicative of a
relatively open nucleotide binding environment and similar
to observations made for the related P-glycoprotein [9].
Control experiments showed that the protein was still
active in ATPase assays after standard ESR experiments,
and that this activity was comparable to that determined
before the titration.
2.3 Determination of SL-ATP Binding to MRP3
in the Absence of Mg2?
Nucleotide binding in the vast majority of ATP-hydrolyz-
ing proteins is dependent upon some type of divalent cat-
ion. Indeed, in ATP hydrolyzing enzymes, Mg2?, Co2? or
Mn2? complexed with ATP are the substrates for hydro-
lysis and the divalent cation is needed for the actual
hydrolysis step to occur. This is often reflected by signifi-
cantly lower dissociation constants for the ion-chelated
nucleotide as compared to ATP in the absence of divalent
cations. To determine ion-dependence of ATP binding to
MRP3, titration experiments were carried out as previously
described, however MgS04 was omitted from the ESR
buffer and 5 mM EDTA was added to the solution to
complex trace amounts of ions. The results are shown in
Fig. 3a. Surprisingly, the shape of the resulting binding
curve did not seem to support typical Michaelis Menton
binding behavior but rather indicated sigmoidal relation-
ship between bound and free nucleotide. When fitting was
attempted using hyperbolic curve analysis, the curve
extrapolated to a maximum binding of about 3 mol/mol
with a Kd of 120 lM. The extremely low R2 (goodness of
the fit) value of 0.71 as well as visual inspection of the
curve shown in Fig. 3a clearly indicated that the data fit
using the hyperbolic equation, y = P1*x/(P2 ? x), was
very poor. The goodness of fit represents the difference
between observed values and the values expected from the
fitted model applied. Therefore, less discrepancy between
the measured and expected values will result in R2 values
close to 1.
Significantly better fit of the experimentally acquired
data was obtained when the Hill equation, see Methods,
was employed. This equation was subsequently used for
data analysis of nucleotide binding to MRP3 in the absence
of divalent cations, see Fig. 3b. Non-linear fit using the Hill
equation with no pre-set Hill coefficient value extrapolated
Fig. 2 SL-ATP binding to wild-type MRP3 in the presence of
2 mM Mg2?. a 18–40 lM MRP3 were titrated with 20, 30-SL-ATP.
The mol of protein-bound nucleotide per mol of protein was plotted
over the concentrations of free SL-ATP that were determined from
the corresponding ESR-spectra. Hyperbolic curve fit resulted in a
maximum binding of 2.3 (±0.15) mol/mol and a dissociation constant
of 103 lM (±17 lM). b ESR-spectra of SL-ATP binding to MRP3.
Full spectrum: 34 lM MRP3 in the presence of 54 lM SL-ATP. The
signals of the bound component can be seen in the low and high field
in the presence of 54 and 104 lM SL-ATP, with the black arrowpointing to the bound signal in both the low and high field
Spin-Labeled ATP to Study Nucleotide Binding Sites 375
123
to a maximum binding of 2.1 (±0.06) mol of SL-ATP per
mol of MRP3 and a dissociation constant of 80 lM
(±4 lM), which was very similar to that determined for
SL-ATP binding to MRP3 in the presence of Mg2?
(Fig. 2a). The Hill coefficient determined under these
conditions, however, was 4, which would indicate at least
four interacting nucleotide binding sites. To investigate this
result further, a series of Hill-analyses was performed
where the Hill coefficient was pre-set to 1 (no coopera-
tivity), or to the values 2, 3 or 4. The corresponding R2 for
the individual fits were 0.7392 for n = 1, 0.8736 for n = 2,
0.9195 for n = 3 and 0.9317 for n = 4, see Fig. 3c. Again,
line-shape analysis allowing for a Hill coefficient of 4
showed the best goodness of fit.
3 Discussion
3.1 ATPase and Stimulation
The basal and E217bG-stimulated ATPase activity of
recombinant MRP3 expressed in Pichia pastoris determined
in this study were similar to what has been previously pub-
lished [7]. One notable difference, however, was the obser-
vation that neither methotrexate nor taurocholate were able
to stimulate the ATPase activity of MRP3 as compared to
results from [7]. This lack of stimulation is potentially due to
differences in the reconstitution state of the protein and the
presence of lyso-PC that was used during solubilization and
purification, while in previous studies lipid reconstituted
Fig. 3 SL-ATP binding to wild-type MRP3 in the presence of 5 mM
EDTA. The experimental setup was identical to Fig. 2, however
Mg2? was excluded and 5 mM EDTA was added. 25–35 lM MRP3
were titrated with 20, 30-SL-ATP. The mol of protein-bound
nucleotide per mol of protein was plotted over the concentrations of
free SL-ATP that was determined from the corresponding ESR-
spectra. Single data points are visually represented as squares;
overlaying data points are represented as rectangles. a, Hyperbolic
curve fit analysis indicated a maximum binding of 3 mol of SL-ATP/
mol of MRP3 and a dissociation constant of 119 lM (R2 = 0.71). b,
SL-ATP binding to MRP3 in the presence of 5 mM EDTA fitted
using the Hill equation. Hill analysis resulted in a maximum binding
of 2.1 (±0.06) mol/mol and a dissociation constant of 80 lM
(±4 lM), with a hill coefficient of 4 (±0.8). c, Hill equation fit
analysis using pre-set n values of: n = 1(black), n = 2 (orange),
n = 3 (blue) and n = 4 (red)
376 A. D. Hoffman et al.
123
protein was used [7]. Protein solubilized in DDM and lyso-
PC was used in all ATPase activity assays to ensure com-
parability with the ESR experiments described.
3.2 ESR Spectroscopy
ESR studies showed that SL-ATP used in the studies
described here is an adequate ATP analog and readily
bound to solubilized MRP3. Independent hydrolysis assays
showed that SL-ATP was hydrolyzed by the enzyme at
about 10% the rate of normal ATP, values that have been
observed also for other ATPases like the human and mouse
P-glycoprotein (9 and unpublished) and F-type ATPases
from different organisms [19, 20, 27].
The outermost splitting of the signals of protein-bound
SL-nucleotides (2Azz-value) is indicative of the relative
mobility of the spin-label within the nucleotide binding
site, for examples see 5, 6, 9, 12, 19–21). Larger 2Azz
values usually result from spin-labels that are relatively
strongly immobilized within the nucleotide binding sites.
The 2Azz-value of SL-ATP bound to MRP3 was hard to
determine due to the relatively low concentration of protein
that was available for each titration experiment and the
comparatively low signal amplitude of the signals of the
bound SL-nucleotide. Estimation of the 2Azz-value was
furthermore complicated by the close spectral proximity of
the signals of the bound nucleotide to those arising from
the freely tumbling, not enzyme bound radicals (sharp
lines). We estimated the 2Azz-value to be between 54 and
55 Gauss, which would indicate relatively high mobility of
the spin-label, consistent with a structure and orientation of
nucleotide binding domains that are rather exposed to the
solvent in the resting state protein. Similar observations
were made in human and mouse P-glycoprotein (9 and
unpublished), where also only one spectral component was
observed in the resting state of the enzyme with a resulting
2Azz value of the spectra of 56 G for the normal mouse
homolog and 53 G for the corresponding cysteine-less
mutant [9]. The significantly higher stability of P-glyco-
protein as compared to the MRP3 used in this study had
allowed us, however, to obtain higher overall protein
concentrations during the ESR experiments that in turn
enabled us to obtain a better resolution of the signals
stemming from enzyme bound SL-nucleotide. In neither
case, P-gp or MRP3, were we able to observe ESR signals
that would suggest different conformations of the nucleo-
tide binding sites. This suggests that even though different
catalytic properties of the nucleotide binding sites are
proposed for MRP3, the overall structure and conformation
of the sites does not differ significantly, at least not within
the ribose-binding pocket of the sites.
Up to two SL-nucleotides bound readily to MRP3 in the
presence of divalent cations like Mg2?. The apparent
dissociation constant was determined to be about 100 lM.
The binding curve could be fitted adequately assuming a
hyperbolic relationship and one single dissociation con-
stant. Interestingly, in the absence of Mg2? and the pres-
ence of EDTA, SL-ATP also bound readily to MRP3 with a
comparable dissociation constant. The binding curve,
however, could not be fitted well using a traditional
Michaelis Menten, hyperbolic relationship of bound
nucleotide versus free. Using the Hill equation with no pre-
set Hill coefficient resulted in a fit with the best goodness
of fit and an R2 = 0.9317. Maximal binding of two
nucleotides per MRP3 were determined and a dissociation
constant of 80 lM. However, the Hill coefficient that
allowed for this fit was determined to be n = 4, indicating
at least 4 interacting nucleotide binding sites. Further Hill
analysis with pre-set Hill coefficients of 3, 2 or 1 (no
cooperativity) resulted in decreasing goodness of the fit.
One possible explanation for this surprising result of more
than two interacting nucleotide binding sites in an enzyme
with supposedly only two nucleotide binding sites may be
that MRP3 dimerized in the absence of Mg2? and other
divalent cation. Although our attempts to show such poten-
tial dimerization using alternative methods, i.e. by native
acryl amide gel analysis or by glutaraldehyde cross-linking
attempts, failed, this result seems of great interest with regard
to potential ion-dependent regulation of this multidrug
resistance pump and should be further investigated.
In order to ensure comparability of the results, we also
analyzed the binding of SL-ATP to MRP3 in the presence
of Mg2? using the Hill equation. Results extrapolated to a
maximum binding of 2 mol of SL-ATP bound per mol of
MRP3 with an apparent Kd of 83 lM (±5 lM), with a n
value of 1, which indicates non cooperative binding (data
not shown).
Collectively, these findings suggest that nucleotide bind-
ing to MRP3 is not Mg2? dependent per se, as similar stoi-
chiometries and affinities were observed in the presence and
absence of Mg2?. Since no detectable ATPase activity was
observed in the absence of Mg2? we conclude that while
magnesium may not be directly involved in nucleotide
binding it is clearly needed to activate the hydrolytic step of
the catalytic cycle. Whether or not Mg2? in addition may act
as a molecular switch by altering the assembly of MRP3
proteins and thereby the cooperativity between the nucleotide
binding domains seems a novel concept worth looking into.
4 Materials and Methods
4.1 Growth of MRP3
The human wild-type mrp3 used in this work was
expressed in the Pichia pastoris strain KM71wtmrp3 as
Spin-Labeled ATP to Study Nucleotide Binding Sites 377
123
described in [7]. Cells were either obtained as described in
[7] or using Fernbach flask cultures. The culture conditions
for growth of P. pastoris in Fernbach flasks were optimized
in order to achieve maximal protein expression. In short,
the cells were grown in 2-L Fernbach flasks in minimal
glycerol medium containing 1.34% yeast nitrogen base, 1%
glycerol, 4 9 10-5% biotin, 0.006% histidine until they
reached an OD600 between 3.0 and 4.0. Then the cells were
switched into minimal methanol medium containing 1.34%
yeast nitrogen base, 1% methanol, 4 9 10-5% biotin,
0,006% histidine and grown for 48 h at 28 �C. Under these
conditions, 20–25 grams of cells were typically obtained
per liter of culture.
4.2 Purification of MRP3
Highly enriched MRP3 was obtained similarly as described
in [7] with the following modifications: Microsomes of cells
expressing wild-type human mrp3 were prepared as in [18]
yielding 6–10 mg of microsomal protein per gram of wet
cells. Solubilization was achieved by suspending the
microsomes in buffer A (50 mM TES-NaOH, pH 7.4 at 4 �C,
50 mM NaCL, 30% (v/v) glycerol, 10 mM imidazole and
1 mM DTT) containing 0.6% n-dodecyl-b-D-maltoside
(DDM) and 0.1% lyso-PC (100%). This suspension was
subjected to sonication in a bath sonicator at 4 �C for 5 min
followed by 5 min incubation on a rocking platform at 4 �C.
These cycles were repeated a total of 3 times. Insoluble
material was removed through ultracentrifugation at 200,000
9g for 90 min. The supernatant containing MRP3 was
applied to a nickel-affinity chromatography column as
described in [18], with a ratio 1 ml of resin per 100 mg of
microsomal protein . The following successive wash steps
for MRP3 included: 15-bed volumes of buffer A containing
0.1% (w/v) DDM (Anagrade, Anatrace), 0.05% (w/v) lyso-
PC and 1 mM DTT, followed by 15-bed volumes of a buffer
containing 50 mM imidazole, 0.1% (w/v) DDM and 0.05%
(w/v) lyso-PC. MRP3 was eluted with 3 bed volumes of a
buffer containing 300 mM imidazole, 0.1% (w/v) DDM and
0.05% (w/v) lyso-PC. The Ni2?-NTA flow-through was
allowed to pass through the column at a rate of 1.0–1.5 ml/
min. The flow rate for the first and second wash step was
1 ml/min. The column was stopped once the elution volume
was added and allowed to sit for 20 min, after which the
column was opened and adjusted to a flow rate of 0.25 ml/
min. The use of pH 7.4 in all buffers (closer to MRP3’s
isoelectric point and substituting TES for Tris) throughout
the procedure drastically increased the solubility and sta-
bility of the protein. 6–12 lg of extremely enriched protein
per g wet cell were obtained. The Ni-NTA chromatography
fractions collected during the purification process were
subjected to immunoblot analysis using the M3II-21 anti-
MRP3 monoclonal antibody (Signet). Western blot verified
that the thick band seen in Fig. 1 was indeed MRP3 (data not
shown). Very little MRP3 was detected in the Ni-NTA flow-
through, as well as the wash steps containing the lower
concentration of imidazole. A considerable amount of MRP3
was detected in the 50 mM imidazole wash step, which was
sacrificed for the sake of purity of the final product.
4.3 ATPase Assays
ATPase assays were performed using a Pi release assay
described in [25]. 100–200 lg of MRP3 were added to
220–500 ll of ATPase cocktail pre-warmed to 37 �C. The
ATPase cocktail contained 50 mM Tris-HCL, pH 7.5,
10 mM ATP and 10 mM MgSO4. 150 lM b-Estradio
l17—(b-D-glucuronide), 800 lM methotrexate, or
150 lM–1 mM taurocholate were added directly to the
cocktail to test for substrate activation of the enzyme. An
equal volume of DMSO was added to control tubes in those
experiments where the substrate/modulator compounds
had been dissolved in DMSO. At different time points,
50–100 ll aliquots were transferred to 1 ml ice cold
20 mM H2S04 to quench the reaction with colorimetric
development carried out as described [25].
Alternatively, a coupled-enzyme assay similar to that
described in [9, 26] was used, where 30–80 lg of MRP3
were added to 1 ml of ATPase cocktail that was pre-
warmed to 37 �C. The ATPase cocktail contained 10 mM
ATP, 12 mM MgSO4, 1 mM PEP, 0.28 mM NADH,
50 mM Tris-HCL, pH 7.5, 10 mM KCl, 0.014 mg/ml
lactate dehydrogenase and 0.0288 mg/ml pyruvate kinase.
150 lM b-Estradiol17–(b-D-glucuronide), 800 lM metho-
trexate and 150 lM–1 mM taurocholate were added
directly to the cocktail where indicated. ATP hydrolysis
was recorded as the absorbance decrease at 340 nm
(e = 6,300 M-1 cm-1). Control experiments showed that
none of the modulators used in this study interfered with
the cocktail components or absorbance decrease. Routinely
0.1 mM EDTA, 4 mM NaN3 and 1 mM ouabain were
added to the cocktail mixture to inhibit non-specific
ATPase activity. Control experiments showed that these
compounds did not affect the assay.
4.4 ESR-Titrations to Study SL-ATP Binding
Nucleotide binding studies were performed using a Bruker
EMX 6/1 ESR spectrometer operating in the X-band mode,
and equipped with a high sensitivity cavity. MRP3 concen-
trations were typically about 20 lM in 40 ll total volume.
Stepwise additions of SL-ATP were made in 1 ll steps.
Samples were gently mixed and exactly 30 ll were pipetted
into a glass capillary and placed into a quartz cuvette which
was then placed in the ESR cavity. The centerfield was set to
3,325 G and an area of 100 G was scanned with a peak-to-
378 A. D. Hoffman et al.
123
peak modulation amplitude of 1 G. The time constant was set
to 10.40 ms and the conversion time of 40.96 ms, which
resulted in a total sweep time of 41.943 s. The signal gain
was adjusted to the SL-ATP concentrations in the different
experiments. Signal amplitudes of the high-field signal were
determined using WINEPR (Bruker) software for each
concentration of SL-ATP used. Special care was taken to
ensure that the signal amplitudes had stabilized, which
included 15–20 individual determinations of signal ampli-
tudes for each concentration of SL-ATP. A standard curve
was generated for every experiment, prior to the actual
titration in the presence of protein. The buffer used for the
standard curve contained the same components as the buffer
used for the measurements in the presence of the protein. To
accommodate for the viscosity changes of the solution due to
extensive concentrating of the protein sample in the presence
of detergent, the corresponding standard curve buffer was
also concentrated down to the same degree. This eliminated
large differences in viscosity between samples, thus allow-
ing more accurate determination of binding stoichiometry.
Finally, the amount of protein-bound spin-labeled nucleotide
was determined as the difference between the known total
concentration of spin-labeled nucleotide added to the ESR
cuvette and the free spin-labeled nucleotide observed. The
free nucleotide was measured directly by comparing the
signal amplitude of the high field signal of the unbound
nucleotide spin label to the standard curve of known con-
centrations of spin-labeled nucleotide that was generated in
the absence of protein as described above. The mol of pro-
tein-bound SL-ATP per mol of protein was plotted over the
concentration of free SL-ATP present and the results were
fitted using the equation y = P1*x/(P2 ? x) for a rectan-
gular hyperbola, where P1 represents the maximum number
of binding sites and P2 represents the dissociation constant
Kd. Nonlinear curve fits were performed using Origin 7
(OriginLab Corp.).
To determine the ATP binding characteristics of MRP3
in the absence of Mg2?, MgSO4 was omitted and 5 mM
EDTA was added. The mol of protein-bound SL-ATP per
mol of protein was plotted over the concentration of free
SL-ATP present. Results were fitted using either hyper-
bolic curve analysis (see above) or with the Hill equation.
In the Hill equation y = Bmax (xn/(kn ? xn)), where B max
represents the maximum number of binding sites, k rep-
resents the dissociation constant, and n is the Hill coeffi-
cient value. Nonlinear curve fits were performed using
Origin 7 (Origin Lab Corp.).
Acknowledgments This work was supported by a grant from the
SMU University Research Council to PDV and a grant from the
Jasper and Jack Wilson Foundation to ILU. The authors wish to thank
J. G. Wise for helpful discussions. The authors wish to thank Dr.
Jurgen Zirkel, Lipoid GmbH, Germany, for providing us with 100%
pure lyso-phosphatidyl cholin.
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