From the Institute of Physiology, Department of
Membrane Transport Biophysics, Academy of Sciences of the Czech
Republic, Prague CZ 14220, Czech Republic, § Institut
für Biochemie, Universität zu Köln, 50674 Köln,
Germany, and ¶ Institute of Experimental Botany, Isotope
Laboratory, Academy of Sciences of the Czech Republic,
Prague CZ 14220, Czech Republic
Received for publication, October 16, 2000, and in revised form, November 14, 2000
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ABSTRACT |
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The electroneutral Pi uptake
via the phosphate carrier (PIC) in rat liver and heart mitochondria is
inhibited by fatty acids (FAs), by
12-(4-azido-2-nitrophenylamino)dodecanoic acid (AzDA) and
heptylbenzoic acid (~1 µM doses) and by lauric,
palmitic, or 12-azidododecanoic acids (~0.1 mM
doses). In turn, reconstituted E. coli-expressed yeast PIC
mediated anionic FA uniport with a similar pattern leading to FA
cycling and H+ uniport. The kinetics of
Pi/Pi exchange on recombinant PIC in the
presence of AzDA better corresponded to a competitive inhibition mechanism. Methanephosphonate was identified as a new PIC substrate. Decanephosphonate, butanephosphonate, 4-nitrophenylphosphate, and other
Pi analogs were not translocated and did not inhibit Pi transport. However, methylenediphosphonate and
iminodi(methylenephosphonate) inhibited both electroneutral
Pi uptake and FA cycling via PIC. AzDA analog
16-(4-azido-2-nitrophenylamino)-[3H4]-hexadecanoic
acid (3H-AzHA) bound upon photoactivation to several
mitochondrial proteins, including the 30- and 34-kDa bands. The latter
was ascribed to PIC due to its specific elution pattern on Blue
Sepharose and Affi-Gel. 3H-AzHA photolabeling of
recombinant PIC was prevented by methanephosphonate and diphosphonates
and after premodification with 4-azido-2-nitrophenylphosphate. Hence, the demonstrated PIC interaction with monovalent long-chain FA
anions, but with divalent phosphonates of short chain only, indicates a
pattern distinct from that valid for the mitochondrial uncoupling
protein-1.
The mitochondrial phosphate carrier
(PIC)1 (1-10) belongs to the
well established gene family of homologous mitochondrial anion carrier
proteins (10-12). It was thought to mediate a stoichiometric Pi*H+ symport (7, 13), but alternatively, a
Pi/OH Indeed, PIC (20) as well as homologous proteins, the ADP/ATP carrier
(AAC; Refs. 19, 21, and 22) and uncoupling proteins UCP1 of brown
adipose tissue mitochondria (19, 23-25), plant uncoupling
mitochondrial protein (26), and recently discovered UCP2 and UCP3 (27)
have been shown to interact with fatty acids (FAs) and are predicted to
mediate the uniport of fatty acid anions (19-23, 26, 27). This allows
for the FA uncoupling cycle, suggested by Skulachev (28, 29). For UCPs,
the FA cycling should represent a major physiological function, whereas
for AAC, PIC, and other carriers, it could be a "tolerated" side
function (19, 20). FAs usually inhibit the other functions of the
carriers. Thus, anion uniport via UCP1 is inhibited by various
FAs (23), including 12-(4-azido-2-nitrophenylamino)dodecanoic acid
(AzDA; Ref. 24). The latter also inhibits ADP uptake on AAC (21) and
the dicarboxylate carrier-mediated transport (30). UCP1 was also found
as a prominently labeled band among PAGE-separated proteins of brown
adipose tissue mitochondria, photolabeled with 3H-AzDA
(24), or with its hexadecanoic acid analog (3H-AzHA; Ref.
25). 3H-AzHA labeling was successfully applied to AAC as
well (21).
PIC has also been found to be partially inhibited by fatty acids;
however, it has been interpreted in terms of a surface charge effect
(31). Besides FAs, the only reported hydrophobic substrate analog was
4-azido-2-nitrophenylphosphate, which inhibited Pi transport by PIC only upon photoreaction when it was covalently attached to PIC after UV irradiation (32). Hence, the specificity of FA
interaction with PIC remains to be elucidated, mainly to determine
whether FAs interfere with a putative H+(OH In this paper, we studied both inhibition of PIC by fatty acids and
other hydrophobic anions, namely hydrophobic phosphate analogs, as well
as fatty acid cycling mediated by PIC. Our results demonstrate that the
pattern of PIC interaction with hydrophobic anions is distinct from
that of UCP1 (33) and other mitochondrial uncoupling proteins.
Materials--
Alkylphosphonates were purchased from Fluka
(Switzerland); materials for electrophoresis, hydroxylapatite (Bio-Gel
HTP), and Affi-Gel were from Bio-Rad. AzDA and 3H-AzHA were
synthesized as described elsewhere (24, 25). The autoradiography
enhancer ENTENSIFY NEF-992 was from PerkinElmer Life Sciences. Other
materials were from Sigma.
Chemical Syntheses--
For 12-azidododecanoic acid, 100 mg of
12-bromododecanoic acid methylester (0.36 mmol) and 116 mg of
NaN3 (1.8 mmol) were stirred in 2 ml of dry
N,N-dimethylformamide at 20 °C for 12 h. The reaction mixture was diluted with 6 ml of water and extracted with
ether. The organic layer was dried and evaporated to an oil, which was
hydrolyzed by stirring with 0.3 ml of 1 N methanolic KOH
(20 °C for 12 h) and subsequently diluted with water and
acidified with 5 N HCl, and 12-azidododecanoic acid
was extracted several times with ether. The organic fraction was washed
with water, dried, and evaporated to a colorless oil (81 mg) that
crystallized in a refrigerator. The product exhibited typical IR (KBr)
spectral peaks at 2097 cm
4-Azido-2-nitrophenylphosphate was synthesized by the modified method
of King and Nicholson (34) from 4-azidonitrophenol prepared according
to Ref. 35, since the original procedures, based upon reaction of
4-azido-2-nitrophenol and 1,4-diaza-bicyclo[2.2.2]octane in
POCl3 or 4-azido-2-nitrophenol and trichloroacetonitrile in phosphoric acid (35), gave very low yield. Thus, 250 µl of
POCl3 (2.8 mmol) was cooled to Biological Material--
Rat liver mitochondria were isolated
from Wistar rats in 250 mM sucrose, 10 mM
Tris-MOPS, 0.1 mM Tris-EGTA, pH 7.4, containing 0.5% BSA.
BSA was omitted in the final washing. Mitochondria from trypsinized rat
heart in 180 mM KCl, 5 mM Tris-Cl, 10 mM Tris-EDTA, pH 7.4, were isolated by differential
centrifugation in 180 mM KCl, 5 mM Tris-Cl, pH
7.4, containing 0.5% BSA (36). Prior to swelling measurement, the KCl
content was minimized by two washings in a sucrose medium.
The yeast phosphate carrier was expressed in Escherichia
coli as described elsewhere (8). The aliquots of inclusion bodies containing about 3 mg of protein were suspended and washed two times in
10 mM Tris-Cl, 0.1 mM Tris-EDTA, pH 7.0. The washed pellet was presolubilized by 1.5 ml of 5 mM
TEA-TES, 30 mM TEA2SO4, 0.1 mM Tris-EDTA, pH 7.2, containing 0.3% sodium
lauroylsarcosinate. After centrifugation at 14,000 × g
for 2 min, the resulting pellet was solubilized in 0.75 ml of 5 mM TEA-TES, 30 mM
TEA2SO4, 0.1 mM Tris-EDTA, pH
7.2 containing 1.67% sodium lauroylsarcosinate and 1% octylpentaoxyethylene.
Phosphate Transport in Mitochondria--
Anion transport in
mitochondria was indicated by osmotic swelling while detecting light
scattering (LS) at 530-550 nm as an apparent absorbance on a
diode-array spectrophotometer (Spectronics 3000). LS intensity,
reflecting the inverse volume, allows measurement at a protein
concentration as low as 0.2 mg/ml in an optimum 40% isotonic medium
osmolarity (270 mosmol as 100%). 44 mM KPi, 54 mM salts of monovalent anions, 36 mM salts of
divalent anions, and 27 mM salts of trivalent anions were
employed, all roughly corresponding to 108 mosmol at full ionization.
The changes in the normalized reciprocal absorbance Phosphate Transport in Proteoliposomes--
The
Pi/Pi antiport mediated by the reconstituted
recombinant yeast PIC was determined by measuring
[33P]phosphate flux using a forward exchange procedure
(8, 9). Kinetics was evaluated by variations of the external
[Pi], while 30 mM [Pi] was kept
constant in the vesicle interior. Proteoliposomes prepared in a medium
containing 30 mM [Pi] were washed on a
Sephadex G25-300 soaked with the desired external [Pi],
while PIC was blocked by mersalyl (0.3 mM). Transport was
then initiated by the addition of 50 mM dithiothreitol. An
inhibitor-stop assay has been employed, so that 64 mM
pyridoxal phosphate was added after a given time. The sample was then
passed through the Dowex column (1-X10, Cl
Reconstitution was performed by 14 cycles of detergent removal on a
single column as described elsewhere (8, 9). Briefly, 94 µl of PIC
(e.g. 0.596 mg/ml) solubilized from inclusion bodies, 224 µl of preformed liposomes (22.4 mg of lipid) from E. coli lecithin (Avanti Polar Inc.), and 140 µl of Triton X114 were vortexed and supplied by stock solutions to obtain 1.4 ml of suspension containing PIC (e.g. 56 µg), 30 mM
KPi, 50 mM K-HEPES, pH 6.8. It was passed 14 times over a column filled with 0.6 g of Bio-Beads-SM2 (Bio-Rad).
Proton Uniport Induced by Fatty Acids in
Proteoliposomes--
PIC reconstitution was performed by the detergent
removal method designed for fluorescent H+ transport
monitoring by SPQ quenching on a Shimadzu fluorometer, RF5301 PC, as
described elsewhere (20). To assay H+ fluxes, monitored as
Photoaffinity Labeling--
Mitochondrial photomodifications
with AzDA and photoaffinity labeling with 3H-AzHA were
performed using protocols described for brown adipose tissue
mitochondria (25). Rat liver or rat heart mitochondria (3 mg of
protein) were resuspended in 10 ml of BSA-free isolation medium and
shaken for 10 min in an ice bath. 3H-AzHA (or
nonradioactive AzDA) was added to reach a final concentration of 0.46 µM (1.5 nmol/mg of protein), and the mixture was shaken in an ice bath, first in darkness for 10 min and then for 10 min under
UV illumination with a 400-watt xenon arc lamp equipped with a WG 8 filter (Schott Glass, Germany) transmitting light above 270 nm. Labeled
mitochondria were washed by three centrifugations with BSA and three
without BSA at 8500 × g. The last pellet was resuspended in 100 µl of the sucrose medium.
Isolation of Rat Heart Phosphate Carrier--
Chromatography on
hydroxylapatite was conducted using stepwise fractionation on spin
columns. Lower loads of mitochondrial octylpentaoxyethylene or Triton
X-100 extract (45 mg of protein per 3 g of dry HTP) gave higher
yields of PIC and AAC content. The labeled mitochondria were either
applied to the HTP column, or proteins contained in the HTP
pass-through (38) were photolabeled in the detergent micelles with
3H-AzHA, using a protocol developed for UCP1 (24, 25).
Labeled proteins were further fractionated on blue Sepharose (2 mg/ml of column) using a modified procedure of Rojo and Wallimann (39). BS
was prewashed, and the first four elutions (1 ml each) were also
performed with 150 mM Na2SO4, 20 mM Na-HEPES, 0.2 mM Tris-EDTA, pH 7.0, containing 0.5% detergent. Thus, a "flow-through" and four
"wash" fractions were obtained. A stepwise NaCl gradient (0.9, 1.5, 2, and 2.7 M) in 20 mM Na-HEPES, 0.2 mM Tris-EDTA, pH 7.0, with 0.5% detergent was subsequently
applied, while the final elutions were performed four times with 1 ml
of 150 mM NaCl containing 0.5% SDS. Since BS retained
mostly AAC, other proteins, including PIC, were contained predominantly
in the flow-through fraction. Proteins of the HTP or BS
flow-through fractions were further separated using organomercurial
affinity chromatography on Affi-Gel 501 (Bio-Rad) soaked in the same
medium as BS. After loading samples, five elutions (0.5 ml each) were
conducted with the BS medium and two subsequent series of five elutions
by the BS medium with 1.5 and 30 mM mercaptoethanol,
respectively. The Affi-Gel 501 was reported to retain the PIC (40), and
hence elutions with 1.5 mM mercaptoethanol should yield
PIC, since the combination of BS and Affi-Gel excludes most proteins of
the HTP eluate.
Laemmli SDS-PAGE was conducted, either on a Mighty Small II apparatus
(Hoefer, minigels), or on a Protean IIxi (Bio-Rad, 15-cm gels). In both
cases, high resolution 17% acrylamide gels were cast with an
acrylamide/bisacrylamide ratio of 150:1, allowing for an expansion of
the 30-40-kDa region (38). Parallel gels to those for autoradiography
were silver-stained using the bichromate method. Coomassie Blue-stained
gels were treated with an autoradiography enhancer ENTENSIFY and dried
between plastic follies and a filter paper under vacuum. Dried gels
with peeled-out follies were exposed in the steel cassettes on Kodak
Scientific Imaging Film X-Omat, AR-5, for 5-10 days (mitochondria) or
10-21 days at Inhibition of Phosphate Transport by Natural and Azido Fatty
Acids--
Rapid electroneutral phosphate uptake was induced in rat
liver (Fig. 1a) or rat heart
(Fig. 1b) mitochondria as a passive swelling initiated by
nigericin in 44 mM potassium phosphate, pH 7.4. Participation of PIC is indicated by electroneutrality and by the
specific inhibitory pattern (1-6) when both NEM and mersalyl are
inhibiting (Fig. 1a). FAs such as lauric (Fig.
2a) and palmitic acid only
inhibited Pi uptake in high concentrations. Lauric acid
exhibited a Ki of 250 µM (Fig.
2a). Contrary to a rather weak inhibition by natural FAs, 10 µM AzDA inhibited Pi uptake by more than 90%
(Fig. 1a), and the estimated Ki was 3.8 µM (Fig. 2a). A substantial inhibitory
strength was exhibited by heptylbenzoic acid (Ki of
89 µM; Fig. 2b). 12-Azidododecanoic acid was
inhibiting with a lower potency (Ki of 310 µM; Fig. 2b). The protein-independent swelling
(induced by nigericin in potassium acetate) was only affected by 10%
with 10 µM AzDA, suggesting that AzDA produces no major
nonselective side effect on the inner mitochondrial membrane. AzDA,
even at 50 µM doses, did not inhibit swelling in sodium
acetate, which reflects the electroneutral
Na+/H+ antiport (Fig. 2a). No
inhibition of the latter by any FA tested was found, nor inhibition of
pyruvate carrier (41), thus demonstrating specificity for PIC.
Interestingly, the FA derivatives, which were previously found (42, 43)
to be unable to flip-flop across the lipid bilayer, such as
12-hydroxylauric (Fig. 2b), phenylvaleric, and
dodecanedioic acid, did not affect Pi transport. Since
the other FAs tested, including 12-azidododecanoic
acid,2 were confirmed to
possess the ability of fast flip-flop, we suggest that it is a specific
(U-shape) conformation in the membrane (42) that prevents these
inactive FAs from interacting with PIC. Photoactivated AzDA also
inhibited Pi uptake in mitochondria with an apparent IC50 of 4 µM. Since the mitochondria were
first irradiated in the presence of AzDA, washed with BSA, and then
reisolated, the apparent IC50 is not directly comparable.
Coincidentally, however, it is the same as the above reported
Ki value; IC50 of 4 µM was
obtained when mitochondria preincubated first with AzDA in the dark
were washed with BSA and reisolated. Consequently, UV illumination does
not seem to strengthen the AzDA inhibition. AzDA binding is apparently
so tight that no effective washing can eliminate it.
Kinetics of Fatty Acid Inhibition of Phosphate
Transport--
To establish the type of inhibition by fatty acids,
kinetic measurements with the reconstituted E. coli-expressed yeast PIC have been performed.
Pi/Pi exchange was measured as a forward 33P uptake into proteoliposomes containing recombinant PIC,
while varying external Pi between 1 and 30 mM.
Kinetics of such Pi/Pi exchange determined in
the absence or presence of 100 µM AzDA was found to agree
better with a competitive mechanism (Fig.
3). Although data cannot definitively
distinguish between the competitive and noncompetitive type of
kinetics, fits of the competitive model gave better agreement. The
derived Vmax in control was 0.55 µmol of
Pi·min Screening of Possible Hydrophobic Substrates of Mitochondrial
Phosphate Carrier--
Interaction of PIC with FAs suggested that PIC
could also interact with some other amphiphilic anions. Therefore, we
evaluated whether hydrophobic phosphate analogs are transported by PIC
or act as competitive inhibitors. Contrary to the alkylsulfonate translocation by UCP1 (33), which is faster with the increasing chain
length, decanephosphonate and butanephosphonate were not transported at
a significant rate by PIC, and neither inhibited Pi
transport up to a 0.7 and 100 mM dose, respectively. We
found that only phosphonate with the shortest chain, methylphosphonate, is the PIC substrate. Methylphosphonate exhibited the same transport characteristics as Pi transport, including NEM sensitivity
(Fig. 4) and inhibition by lauric acid,
12-azidododecanoic acid, and AzDA (Fig. 4).
We also attempted to evaluate whether some other amphiphilic compounds
are transported by PIC or inhibit Pi transport in
mitochondria. While screening various mono-, di-, and
trialkylsulfonates and benzene mono-, di-, and trisulfonates or
anions derived from phosphate and phosphonate, such as phosphoformate,
phosphopyruvate, phosphogluconate, and 4-nitrophenylphosphate, we found
no such case. Particularly, we confirmed that phosphoformate does not
inhibit the net electroneutral Pi uptake, as reported
previously (8). On the contrary, methylenediphosphonate and
iminodi(methylenephosphonate) were found to be strong inhibitors (Fig.
5a and b). The
Ki values derived for their inhibition were 4.9 and 5.2 mM, respectively. However, they were not
transported by PIC. Also, although 4-azido-2-nitrophenylphosphate
(AzNPPi) was previously found to inhibit swelling in
NH4-Pi only when photoactivated (32), in our
study it inhibited the nigericin-induced Pi transport via
PIC in the dark with a Ki of 1.5 mM
(Fig. 5a). Its analog, lacking the azido group,
4-nitrophenylphosphate, did not exhibit an inhibitory effect.
Possible Fatty Acid Cycling Mediated by Phosphate
Carrier--
Although inhibitory to Pi transport, FAs were
previously found to induce H+ uniport in proteoliposomes
containing PIC, sensitive to diphosphonates (20). As for AAC and UCPs,
it has been interpreted in terms of FA cycling (23, 28), in
which uniport of anionic FA is mediated by PIC and the neutral FA
diffuses back across the lipid bilayer, thus carrying H+.
Fig. 6a illustrates the dose
responses for lauric acid-induced H+ uniport (lauric acid
cycling) in proteoliposomes with the reconstituted recombinant PIC in
the absence or presence of 10 mM methylenediphosphonate (MDPh). The difference between them gives a net H+ flux
density sensitive to MDPh, hence a portion of transport that can be
ascribed to PIC. It follows a Michaelis-Menten kinetics (Fig.
6b). The FA cycling via UCP1 was found2 to be
insensitive to MDPh. The residual H+ uniport is very
similar to the one observed in protein-free liposomes (20). Other FAs
tested gave similar kinetics (Fig. 6c and d), with Vmax (derived from MDPh-sensitive fluxes,
in nmol of H+·s 3H-AzHA Labeling of Phosphate Carrier in
Mitochondria--
Using a rather small amount (1.5 nmol/mg of protein)
of highly tritiated azido-FA (3H-AzHA) incubated with rat
heart or rat liver mitochondria and subsequently illuminated by UV
light, only a small portion of numerous mitochondrial proteins were
labeled with 3H-AzHA (Fig.
7). This is very similar to the
photolabeling of brown adipose tissue mitochondria, which yielded UCP1
as the major labeled band (25). Among the labeled proteins, the most
apparent were 30- and 34-kDa bands as illustrated by a typical
autoradiogram (Fig. 7). These two most likely correspond to AAC (22)
and PIC, respectively. Our further steps led to the verification of
this assumption.
3H-AzHA Labeling of Partially Purified Phosphate
Carrier--
To confirm that PIC is the protein interacting with
3H-AzHA, we chose to separate proteins of the
hydroxylapatite eluate by two subsequent affinity chromatography steps,
selective enough to determine the final product as PIC. We solubilized
rat heart and liver mitochondria by octylpentaoxyethylene or Triton
X-100, passed the extracts through HTP, and attempted to photolabel
containing proteins. As a result, the 30- and 34-kDa bands were
again photolabeled with 3H-AzHA (Fig.
8a). In a further experiment,
just the 34-kDa band but not the 30-kDa band was found in the
flow-through fraction of the Cibacron blue affinity agarose column (BS
column) and still retained the 3H-AzHA label attached (Fig.
8a). In turn, AAC (30-kDa monomer) was tightly bound to the
BS column and could be eluted as reported before (39), either at higher
NaCl concentrations or with SDS (Fig. 8a). Also, the 30-kDa
band in these fractions retained the 3H-AzHA label. It was
identified as AAC by Western blots (22). The "upper" bands were
found only in minute amounts in the intermediate NaCl fractions of the
BS column.
When we further fractionated the BS flow-through fraction on Affi-Gel
501 (an organo- mercurial affinity matrix) and eluted first with a
medium containing low and then high mercaptoethanol concentration, the
intermediate fractions yielded only a 34-kDa band, which still retained
the 3H-AzHA label (Fig. 9,
sample 1). Also, when the HTP pass-through containing the labeled proteins was applied on the Affi-Gel, the resulting flow-through contained most of the typical HTP bands, among
which only AAC was labeled with 3H-AzHA (not shown). Hence,
one may expect that the protein retained in the Affi-Gel was
predominantly the PIC protein (40). Identical results were obtained
when rat liver or heart mitochondria was first labeled with
3H-AzHA and then fractionated on HTP, BS column (Fig.
8b), and Affi-Gel (Fig. 9).
Prevention of 3H-AzHA Photolabeling by
4-Azido-2-nitrophenylphosphate, Methanephosphonate, and
Diphosphonates--
To evaluate whether the Pi
binding domain of PIC is close or overlaps with the putative FA binding
site, we performed competition studies and tested which phosphate
analogs would prevent the 3H-AzHA photoaffinity labeling of
recombinant yeast PIC. Percentages of the remaining 3H-AzHA
label were quantified from the band density compared with controls.
There was no change in the 3H-AzHA photolabeling when
preincubations with phosphonoformate or undecanesulfonate were
performed (Fig. 10). Pyrophosphate and 4-nitrophenylphosphate partly prevented the 3H-AzHA
label. Photolabeling was prevented by preincubations with the newly
identified substrate, methanephosphonate (10 mM), and was
completely prevented by 10 mM methylenediphosphonate and
iminodi(methylenephosphonate) (Fig. 10). The amount of the
3H-AzHA label on yeast PIC also decreased after the
preceding photolabeling with 4-azido-2-nitrophenylphosphate (Fig.
10).
We have demonstrated the ability of fatty acids to inhibit
(strongly for azidonitrophenyl-FAs) the mitochondrial phosphate carrier
(PIC) and to function as its monovalent anionic substrates in a FA
cycling process. Furthermore, photolabeling of PIC with azidonitrophenyl-FAs has also been performed. In this way, we have
tried to characterize the corresponding FA binding site and its
relationship to the Pi binding site. We have found that
these two sites reside close to each other (or might overlap), since the inhibition of Pi/Pi exchange by FAs is most
likely of a competitive type, and covalently bound AzNPPi,
the newly identified substrate methanephosphonate, and the inhibitory
diphosphonates did prevent 3H-AzHA photolabeling.
A question now arises concerning the nature of FA interaction with PIC.
An identity of the FA binding site and the Pi binding site
could be excluded by the lack of PIC interaction with long-chain alkylphosphonates. On the contrary, a proximity of these two sites may
be the origin for the observed competitive inhibition of
Pi/Pi exchange by FAs, for prevention of
3H-AzHA binding by some Pi derivatives, and for
the correlation between Ki values of FAs inhibiting
Pi uptake and the apparent affinity of cycling FAs. We have
intentionally selected FAs covering a range of affinities or
Ki values. FA cycling occurred at higher FA
concentrations as suggested by higher Km values
(~100 µM) when compared with UCP1 (~10
µM; Ref. 23). The presence of a 4-azido(2-nitrophenyl)
group enhanced the inhibitory ability of FAs, but it is not exclusively
this portion of AzDA that mediates the inhibitory effect, since the
closest analog, Our findings extend the previous report of Wojtczak and Za Concerning the specificity of FA interaction, it is interesting to ask
whether the demonstrated photolabeling of PIC with 3H-AzHA
or FA cycling resulted from the existence of a specific, preformed, FA
binding site or whether it instead reflects the overall carrier
hydrophobicity and positive charges inside the membrane. This calls for
further studies. Nevertheless, prior to being combusted, natural FAs
will be present in the membrane due to their high partition coefficient
and may potentially inhibit PIC and may cycle via PIC in either of the
two cases described above. Consequently, FAs should be considered as
important regulators of oxidative phosphorylation (29), since they
affect PIC (Ref. 20 and this work) and AAC (18, 19, 21, 22), besides the other mitochondrial carriers (30) and uncoupling proteins (19,
23-27).
We have also revealed for the first time that the "hydrophobic"
Pi analog, methanephosphonate, is a good PIC substrate.
This is unrelated to the FA inhibition, since alkylphosphonates with a
longer chain were found not to be transported. They did not inhibit
Pi uptake as well. These findings suggest that the putative hydrophobic part of Pi binding site cannot accommodate
larger alkylphosphonate analogs. However, we found that the
Pi binding site also interacts with Pi analogs
of medium size such as methylenediphosphonate, iminodi(methylenephosphonate), and AzNPPi, which inhibited
Pi uptake. This confirms the previous finding of inhibition
by photoactivated AzNPPi (32). We found that
AzNPPi together with diphosphonates belongs to
nontransportable Pi analogs. Thus, a specific gate of PIC
does not allow sulfate and tungstate to pass (7), and, on the other
hand, it is able to accommodate the methyl group and part of the
phosphonomethyl and phosphonomethylimino groups, or a phenyl
attached to the phosphate. Wohlrab et al. (7) hypothesized that some acid residues of yeast PIC, such as Glu163,
Glu164, Glu192, and Glu196 might
ensure specificity for phosphate and exclude interaction with sulfate.
Interestingly, glutamate Glu190 on UCP1 conveys the pH
dependence of nucleotide di- and triphosphates binding to this protein
(44). Phosphonoformate, previously reported to inhibit
Pi/Pi exchange (8), seems not to fit in the
revealed pattern, since it is not transported and does not inhibit the net electroneutral Pi transport. Perhaps the short formyl
(carboxyl) group attached on phosphonate cannot cause the inhibition as
methylenephosphonate does.
Diphosphonates that inhibit FA cycling enabled by PIC were also
identified as the first known nontransported substrate analogs that
inhibit the putative FA anion uniport. No such inhibition was found in
the case of uncoupling proteins. UCP1- or PUMP-mediated FA cycling is
inhibited by undecanesulfonate, which is the translocated anionic
substrate (19, 23-27, 33). The existence of FA cycling via PIC
indicates that this is a more general phenomenon not related exclusively to UCPs. However, it is a ligand-gated (purine
nucleotide-gated) regulation of FA cycling, what is distinct for UCPs
(23, 27). It is known that nucleotides, as intermediate size ligands,
interact with several membrane In conclusion, FAs interact with PIC in a hydrophobic binding site that
lies in proximity to or overlaps the Pi binding site, which
might represent a slightly hydrophobic internal domain in PIC. When
amphiphiles such as AzDA or native FAs interfere with the domain, the
transport process is inhibited. Upon interaction with PIC, FAs might
also reach the opposite side of the membrane, which leads to FA cycling
and uncoupling. Both inhibitory and cycling effects could lead in
vivo to a fine regulation of oxidative phosphorylation efficiency.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
antiport has been proposed to be the
most plausible mode (13, 14). In addition to monovalent phosphate (15),
arsenate and divalent monofluorophosphate (16) are also translocated.
With the human genome available, a major effort will be made in the coming years to ascribe particular phenotypes to the revealed genes.
Some proteins possess several functions; consequently, a complete
spectrum of functions for a given protein should be known. It is not
uncommon that a carrier fulfills several functions. For example, the
ADP/ATP carrier is thought to participate in the so called
mitochondrial permeability transition (17), which is also activated by
fatty acids (18). Although they share a similar
trans-membrane folding (10-12), MACPs exert diverse
functions and conduct anions of different charge and by different modes (symport, antiport, uniport). They all contain positively charged and
membrane-embedded arginines or lysines located on
trans-membrane
-helices (11, 12), which may contribute to
the putative anion binding sites for fatty acid anions and other
hydrophobic anions (12, 19). Interaction of some MACPs with fatty acids
seems to represent another common feature, probably their second
phenotype. Consequently, probing carriers with artificial hydrophobic
substrate analogs might reveal some new structure/function relationships.
)
translocation pathway or with a Pi binding site or
translocation pathway in the PIC structure. It is not known whether an
H+(OH
) translocation pathway is identical or
overlapping with the Pi pathway. A possibility that
H+ flux concomitant to Pi flux during the
physiological electroneutral Pi transport on PIC would be
ensured by the FA cycling mechanism, as in UCP1 (23), is unlikely,
since in this hypothetical case, when PIC would act as a
phosphate/fatty acid antiporter, the Pi transport should be
activated by FAs.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 (N3)
and 1710 cm
1 (COOH). When esterified with
diazomethane and analyzed on a gas chromatograph with mass detector,
the chemical purity found was 96%, MS: 228 (M + 1
28).
5 °C, and the solution
of 180 mg (1 mmol) of 4-azido-2-nitrophenol in 2 ml of dry pyridine was
slowly added at
5 °C. The solution was stirred at 20 °C for 15 min and at 110 °C for 15 min. After cooling to 0 °C, 2 ml of
water was added, and the reaction mixture was stirred at 30 °C for
90 min. Pyridine was removed in vacuo; rest was dissolved in
a methanol/water mixture (1:1) and filtered through a small column of
Dowex 50 (H+). The column was thoroughly washed with the
same solvent, the washes were collected and evaporated, and the rest
was evaporated two times with dry EtOH. The crude product was then
extracted to CHCl3 and alkalinized in chloroform while
saturated with NH3. The yellow precipitate crystallized in
a refrigerator to give yellow powder. The powder was extracted several
times with hot MeOH and evaporated to dryness to give a crude product.
TLC (silica gel 60 F254 (Merck), MeOH-NH4OH, 9:1)
exhibited the principal spot at RF = 0.6, a spot of
RF = 0.95 (slightly longer versus
starting 4-azido-2-nitrophenol), and three spots of
RF < 0.45. The crude product was further purified on a silica gel column (Merck; 0.04-0.063 mm, 4 × 10 cm) in
CHCl3-MeOH-concentrated NH4OH, 8:2:0.2
(100 ml), 5:5:0.5 (100 ml), and MeOH-concentrated NH4OH, 9:1. The first eluate, showing
RF = 0.95 on TLC (MeOH-NH4OH, 9:1) and
obtained as yellow crystals (5 mg): mp 135-140 °C, dec. MS: 421 (M-1), 393 (M-29), was identified as
bis(4-azido-2-nitrophenyl)phosphoric acid, which is in good accordance
with its MS and chromatographic behavior. A second fraction (TLC
RF = 0.6) gave the final product, i.e.
4-azido-2-nitrophenylphosphoric acid, isolated as yellow crystals (35 mg): mp189-191 °C dec. TLC:
CHCl3-MeOH-concentrated HCOOH, 7:3:0.5,
RF = 0.55, single spot. MS: 261 (M + 1).
are directly
proportional to the transport rates in min
1
(37) as follows,
where P is protein concentration,
(P1 = 1 mg/ml), and
(Eq. 1)
is a machine constant,
0.1163 for the Spectronics 3000.
form) to
remove the external label. Forward exchange rates were determined by
fitting the time course of isotope equilibration to a single
exponential, Y = A(1
e
kt) + B, leading to the
first order rate constant k
(min
1). A specific activity in
µmol·min
1(mg of
protein)
1 was calculated using k,
the known external [Pi] (the total Pi amount
in the sample volume) and the protein amount in the sample in mg
(usually 4 µg of PIC protein, which equals a lipid/protein ratio of
400 or a molar lipid/PIC monomer ratio of 18,100.
H+, i.e. changes in internal
[H+] relative to the initial state, FAs were added to
proteoliposomes containing recombinant yeast PIC, and after their
redistribution to both sides of the membrane apparent as interior
acidification, 1 µM valinomycin was added to initiate
H+ efflux. Measured fluorescence traces were converted into
"H+ traces" (20), from which the derived rates in
mM·s
1(mg of
lipid)
1 were multiplied by the internal
vesicle volume (estimated from SPQ volume distribution) and divided by
a surface of 1 mg of liposomes (in µm2) so that an
H+ flux density per µm2 was obtained in pmol
of
H+·s
1·µm
2.
70 °C. Films were developed by Dektol (Kodak) for
25 min and fixed in Kodak fixer for 5 min, both at 21 °C.
Percentages of the remaining 3H-AzHA label were quantified
from the band density compared with controls using one-dimensional
image analysis software (EDAS 40, Digital Science, Kodak).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (9K):
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Fig. 1.
The effect of AzDA, NEM, and mersalyl on
phosphate uptake in rat liver (a) and rat
heart mitochondria (b). Nigericin
(Nig)-induced swelling of rat liver mitochondria in
KPi is illustrated by traces expressed in the normalized
reciprocal absorbance for control (bottom traces with no
label), 10 µM mersalyl (upper traces), and 10 µM AzDA (middle traces). The trace marked
NEM was recorded for mitochondria preincubated with 400 µM NEM (2 µmol/mg of protein) for 30 s.
Measurements were performed in 1 ml of 44 mM
KPi, 5 mM Tris-MOPS, pH 7.2, containing 2 µM rotenone, 0.25 µg/ml antimycin, 50 µM
atractyloside. For the assay, 0.2 mg of mitochondria were added to the
medium, and, after 10 s, transport was initiated by 2 µM nigericin.
View larger version (14K):
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Fig. 2.
Dose responses for FA inhibition of phosphate
uptake in rat liver mitochondria. a, inhibition by lauric
acid ( ) and AzDA (
); b, inhibition by heptylbenzoic
(
) and 12-azidolauric (
) acids versus no effect of
12-hydroxylauric acid (
). All FAs were added directly to the assay
medium. a, the inhibitory dose responses are compared with
the data illustrating no effect of AzDA on the swelling of rat liver
mitochondria in sodium acetate, monitoring the function of the
Na+/H+ antiporter (
). Theoretical fits
(solid lines) to the Hill equation are also shown
(for which the Ki values of 3.8 µM for
AzDA, 250 µM lauric acid, 89 µM
heptylbenzoic acid, and 310 µM 12-azidolauric acid and
the Hill coefficients (nH) of 0.93, 1.07, 1.02, and 1.00, respectively, were derived on the assumption of 100%
inhibition at infinite concentration). Measurements were performed as
described in the legend to Fig. 1 with the exception of
Na+/H+ antiporter testing, for which 54 mM sodium acetate, 5 mM Tris-MOPS, pH 7.2, containing 2 µM rotenone and 0.25 µg/ml antimycin, was
used.
1·(mg of
protein)
1, Km was 6.5 mM, and derived Ki from the data measured with 100 µM AzDA was 99.5 µM. When
the direct plots V versus [Pi] were
fitted by nonlinear regression to the Michaelis-Menten equation,
the derived Vmax values in control and with 100 µM AzDA were 0.74 and 0.77 µmol of
Pi·min
1·(mg of
protein)
1, respectively, and
Km was 9 mM in control.
View larger version (10K):
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Fig. 3.
AzDA inhibitory effect on kinetics of
Pi/Pi exchange in PIC proteoliposomes.
Double-reciprocal plots illustrating the kinetics of
Pi/Pi exchange measured as a forward
33P uptake into proteoliposomes containing E. coli-expressed yeast phosphate carrier are shown for controls
( ) or samples measured in the presence of 100 µM AzDA
(
). Each point represents an average from 2-4 determinations. Data
were fitted to the competitive inhibition model by linear regressions
using the Marquardt algorithm. The derived Vmax
in control was 0.55 µmol of
Pi·min
1·(mg of
protein)
1, Km was 6.5 mM, and derived Ki from the data
measured with 100 µM AzDA was 99.5 µM.
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Fig. 4.
Methanephosphonate uptake in rat liver
mitochondria. Swelling of rat liver mitochondria in 44 mM potassium methanephosphonate, 5 mM
Tris-MOPS, pH 7.2, containing 2 µM rotenone, 0.25 µg/ml
antimycin, and 50 µM atractyloside was induced by 2 µM nigericin (Nig). The effects of 10 µM AzDA and 10 µM mersalyl are illustrated.
The +NEM trace was recorded with mitochondria
preincubated with 400 µM NEM (2 µmol/mg protein) for
30 s.
View larger version (12K):
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Fig. 5.
Dose responses for inhibition of phosphate
uptake in rat liver mitochondria by 4-azido-2-nitrophenylphosphate and
methylenediphosphonate (a) and
iminodi(methylenephosphonate) (b). Pi
uptake was assayed as described in the legends to Figs. 1 and 2.
Theoretical fits (solid lines) to the Hill
equation are also shown, from which the Ki values of
1.5 (AzNPPi, ) or 4.9 mM
(a, filled hexagons, MDPh) and 5.2 mM (b) and Hill coefficients
(nH) of 1.7, 2.4, and 1.97, respectively, were
derived.
1· (mg of
lipid)
1) highest for oleic (1.55) and
decreasing for heptylbenzoic (1.49), myristic (1.35), lauric (0.96;
Fig. 6b) and 12-azidolauric (0.55) acids. Note that FAs that
are unable to flip-flop, such as 12-hydroxylauric acid, gave a
background H+ flux of 0.17 nmol of
H+·s
1·(mg of
lipid)
1 that was identical to the background
H+ flux in the absence of FAs (Fig. 6c). The
apparent affinity to PIC (inverse Km) decreased in
nearly the same order as Vmax
(Km values in parentheses): oleic acid (72 µM) > myristic acid (89 µM) > heptylbenzoic acid (207 µM) > lauric acid (278 µM) > 12-azidolauric acid (364 µM);
i.e. only the order of myristic and heptylbenzoic acid is
switched when compared with the order of Vmax
values. Moreover, the pattern of FA cycling is similar to the FA
inhibitory effect on PIC. Nevertheless, the Km
values for PIC are much higher than those reported for UCP1 (23) or
UCP2 and UCP3.2
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Fig. 6.
Fatty acid-induced H+ uniport in
proteoliposomes containing PIC. a, H+ flux
density J is a function of total lauric acid
concentration in the absence ( ) and presence of 10 mM
methylenediphosphonate (
). The H+ efflux induced by
various lauric acid concentrations, expressed as flux density in
10
6 pmol of H+
s
1 µm
2 is plotted
versus total lauric acid concentrations. Error
bars represent S.D. values of two or three estimations; when
omitted, S.D. was the size of the symbol. b,
Eadie-Hofstee plot for the differential methylenediphosphonate-
sensitive flux calculated from the data of a. The derived
Vmax amounted 168 × 10
6 pmol of H+
s
1 µm
2, which is
0.962 nmol of H+ s
1 (mg of
lipid)
1. The apparent Km
was about 278 µM. c, H+ flux
density J as a function of total myristic (
), oleic
(
), and 12-hydroxylauric acid concentration (
). d,
H+ flux density J as a function of total
heptylbenzoic (filled hexagon) and 12-azidolauric
acid (
) concentration, in the absence and presence of 10 mM methylenediphosphonate (open
symbols). Vmax values (from
Eadie-Hofstee plots for MDPh-sensitive fluxes, in
10
6 pmol of H+
s
1 µm
2) were 205 (oleic acid), 158 (myristic acid), 169 (heptylbenzoic acid), and 62 (12-azidolauric acid), while Km values were 72 µM for oleic, 89 µM for myristic, 207 µM for heptylbenzoic, and 364 µM for
12-azidolauric acid.
View larger version (51K):
[in a new window]
Fig. 7.
Photoaffinity labeling of rat heart
mitochondria with 3H-AzHA. Photographs of SDS-PAGE
(left panel) and the corresponding autoradiograms
(right panel) of rat heart mitochondria (two samples,
A and B), photolabeled with 3H-AzHA
(3 µmol/mg protein) are shown. The positions of the molecular mass
(M) standards are marked by the displayed scale. The Laemmli
system of SDS-PAGE using 17.5% acrylamide with an
acrylamide/bisacrylamide ratio of 150:1 was employed, and
autoradiography was performed as described under "Experimental
Procedures."
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Fig. 8.
Phosphate carrier and ADP/ATP carrier as two
proteins photolabeled by 3H-AzHA. Shown are
autoradiograms (A) and the corresponding PAGE gels
(P) run in parallel. Either partially purified proteins of
the HTP pass-through (a) or the rat heart mitochondria
(b) were photolabeled. a, 3H-AzHA was
preincubated with the HTP pass-through fraction, prepared from Triton
X-100- extracted rat heart mitochondria, and the sample was irradiated
by UV light and passed over Sephadex G25-300. The labeled sample (1.5 mg of protein) was further fractionated on blue Sepharose.
b, labeling of rat heart mitochondria was performed first
(cf. Fig. 7), and subsequently separations on HTP and blue
Sepharose columns were conducted. Lanes in both
panels illustrate hydroxylapatite pass-through
(HTP) of Triton X-100-extracted rat heart mitochondria (3 µg of protein per lane), fractions of the blue Sepharose
column (flow-through; BS), a 2.7 M NaCl fraction
containing 0.5% Triton X-100 (BS 2.7M NaCl), and 150 mM NaCl fraction containing 0.5% SDS (BS & SDS). Positions of molecular mass (M) standards are
indicated.
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Fig. 9.
Affi-Gel purification of phosphate carrier
photolabeled with 3H-AzHA. Samples were photolabeled
with 3H-AzHA in the stage of hydroxylapatite pass-through
(1) (sample of Fig. 8a) or as isolated rat heart
mitochondria (2) (sample of Fig. 8b). Both
samples were fractionated on three columns: first on hydroxylapatite,
its pass-through fraction (1.5 mg of protein) was loaded onto a blue
Sepharose column, and the resulting flow-through was loaded onto the
Affi-Gel 501 column, which was washed and then eluted with 1.5 mM mercaptoethanol. Shown are autoradiograms (A)
and the corresponding PAGE gels (P) run in parallel.
Positions of molecular mass (M) standards are
indicated.
View larger version (69K):
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Fig. 10.
Photoaffinity labeling of recombinant yeast
phosphate carrier with 3H-AzHA and its prevention by
various phosphocompounds. Autoradiograms of PAGE-separated yeast
PIC solubilized from inclusion bodies (equal amounts, three
experiments, A, B, and C) as described
under "Experimental Procedures" are shown. Samples were
photolabeled with 3H-AzHA (5.2 nmol/mg total protein,
corresponding to a stoichiometry of 0.3 per dimer) in controls
(C, 100%) or after preincubations with 10 mM
methanephosphonate (C1Ph, remaining 37%
of the label), 10 mM phosphonoformic acid (PFA,
99%), 50 mM sodium pyrophosphate (PPi,
44%), 1 mM sodium undecanesulfonate
(C11SO3, 99%), 10 mM
methylenediphosphonate (MDPh, 9%), 10 mM
iminodi(methylenephosphonate) (IDPh, 12%), 10 mM 4-nitrophenylphosphate (NPPi,
40%), or after preceeding photolabeling with nonradioactive 10 mM 4-azido-2-nitrophenylphosphate
(AzNPPi, 30%). For details of SDS-PAGE (17.5%
acrylamide, its ratio to bisacrylamide 150:1) and autoradiography, see
"Experimental Procedures."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-azido-dodecanoic acid, and natural FAs inhibit as
well. The enhancement of the inhibitory strength could originate from
the interaction within the Pi binding site (anion pathway)
of PIC, since AzNPPi, attached by photoreaction to the
carrier, does interfere with 3H-AzHA photolabeling. This
fact again confirms the proximity of the Pi and FA binding sites.
uska (31),
who interpreted the observed FA inhibition of Pi transport in rat liver mitochondria as a surface charge effect. Our data indicate
a rather specific effect of FAs on Pi transport via PIC. A
surface charge should also probably inhibit the H+ efflux
coupled to the Na+ uptake by the
Na+/H+ antiporter or the
pyruvate*H+ symport via the pyruvate carrier. None of these
effects was observed. Also, several inactive FA derivatives (42,
43), such as 12-hydroxydodecanoic acid, if causing the surface charge
effect, should inhibit the PIC as well. Again this was not observed.
Finally, butyl- and decylphosphonate should cause even higher
inhibition due to a surface charge, but they did not, even at
millimolar concentrations.
-helices of UCP1 (45). Also,
phosphate might interact with several trans-membrane
-helices of PIC, as shown by the site-directed mutagenesis (7),
revealing five critical residues for yeast PIC spread along the whole
sequence: His32 and Asp39 on the first;
Glu126 and Glu137 on the second; and the
Asp236 on the third trans-membrane
-helix,
respectively. Among them, His32, Glu126, and
Glu137 were proposed to form a putative proton cotransport
pathway (7). However, due to functional reasons, we cannot conclude
that the FA binding site is identical with the
H+/OH
binding site of PIC, as we did for
UCP1, for which FAs were documented to enter into its anion binding
site (24, 25).
![]() |
ACKNOWLEDGEMENTS |
---|
A contribution to transport measurements by
Jitka Polechová and Dr. Milo Nekvasil, the figure design
by Dr. Ji
í Borecký, and the excellent technical
assistance of Jana Brucknerová and Jana
Ko
a
ová are gratefully acknowledged.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant Agency of the Czech Republic Grants 301/95/0620 and 301/98/0568, U.S.-Czechoslovak Science and Technology Program Grant 94043, and Czech Ministry of Education Programs Kontakt ME085 and ME389.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.
To whom correspondence should be addressed: Dept. 375, Institute of Physiology, Academy of Sciences of the Czech Republic, Víde
ská 1083, CZ 14220 Prague, Czech Republic.
Fax: 011-4202-4752488 or 011-4202-44472269; E-mail:
jezek@biomed.cas.cz.
Published, JBC Papers in Press, November 20, 2000, DOI 10.1074/jbc.M008945200
2 P. Jezek, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PIC, mitochondrial phosphate carrier; AAC, ADP/ATP carrier; AzDA, 12-(4-azido-2-nitrophenylamino)dodecanoic acid; 3H-AzHA, 16-(4-azido2-nitrophenylamino)-[3H4]hexadecanoic acid; AzNPPi, 4-azido-2-nitrophenylphosphate; BSA, bovine serum albumin; BS, blue Sepharose (Cibacron blue affinity agarose); FA, fatty acid; HTP, hydroxylapatite; LS, light scattering; MDPh, methylenediphosphonate; MOPS, 3-(N-morpholino)propanesulfonic acid; NEM, N-ethylmaleimide; PUMP, plant uncoupling mitochondrial protein; UCP, uncoupling protein (UCP1, brown adipose tissue-specific UCP; UCP2, ubiquitous UCP; UCP3, predominantly muscle-specific UCP; BMCP and UCP4, brain-specific UCPs); PAGE, polyacrylamide gel electrophoresis; TEA, tetraethylammonium; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid.
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