From the
Plant Research Department, Risø
National Laboratory, P. O. Box 49, DK-4000 Roskilde and the
¶Department of Biochemistry and Molecular
Biology, University of Southern Denmark, DK-5230 Odense, Denmark
Received for publication, January 9, 2003 , and in revised form, March 7, 2003.
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ABSTRACT |
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INTRODUCTION |
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Although the PDH kinase and phosphatase system is the only fully
characterized system for reversible protein phosphorylation in mitochondria,
there are clear indications that others are present. Incubating intact
mitochondria
(35)
or submitochondrial fractions like the outer membrane
(6) or the inner membrane
(79)
with [-32P]ATP gives labeling of up to 30 proteins (as
detected by one-dimensional gel electrophoresis) indicating that kinases are
present in these fractions and in contact with target proteins. The matrix
appears to contain one of more protein phosphatases responsible for
dephosphorylation of inner membrane phosphoproteins
(8,
9).
Only eight mitochondrial phosphoproteins have been identified to date. In
addition to the E1-subunit of PDH, these are band
'-subunits of the ATP synthase
(10), HSP70
(11,
12) and MTSHP
(13,
14), nucleoside diphosphate
kinase (15,
16), E1 of the branched-chain
-ketoacid dehydrogenase
(2), and the 18-kDa iron
protein subunit of complex I in mammalian mitochondria
(17). In a very recent study
(18) a further 14
mitochondrial phosphoproteins were identified, all household proteins involved
in the citric acid cycle and associated reactions, the respiratory chain
complexes, in heat shock proteins, and the detoxification of reactive oxygen
species.
Formate dehydrogenase (FDH) catalyzes the oxidation of formate to CO2 reducing NAD+ to NADH in the process. Formate may have a role in biosynthesis of numerous compounds, such as in energetic metabolism and in signal transduction pathways related to stress response. The enzyme is induced in potato leaves by a variety of stresses including hypoxia (19, 20). It is one of the most abundant proteins in the matrix of potato tuber mitochondria (19), but its function is unknown partly because the source of formate is unknown (19, 21).
In the present study we use two-dimensional gel electrophoresis and mass spectrometry to show that a major part of the phosphorylation in potato tuber mitochondria that was ascribed previously to PDH is due, in fact, to labeling of FDH. We identify the site of FDH (and PDH) phosphorylation and describe some properties of FDH phosphorylation. Finally, we show that FDH activity is highest in intact sprouting tubers where the oxygen concentration is relatively low implying that FDH has a role in hypoxic metabolism.
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EXPERIMENTAL PROCEDURES |
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Phosphorylation AssayProtein phosphorylation in intact
plant mitochondria and in the mitochondrial subfractions was investigated by
in vitro labeling analyses with [-32P]ATP.
Phosphorylation assays were carried out at 25 °C for 10 min in a volume of
200 µl containing 1 mg of protein, 0.3 M mannitol, 10
mM MOPS-KOH, pH 7.2, 5 mM MgCl2, 0.1
mM CaCl2, 1 mM phenylmethylsulfonyl fluoride,
150 µCi of [
-32P]ATP (AA0068, Amersham Biosciences), and
0.2 mM ATP. The reactions were terminated, and
[
-32P]ATP was removed from the mitochondria by addition of
ice-cold reaction buffer and centrifugation at 100,000 x g for
1 h or for the matrix fraction by precipitation with 80% ice-cold acetone
containing 35 mM mercaptoethanol followed by centrifugation at
10,000 x g for 20 min.
Two-dimensional Blue Native/PAGEIn the first dimension blue native electrophoresis was used in order to separate protein complexes according to their size (23) followed by gradient 1015% Tricine SDS-PAGE in the second dimension. For blue native gels 1 mg of matrix protein was supplemented with a buffer to give the following final concentrations: 750 mM aminocaproic acid, 0.5 mM EDTA, 50 mM Bis-Tris, pH 7.0 and 1.5% w/v n-dodecyl maltoside. Coomassie Blue solution, 15 µl of 5% (w/v) Serva Blue G, 750 mM aminocaproic acid, was added to the solution and loaded directly onto a 4.5% to 16% (w/v) acrylamide gradient blue native gels. BN-PAGE was carried out as described (23, 24).
Two-dimensional Isoelectric Focusing/Tricine SDS-PAGEThe isoelectric focusing was conducted with the IPGphor system (Amersham Biosciences) using 17-cm ReadyStrip IPG strips with a linear pH gradient range 58 according to the manufacturer's instructions (25). Mitochondrial proteins were solubilized in 300 µl of rehydration solution containing 7 M urea, 2 M thiourea, 2% w/v CHAPS, 2% w/v n-dodecyl maltoside, 20 mM dithiothreitol, 1% v/v IPG buffer, pH 3.010.0. For rehydration with matrix proteins, the solution contained 7 M urea, 2 M thiourea, 4% CHAPS, 20 mM dithiothreitol, 0.5% IPG buffer, pH 3.010.0. Tricine SDS-PAGE was carried out as described (26). The pI and molecular mass scales of the two-dimensional maps were calibrated using the ImageMaster two-dimensional Elite software (Amersham Biosciences). The 32P-containing phosphoproteins were visualized by PhosphorImaging using Quantity One software (Bio-Rad).
Tandem Mass Spectrometry Analysis of Tryptic PeptidesMS/MS analysis of peptides generated by in-gel digestion as described previously (27) was performed on a Finnigan LCQ (Classic) quadrupole ion storage mass spectrometer (San Jose, CA) equipped with a nanoelectrospray source (Protana Engineering, Odense, Denmark). Prior to MS analysis samples were purified on reverse phase POROS R2 or OligoR3 (2030 µm bead size, PerSeptive Biosystems, Framingham, CA) nanocolumns (28, 29) and eluted with 1 µl of 50% (v/v) methanol, 5% (v/v) formic acid directly into the precoated borosilicate nanoelectrospray needles (Protana Engineering, Odense, Denmark).
Phosphopeptide Purification by Nanoscale Fe(III)-IMACPhosphopeptides present in tryptic digestion mixtures generated from phosphoproteins were purified by miniaturized Fe(III)-IMAC columns as described (29). Peptide samples were redissolved in 0.1 M acetic acid containing 10% (v/v) acetonitrile and slowly loaded onto the IMAC column (loading time 120140 s). The column was then washed with 0.1 M acetic acid, 10% (v/v) acetonitrile in 0.1 M acetic acid, Milli-Q water. Peptides were eluted from nanoscale IMAC columns by using pH 10.5 solvent (Milli-Q water adjusted to pH 10.5 by addition of 25% (v/v) ammonia). The eluate was acidified to 5% (v/v) formic acid and loaded directly onto a nanoscale column (28) packed with Oligo R3 material. The column was rinsed with 5% formic acid and then eluted with saturated matrix solution (2,5-dihydroxybenzoic acid in 50% (v/v) acetonitrile, 5% formic acid). The matrix/analyte eluate was spotted as a series of nanoliter volume droplets onto the MALDI probe.
Peptide Mass Mapping by MALDI-TOF-MSDelayed extraction
MALDI mass spectra were recorded on a Reflex IV time-of-flight mass
spectrometer (Bruker-Daltonics, Bremen, Germany) equipped with a nitrogen
laser ( = 337 nm). Mass spectra were acquired in both positive ion
linear mode and positive ion reflector mode. Instrument mass calibration was
performed by using a tryptic peptide mixture derived from bovine lactoglobulin
(external calibration). Internal mass calibration of peptide mass spectra
using digestion products of trypsin or from the identified proteins resulted
in mass errors of less than 50 ppm, typically 1525 ppm.
Automated Nano-flow LC-MS/MS Analysis of Tryptic Peptides Tryptic peptide mixtures were separated and analyzed using a nanoscale capillary high pressure liquid chromatography system (Ultimate, LC-Packings) interfaced directly to a Q-TOF tandem mass spectrometer (Q-TOF Ultima API, Micromass, UK). Peptide mixtures were separated on a C18 reverse phase column (75 µm (inner diameter) x 90.0 mm, Zorbax SB-C18 3.5 µm) during a 1.5-h gradient of 090% acetonitrile (v/v) containing 1% (v/v) formic acid, 0.6% (v/v) acetic acid, and 0.005% (v/v) heptafluorobutyric acid at a flow rate of 175 nl/min. The mass spectrometer was calibrated by using NaI (8-s scan time, m/z 4002000).
Data Base Searching and Protein Sequence AnalysisIon trap tandem mass spectrometry data were interpreted by using the Mascot MS-MS Ions Search software (www.matrix-science.com). The Sequest program (Thermoquest, San Jose, CA) was used to convert ion trap MS/MS spectra into DTA format. Only matching peptides with precursor ion mass tolerance of less that 1 Da and individual ion scores indicating identity to the protein were taken into consideration and used for estimation of a minimum percentage sequence coverage. Mass spectra obtained by automated nano-flow LC-MS/MS using data-dependent acquisition modes were analyzed using the MassLynx 3.5 software (Micromass, UK) and Mascot MS-MS Ions Search. MALDI mass spectra were analyzed by using the software package m/z (Proteometrics Ltd., New York). The differential peptide mass mapping and analysis of theoretical tryptic peptide sequences were performed using GPMAW 5.01 (Lighthouse Data, Odense, Denmark). Tandem mass spectra obtained from phosphopeptides were manually inspected.
Computer Modeling of S. tuberosum FDHSequence of S. tuberosum FDH was from NCBI-CAA79702 (19). The three-dimensional structural homology model was constructed with Swiss model 3.5 protein modeling server and the Swiss-PdbViewer version 3.7b2 (30, 31).
Measuring the Oxygen Concentration in Intact Potato TubersTubers of 40200 g fresh weight (average weight 109 g after peeling; 46 cm in diameter) stored at 4 °C were incubated at room temperature (2224 °C) in darkness for 23 weeks and had started sprouting.
An OX100 microelectrode from Unisense, Århus, Denmark, with a tip diameter of 100 µm was calibrated in air-saturated water immediately before use. A tuber was immobilized on a platform, and the electrode was inserted into the tuber using a micromanipulator. Two different insertion methods were used. (a) The electrode was inserted 25 mm into the tuber stopping for 1 min to take a reading just below the surface and at 5, 10, 15, 20, and 25 mm depth in the same hole. (b) Alternatively, the oxygen concentration at each depth was measured by sticking the electrode in at a new position. The read-out usually stabilized well before the minute was up, and the values were normally similar with the two insertion methods indicating that leakage of air into the tuber along the electrode did not occur. The oxygen concentration was calculated assuming that the water in equilibrium with air contains 270 µM oxygen at 22 °C (32).
After the oxygen concentration measurements the tuber was peeled and homogenized to give a soluble fraction where the activity of FDH and CCO was measured (see below).
Enzyme Activity Measurements in Whole Tuber ExtractsEach tuber was homogenized separately using a Braun juice extractor and 17 ml of medium/100 g fresh weight containing 0.9 M mannitol, 30 mM MOPS, 3 mM EDTA, 0.3% (w/v) bovine serum albumin, and 25 mM cysteine, pH 7.2. Large particles were removed by filtration through nylon cloth (mesh 120 µm) and centrifugation at 1600 x g for 10 min. After decantation and volume measurement, the supernatant was frozen in liquid nitrogen and stored at 80 °C until use.
CCO activity was measured as described previously (33). FDH activity was measured as NADH production at 340 nm in a medium containing 0.3 M sucrose, 20 mM MOPS, pH 7.2, 1 µg/ml antimycin A, 15 mM formate, 1 mM NAD+, and 0.025% (v/v) Triton X-100. The reaction was started by the addition of sample, and it was linear for at least 5 min.
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RESULTS |
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Identification of Site of Phosphorylation on PDHMammalian
mtPDH is phosphorylated on three serines, only one of which is conserved in
plant mtPDH. However, the site of plant mtPDH phosphorylation has not been
identified previously. By using automated nano-flow LC-MS/MS, we identified a
14-amino acid-long phosphopeptide, Tyr290Arg303
(Fig. 3) in the tryptic peptide
mixture derived from protein spot 13 (Fig.
2). The same phosphopeptide was found in protein spots 1 and
1214 (Figs. 1 and
2). The MS/MS scan in
Fig. 3B derived from
the peptide eluting at the time point is indicated with an arrow in
Fig. 3A. The mass
difference between the doubly protonated precursor ion ((M +
2H+)2+, m/z 837.94) and the
ion of m/z 788.94 is a signature for the loss of phosphoric
acid from the phosphopeptide via a gas-phase -elimination mechanism. The
mass difference between the peptide fragment ions y9 and y10 (69 Da)
corresponds to the dehydroalanyl residue formed by loss of phosphoric acid
from phospho-Ser294
(35). Thus, Ser294
in PDH is phosphorylated.
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Identification of the Phosphorylation Sites on FDHInitial
phosphorylation analysis of FDH was performed by using a combination of
nanoscale Fe(III)-IMAC for enrichment of tryptic phosphopeptides and OligoR3
column for phosphopeptide concentration and desalting
(29). Identical phosphopeptide
mass maps were obtained for FDH protein spots 1 and 2 on BN/SDS-PAGE
(Fig. 1A and
Table I). Fig. 4A shows a MALDI
mass spectrum of an IMAC-retained phosphopeptide from FDH. The ion at
m/z 2215.0 is the unphosphorylated, singly charged 19-amino
acid peptide Gly70Lys88, whereas the ion at
2295.0 Da is the phosphorylated form of this peptide (HPO3, +80
Da). The peptide Gly70Lys88 has two candidate
phosphorylation sites, viz. residues Tyr73 and
Thr76. Closer inspection of the mass spectrum reveals several ion
signals indicative of -elimination of phosphoric acid (see figure
legend), making Thr76 the prime candidate for phosphorylation
because Tyr(P) cannot undergo
-elimination.
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To confirm this hypothesis the Fe(III)-IMAC enriched phosphopeptide sample was next subjected to amino acid sequencing by nanoelectrospray ion trap tandem mass spectrometry using the triply protonated precursor ion (m/z 766.24) (Fig. 4B). About 10 fragment ions can be matched to the peptide, and in all cases the masses match a sequence containing dehydro-2-aminobutyryl residue instead of Thr76. The peptide gave quite a high prediction score 0.82 for phosphorylation when tested with the NetPhos 2.0 software (Table II). Thus, these experiments strongly indicate that Thr76 in FDH is phosphorylated.
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In summary, we have identified two sites of phosphorylation on FDH, Thr76 and Thr333. To get an idea of their location in the native FDH conformation, we applied a structural homology three-dimensional model of S. tuberosum FDH (Fig. 6). S. tuberosum FDH shares high sequence and structural similarity to the bacterial FDH from Pseudomonas sp. 101. A backbone alignment of the mitochondrial FDH and bacterial FDH is shown in Fig. 6A and underlines their similarity. The image shown in Fig. 6B indicates the positions of the NAD+-binding site and the active site, both of which lie close to a groove that can accommodate the formate molecule. Thr76 and Thr333 are both on the outer surface of the protein and thus easily accessible to kinases and phosphatases.
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Regulation of FDH (and PDH) PhosphorylationTo investigate the regulation of protein phosphorylation, intact mitochondria or matrix fraction was incubated with [32P]ATP and various compounds either alone or in combination. In Fig. 7 the proteins were separated by SDS-PAGE, and phosphorylation was quantified by PhosphorImager scanning. Fig. 7C shows the protein bands that were cut out and identified by ESI-MS/MS (Table I).
In intact mitochondria the labeled band 20 contained both PDH and FDH. In the matrix fraction, band 21 contained PDH + FDH, whereas the lower band 22 contained only FDH (and isocitrate dehydrogenase, not shown) (Fig. 7B and Table I). The resolution on the PhosphorImager did not allow us to exclude any of these proteins from contributing to the labeling pattern. However, when bands 21 and 22 were cut out of the gel and read by PhosphorImaging separately, they both displayed 32P incorporation, and the labeling intensity in spot 21 was about 7 times higher than that in spot 22 (results not shown). Formate had almost no effect on the amount of 32P incorporation in intact mitochondria but clearly depressed phosphorylation in the matrix fraction. Formate and NAD+ inhibited phosphorylation in the matrix fraction in an additive manner by reducing labeling by 36 and 60% when added separately and by 90% when added together. Pyruvate also inhibited phosphorylation very strongly both in intact mitochondria (Fig. 7, A and B) and in the matrix fraction (not shown), whereas NADH only inhibited labeling in the matrix fraction by about 40% (Fig. 7).
A matrix fraction was also labeled either without additions (control) or in the presence of the FDH substrates formate + NAD+, and these two fractions were separated on IEF-PAGE two-dimensional gels. In the presence of formate + NAD+, two protein spots disappeared (Fig. 8, A and C), spot 12 and spot 13 containing both PDH and FDH (Table I), and labeling decreased markedly in that area (Fig. 8, B and D). This indicates that these spots contained phosphorylated FDH and PDH formed during the labeling incubation. The Coomassie staining of spot 16 containing only FDH did not appear to alter when labeling was inhibited, whereas spots 14 and 15 containing both enzymes increased in size (Fig. 8, A and C; Table I); but the labeling of all three spots almost completely disappeared (Fig. 8, B and D). The amount of protein in the unlabeled spots 1719 was apparently unaffected by formate + NAD+. The results indicate clearly that formate + NAD+ inhibited the labeling of both PDH and FDH.
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FDH Activity and the Oxygen Concentration Inside Sprouting Potato Tubers Are Negatively CorrelatedFDH is induced by a variety of stresses including hypoxia and anoxia (20, 36). The oxygen concentration inside actively growing potato tubers is so low as to limit respiration (37). For this reason we measured the activity of FDH in extracts from whole sprouting potato tubers and compared that to the activity of CCO, as an indicator of aerobic respiratory capacity, measured in the same extracts. These values were correlated with the concentration of oxygen measured in the same tubers before making the extracts. The oxygen concentration in the tubers was 3075% that in air-saturated water or 80200 µM similar to what was reported for growing tubers (37). However, unlike Geigenberger et al. (37), we did not observe any oxygen gradients in the tubers, the oxygen concentration was very similar whether measured immediately below the cork layer or at depths of 525 mm (the diameter of the tubers being 45 cm).
FDH activity increased and CCO activity decreased in the tuber extracts with decreasing oxygen concentration in the intact tubers (Fig. 9, A and B). As a result, FDH activity increased 4-fold relative to CCO activity with decreasing oxygen concentration (Fig. 9C). For a comparison, tubers stored at 4 °C with a low metabolic activity had an oxygen concentration above 300 µM, FDH and CCO activities of 6.8 ± 1.1 and 287 ± 49 nmol min1 g fresh weight1, and an FDH/CCO ratio of 0.025 ± 0.006 (n = 9) or very similar to that observed for sprouting tubers with oxygen concentrations above 200 µM (Fig. 9).
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DISCUSSION |
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Phosphorylation of PDHNo PDH phosphopeptide was found in
spots 15 and 17 (Fig. 8, A and
C) indicating that potato tuber mitochondria contain two
unphosphorylated forms of the E1-subunit of PDH. Whether these are
products of different genes or caused by post-translational modification, we
do not know. We identified Ser294 as a site of phosphorylation in
plant PDH (Fig. 3) consistent
with the fact that it is the only one of three serines phosphorylated in
mammalian PDH that is conserved in plants
(2). However, the
phosphopeptide was found in three protein spots (spots 1214;
Fig. 8, A and
B), two of which disappeared when phosphorylation was
inhibited (spots 12 and 13,
Fig. 8, A and
C). It is therefore possible that both PDH forms are
phosphorylated on Ser294, but we cannot exclude that there are
more, as yet undetected and unidentified, sites of phosphorylation in PDH.
Phosphorylation of FDHPreviously it was assumed that 32P labeling was observed around 4042 kDa for potato tuber mitochondria separated on one-dimensional gels derived from PDH (2, 3, 38). We now show that FDH is phosphorylated and that FDH phosphorylation is also complex.
Spots 18 and 19 contained only unlabeled FDH, and unlabeled FDH could have been present in other spots (Fig. 8, A and B). Because only one FDH gene product was detected in potato callus (19), it seems likely that either spot 18 or 19 contains post-translationally modified FDH. We did detect several post-translational modifications in the form of deamidations and methionine oxidation in one of the identified phosphopeptides from FDH (Fig. 5). A protein with these deamidations would have a lower pI than the unmodified protein, and this could be one of the reasons for finding FDH in a number of spots with pI ranging from pH 6.74 to 7.19 (Fig. 8 and Table I).
We identified both Thr76 and Thr333 as sites of phosphorylation on FDH (Figs. 4 and 5). We cannot at the moment exclude the possibility that there are more phosphorylation sites. A structural homology three-dimensional model of the enzyme indicates that the two phosphorylated residues are found on the outer surface of the protein (Fig. 6). The residues are therefore conveniently located to be reached by kinases and phosphatases.
Regulation of FDH PhosphorylationPyruvate, formate, and NAD+, the substrates of FDH and PDH, all reduced the phosphorylation level of FDH + PDH strongly (Fig. 7). PDH kinase is inhibited by pyruvate (39), so a pyruvate inhibition of PDH labeling was expected. However, it was unexpected that pyruvate completely abolished all phosphorylation in the 40 43-kDa region because this implies that pyruvate also inhibits the kinase responsible for FDH labeling (Figs. 7, A and B, and 8). By analogy with PDH kinase, the inhibition of FDH phosphorylation by formate and by formate + NAD+ was expected. However, the complete inhibition of all phosphorylation in the 4143-kDa region by formate + NAD+ (Figs. 7 and 8) was also unexpected because it implies that PDH kinase is inhibited by formate + NAD+.
The results indicate that FDH phosphorylation and PDH phosphorylation are regulated in the same way, which can only be interpreted after we have considered the physiological role of FDH. It appears clear, however, that PDH kinase can not be responsible for FDH phosphorylation on Thr76 or Thr333, because the PDH kinase is specific for serine residues (2).
Other Post-translational Modifications of FDHThe identified phosphopeptide Tyr326Arg346 in FDH contained several other modifications, oxidation of methionine and deamidation of asparagine and glutamine (Fig. 5). The oxidized methionines were probably, but not certainly, artifacts. Amino acid residues containing thioethers are easily oxidized during protein purification, derivatization, and/or digestion (40, 41).
However, the two deamidations in the phosphopeptide Tyr326Arg346 could be biologically relevant. Deamidation is a modification formed both naturally and artificially. As a natural post-translational modification, it has been described in the context of protein aging and cataract formation (42, 43) and G-protein regulation (44). A deamidating enzyme has been described in Escherichia coli (45). Artificial deamidation can occur spontaneously under both acidic and alkaline conditions (46). We attempted to test the biological relevance by comparing the prediction score for threonine phosphorylation for the predicted peptide with Asn and Gln and the modified peptide containing Asp and Glu (Table II). The modified peptide with two extra negative charges gave a much higher prediction score than the encoded peptide implying that the modification in vivo could facilitate the recognition by a specific protein kinase.
The Physiological Role of FDHWe found a marked increase of FDH activity relative to aerobic respiration (monitored as CCO activity) at lower oxygen concentrations in the potato tubers (Fig. 9). This implies that FDH has a role in hypoxic metabolism, which is consistent with the observation that FDH synthesis is induced under hypoxic (36) or anoxic (20) conditions.
The physiological role of FDH in plants is uncertain as discussed by Hourton-Cabassa et al. (36) mainly because the source of formate is uncertain (21). One possible source of formate is pyruvate provided that the enzyme formate C-acetyltransferase (EC 2.3.1.54 [EC] ), also called pyruvate formate-lyase (PFL), is present. In microorganisms and mitochondria from unicellular algae, the enzyme is strictly anaerobic, but although fairly strongly hypoxic conditions are observed inside potato tubers (Fig. 9) (37), the conditions in the tubers are far from anoxic. Still, we searched the Arabidopsis data base for genes that might encode PFL. That did not give significant hits so instead we searched for the conserved local sequence motif in PFL and found a gene containing it in Arabidopsis (Fig. 10).
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If PFL is present in potato tubers, the combined action of PFL and FDH
gives the same net result as that of PDH alone as shown in Reactions
1,
2,
3,
![]() | (1) |
![]() | (2) |
![]() | (3) |
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FOOTNOTES |
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Present address: Dept. of Physics and Astronomy, University of Manitoba,
301 Allen Bldg., Winnipeg, Manitoba R3T 2N2, Canada.
|| To whom correspondence should be addressed: Plant Research Dept., Risø National Laboratory, Bldg. 301, P. O. Box 49, DK-4000 Roskilde, Denmark. Tel.: 45-46-77-42-13; Fax: 45-46-77-41-22; E-mail: ian.max.moller{at}risoe.dk.
1 The abbreviations used are: PDH, pyruvate dehydrogenase; BN, Blue Native;
CCO, cytochrome c oxidase; FDH, formate dehydrogenase; IEF, isoelectric
focusing; LC, liquid chromatography; MOPS, 4-morpholinopropanesulfonic acid;
PFL, pyruvate formate-lyase; Tricine,
N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; LC/MS, liquid
chromatography/mass spectrometry; ESI, electrospray ionization; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MALDI-TOF,
matrix-assisted laser desorption ionization time-of-flight; IMAC, immobilized
metal affinity chromatography.
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ACKNOWLEDGMENTS |
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REFERENCES |
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