Download Partial Purification and Characterization of the Maize Mitochondrial

Plant Physiol. (1998) 116: 1443–1450
Partial Purification and Characterization of the Maize
Mitochondrial Pyruvate Dehydrogenase Complex1
Jay J. Thelen, Jan A. Miernyk, and Douglas D. Randall*
Department of Biological Sciences (J.J.T.), and Department of Biochemistry (J.A.M., D.D.R.),
University of Missouri, Columbia, Missouri 65211
The pyruvate dehydrogenase complex was partially purified and
characterized from etiolated maize (Zea mays L.) shoot mitochondria. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed proteins of 40, 43, 52 to 53, and 62 to 63 kD.
Immunoblot analyses identified these proteins as the E1b-, E1a-,
E2-, and E3-subunits, respectively. The molecular mass of maize E2
is considerably smaller than that of other plant E2 subunits (76 kD).
The activity of the maize mitochondrial complex has a pH optimum
of 7.5 and a divalent cation requirement best satisfied by Mg21.
Michaelis constants for the substrates were 47, 3, 77, and 1 mM for
pyruvate, coenzyme A (CoA), NAD1, and thiamine pyrophosphate,
respectively. The products NADH and acetyl-CoA were competitive
inhibitors with respect to NAD1 and CoA, and the inhibition constants were 15 and 47 mM, respectively. The complex was inactivated by phosphorylation and was reactivated after the removal of
ATP and the addition of Mg21.
The PDC catalyzes the oxidative decarboxylation of
pyruvate to form acetyl-CoA and NADH. The PDC is
composed of three fundamental enzymatic components:
PDH (E1, EC 1.2.4.1), dihydrolipoyl transacetylase (E2, EC
2.3.1.12), and dihydrolipoamide dehydrogenase (E3, EC
1.8.1.4). The PDC from mammals (Gopalakrishnan et al.,
1989), yeast (Behal et al., 1989), and perhaps plants (Taylor
et al., 1992) contains an associated E3-binding protein.
mtPDCs also contain two regulatory enzymes, PDH kinase
and P-PDH phosphatase, which regulate PDC by reversible
phosphorylation of the a-subunit of PDH (E1a). Mammalian and yeast mtPDC have a central pentagonal dodecahedryl core of E2-subunits to which the E1- and E3subunits attach (Patel and Roche, 1990; Stoops et al., 1997).
This E2 core comprises 20 trimers of a single polypeptide
(Patel and Roche, 1990). Six to 12 E3 dimers, 6 to 12 E3binding protein monomers, and 20 to 30 E1-a2b2 heterotetramers bind noncovalently to the E2 core (Patel and Roche,
1990).
The metabolic location of mtPDC and the irreversible
nature of the reaction suggest that it is a site for regulation
of mitochondrial carbon metabolism (Randall et al., 1996).
1
This research was supported by a National Science Foundation
grant (no. IBN-9419489) and by a Maize Training Grant Fellowship
awarded to J.J.T. This is journal report no. 12,648 from the Missouri
Agricultural Experiment Station.
* Corresponding author; e-mail [email protected].
edu; fax 1–573– 883–5635.
All PDCs studied thus far are regulated by product inhibition (Patel and Roche, 1990; Luethy et al., 1996). In higher
eukaryotes mtPDC activity is also regulated by reversible
phosphorylation catalyzed by a PDH-specific protein kinase and a P-PDH-specific phosphatase (for review, see
Patel and Roche, 1990; Randall et al., 1996).
The importance of mtPDC in controlling primary carbon
metabolism is reflected by the many literature reports.
However, there are a limited number of reports describing
research on plant mtPDCs (for review, see Randall et al.,
1996). Furthermore, our understanding of the regulation of
plant mtPDC is derived from a limited number of C3
species (e.g. pea, broccoli [Rubin and Randall, 1977], and
castor bean [Rapp et al., 1987]).
In most C3 species, leaf mtPDC is reversibly inactivated
in the light in a photosynthesis- and photorespirationdependent manner (Budde and Randall, 1990; Gemel and
Randall, 1992). This is most likely the result of photorespiratory Gly metabolism that occurs in the leaf mitochondria
during photosynthesis. Gly oxidation generates large
amounts of NADH to support the necessary mitochondrial
ATP production and NH41 to stimulate PDH kinase
(Schuller et al., 1993). Consequently, mtPDC is negatively
regulated as Gly oxidation increases, and all indications are
that this light inactivation is caused by reversible phosphorylation of mtPDC. Pyruvate is the most effective inhibitor of mtPDC phosphorylation/inactivation (Schuller
and Randall, 1990). In many C4 species such as maize (Zea
mays L.), pyruvate is a major metabolite in the photosynthetic CO2-fixation process, and C4 species lack significant
photorespiration (Hatch, 1987). Therefore, light-dependent
inactivation of mtPDC would not be expected. However,
light-dependent inactivation of mtPDC was observed in
maize leaves (Gemel and Randall, 1992), suggesting that
the regulation of mtPDC may be different in maize and
other C4 plants.
To establish the properties and regulation of mtPDC in
maize as a representative C4 plant, we have undertaken a
thorough examination of maize mtPDC beginning with
nonphotosynthetic tissue to establish a baseline before proceeding to the characterization of the leaf mtPDCs, which
Abbreviations: 400K enzyme, the pellet from the 400,000g centrifugation; GDC, Gly decarboxylase complex; mtPDC, mitochondrial pyruvate dehydrogenase complex; PDC, pyruvate dehydrogenase complex; PDH, pyruvate dehydrogenase; P-PDH,
phosphopyruvate dehydrogenase; TPI, triose phosphate isomerase; TPP, thiamine pyrophosphate.
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1444
Thelen et al.
will involve the two different cell types involved in C4
photosynthesis. This report describes the partial purification and characterization of mtPDC from etiolated shoots
of maize.
MATERIALS AND METHODS
Maize (Zea mays B73) seeds were obtained from the
Illinois Seed Foundation (Urbana). Mitochondria were isolated from etiolated shoots as previously described (Hayes
et al., 1991). Protein was quantified according to the
method of Bradford (1976) using BSA as the standard. All
other materials were from Sigma or Fisher Scientific.
Plant Physiol. Vol. 116, 1998
7.5, 2 mm DTT buffer, clarified by centrifugation at 13,000g
for 15 min, and designated the 400K enzyme. The 400K
enzyme (1 mL) was layered onto a 40-mL, 10 to 50% (v/v)
linear glycerol gradient. The glycerol stock solutions contained 50 mm Tes-KOH, pH 7.5, 1.5 mm pyruvate, 1 mm
MgCl2, and 14 mm 2-mercaptoethanol. Gradients were centrifuged for 18 h at 25,000 rpm using an SW-28 rotor in an
L8-55 ultracentrifuge (Beckman). Gradients were fractionated from the bottom, and the glycerol concentration was
determined using a refractometer.
RESULTS
SDS-PAGE, two-dimensional gel electrophoresis, and
immunodetection of proteins bound to nitrocellulose membranes were performed as previously described (Luethy et
al., 1995a). E1a monoclonal antibodies were raised against
maize protein (Luethy et al., 1995a). E1b polyclonal antibodies were raised against recombinant Arabidopsis thaliana
protein (M. Luethy, unpublished data). E3 polyclonal antibodies (generously provided by Dr. Steve Rawsthorne,
John Innes Institute, Norwich, UK) were raised against the
pea (Pisum sativum) L-protein of the GDC (Turner et al.,
1992). E2-specific antibodies were affinity purified from
total PDC antibodies raised against broccoli PDC (Randall
et al., 1981) by incubating nitrocellulose-immobilized pea
E2 with the antibodies and eluting as described by Smith
and Fisher (1984).
Plants are unique in that they contain a plastid PDC
isoform (Williams and Randall, 1979; Camp and Randall,
1985) in addition to mtPDC. Therefore, to study the mitochondrial isoform, it was necessary to demonstrate that the
purified mitochondria had low plastid contamination. Using TPI as the plastid marker enzyme, it was established
that only 0.05% of the total TPI activity was present with
the purified mitochondria.
Starting with 955 g fresh weight of etiolated maize
shoots, 0.4 mg of highly enriched PDC was obtained, corresponding to 1.4% of the total mitochondrial protein (Table I). Almost 30% of total PDC activity was recovered,
with a 21-fold enrichment. The specific activity of the partially purified maize mitochondrial PDC (0.81 mmol min21
mg21) was lower than values previously reported for purified cauliflower mtPDC (5.4 mmol min21 mg21, enriched
approximately 100-fold; Randall et al., 1977) and purified
broccoli mtPDC (6.3 mmol min21 mg21, enriched approximately 200-fold; Rubin and Randall, 1977). The peak of
PDC activity consistently sedimented at 30% glycerol (Fig.
1A), which is similar to the peaks of other mtPDCs but
larger than the peak of either plastid or Escherichia coli PDC
(Camp and Randall, 1985). Compared with the sedimentation profiles of other mtPDCs, the molecular mass of the
maize mtPDC was estimated at about 8000 to 9000 kD
(Patel and Roche, 1990).
Purification of Mitochondrial PDC
PDC Subunit Composition
Purified mitochondria were resuspended in 30 mm TesKOH, pH 7.5, 2 mm DTT, lysed with a Polytron homogenizer (30 s at the 70% setting), and centrifuged for 15 min
at 100,000g at 4°C in a rotor (model TL-100.3, Beckman) to
remove membranes. The supernatant was subsequently
centrifuged for 6 h at 400,000g. The resulting pellets were
resuspended in a minimal volume of 30 mm Tes-KOH, pH
SDS-PAGE analysis of the pooled mtPDC activity fractions from the glycerol gradient showed proteins at 40, 43,
52 to 53, 62 to 63, and 110 kD (Fig. 2, lane 4). Subunitspecific antibodies showed the enrichment of the putative
mtPDC components through the purification (Fig. 2B), accounting for all of the major polypeptides observed in the
gel except the 110-kD protein, which was probably a con-
Activity Assays
mtPDC activity was measured by monitoring NAD1
reduction at 340 nm (Randall et al., 1977) using a Response
UV/visible spectrophotometer (Gilford, Oberlin, OH). The
plastid marker TPI (EC 5.3.1.1) was assayed according to
the method of Eisenthal and Danson (1992).
Electrophoresis and Immunoblot Analysis
Table I. Summarized purification of maize mtPDC
Fraction
Specific Activity
mmol NADH formed min
Lysed mitochondria
100K Enzymea
400K Enzyme
Glycerol gradientb
a
0.039
0.12
0.16
0.81
Supernatant from 100,000g centrifugation.
b
21
mg
Total Activity
21
protein
mmol min
1.2
1.0
0.72
0.35
21
Enrichment
Yield
Protein
-fold
%
mg
1.0
3.1
4.1
21
100
83
60
29
30
8.5
4.4
0.43
Pooled mtPDC activity fractions from glycerol-gradient fractionation.
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Maize Mitochondrial Pyruvate Dehydrogenase Complex
1445
The identity of the 52- to 53-kD doublet was further
established by microsequencing the N terminus of these
two proteins. The N-terminal amino acid sequence of the
52-kD protein (Fig. 4) had the highest similarity to a mammalian dihydrolipoamide transacetylase (E2), according to
BLAST (Altschul et al., 1990), an amino acid alignment
algorithm. N-terminal sequencing of the 53-kD protein revealed that it is related to the 52-kD protein (Fig. 4). Aligning the N-terminal sequence of the maize 52-kD protein
with yeast (Niu et al., 1988), Arabidopsis (Guan et al.,
1995), and human (Coppel et al., 1988) deduced E2 amino
acid sequences showed the highest similarity within the
lipoyl domains.
Reaction Requirements and Kinetic Properties
Maize mtPDC activity showed a sharp optimum at pH
7.5, similar to that for pea mtPDC (Miernyk and Randall,
Figure 1. A, Fractionation profile for a typical rate-zonal glycerol
gradient. Approximately 3 mg of 400K enzyme was loaded onto this
gradient. Fractions 19 through 30 were pooled and concentrated for
the SDS gel shown in Figure 2. B, Coomassie blue-stained SDS-PAGE
of odd-numbered glycerol-gradient fractions 7 to 33. Positions of
protein standards are indicated on the left (in kilodaltons). The
position of the 110-kD protein is indicated on the right with an
arrowhead. U, Units.
taminant because it did not react with PDC antibodies and
peaked at a higher point in the glycerol gradient (Fig. 1B).
Monoclonal antibodies to maize E1a recognized the
43-kD band in immunoblots (Fig. 2B). However, this single
43-kD band presented six isoelectric forms on immunoblots
after two-dimensional IEF/SDS-PAGE separation (Fig. 3B).
These multiple isoelectric forms may reflect the phosphorylation of multiple residues creating a gradient of phosphoproteins. Polyclonal antibodies to recombinant Arabidopsis E1b recognized a 40-kD polypeptide (Fig. 2B) highly
enriched in the pooled glycerol-gradient fraction. In light
of the strong evidence that PDC and GDC in pea mitochondria share identical E3 components (Bourguignon et al.,
1996), antibodies raised against the pea L-protein of GDC
were used to probe maize mtPDC. These antibodies recognized a 62- to 63-kD doublet that was enriched in the
glycerol-gradient-purified fraction (Fig. 2B). Antibodies
that recognize the 76-kD pea E2-subunit reacted with a 52to 53-kD maize doublet that was enriched throughout the
PDC purification (Fig. 2B).
Figure 2. SDS-PAGE and corresponding immunoblots of lysed mitochondria (total mito), the supernatant from the 100,000g centrifugation (100K super), the 400K enzyme (400K pellet), and the pooled
mtPDC activity fraction from the glycerol-gradient fractionation
(glycerol). A, Coomassie blue-stained SDS-PAGE gel loaded with 5
mg of protein per lane. Positions of protein standards are indicated on
the left and calculated molecular mass values of the predominant
bands in the glycerol fraction are indicated on the right (in kilodaltons). B, Four replica protein blots of the fractions in A were probed
with anti-subunit antibodies. One microgram of protein was loaded
per lane. The molecular masses of the protein bands are indicated on
the left (in kilodaltons).
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1446
Thelen et al.
Plant Physiol. Vol. 116, 1998
Figure 4. Amino acid alignment of N-terminal amino acid sequences
for maize 52- and 53-kD proteins and deduced amino acid sequences for yeast, Arabidopsis, and human E2-subunits. The number
of amino acid residues (aa) before the homologous region is indicated to the left of the sequences. Shading indicates an identical
amino acid. X indicates a cycle of Edman degradation for which no
determination was made.
Km values were determined under optimal conditions of
pH and saturating concentrations of nonvariable substrates. Most of the Km values for maize mtPDC were in the
range of those reported for other plant PDCs (Table II). The
exception was TPP; its Km value was 10-fold higher than
that of pea. This may explain why the maize complex,
unlike the pea complex (Miernyk and Randall, 1987), required exogenous TPP for activity. The Ki for NADH was
5-fold lower than the Km for NAD1, whereas the Ki for
acetyl-CoA was much higher than the Km for CoA, suggesting that NADH could be a more potent product inhibitor (Table II). Both products were competitive inhibitors
with respect to their substrates (data not shown).
Intermediates of the Krebs cycle, amino acids, and polyamines had little effect on the activity of the 400K enzyme
when tested at 2 mm. Hydroxypyruvate, previously shown
Figure 3. Two-dimensional gel electrophoresis of glycerol-gradientenriched maize mtPDC and corresponding immunoblots. A, Ten
micrograms of glycerol-gradient-enriched mtPDC was resolved by
IEF in the first dimension followed by SDS-PAGE. B and C, One
microgram of protein was resolved as in A, transferred to nitrocellulose, and probed with antibodies (Ab) to the E1a- and E1b-subunits.
The molecular masses of the protein bands are indicated on the left
(in kilodaltons).
1987), but lower than that for plastid PDC (pH 8.2; Williams and Randall, 1979). Maize mtPDC activity was sensitive to high ionic strength, similar to porcine PDC (Pawelczyk et al., 1992), with buffer concentrations higher than 75
mm and NaCl concentrations greater than 50 mm reducing
mtPDC activity; NaCl, KCl, and NH4Cl all gave similar
patterns of inhibition.
Maize mtPDC required CoA, NAD1, thiamine pyrophosphate, and divalent cations for activity and did not use
NADP1. The 400K enzyme specifically decarboxylated
pyruvate and exhibited only minor activity with 2-oxobutyrate (15%), 3-hydroxypyruvate (6%), and 3-hydroxybutyrate (5%). No activity was seen with the branchedchain keto acids 2-oxoisovalerate and 2-oxoisocaproate, or
with 2-oxoglutarate. The dialyzed maize 400K enzyme had
an absolute requirement for divalent cations (Fig. 5), with
Mg21, Mn21, and Ca21 all able to restore activity.
Figure 5. Divalent cation requirement for the 400K enzyme. The
400K enzyme was dialyzed for 2 h in 2 L of 2 mM EDTA and 2 mM
EGTA to remove endogenous cations, and then dialyzed twice in 2 L
of 20 mM Tes, pH 7.5, and 0.5 mM DTT to remove the chelators.
Enzyme was added to an assay vessel that contained divalent cations
and necessary components. Rates are expressed as relative percentages of the maximum rate (0.093 mmol min21 mg21).
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Maize Mitochondrial Pyruvate Dehydrogenase Complex
1447
Table II. Km for PDCs
The maize 400K enzyme was used in this study. Values are the mean of at least three separate
preparations 6 SD.
Plant Tissue
Km
Pyr
NAD
Ki
CoA
MgTPP
NADHa
AcCoAb
mM
MtPDCs
Etiolated maize shoots
Pea leafc
Broccoli floretd
Plastid PDC
Pea leaf f
47 6 3
57
250
77 6 18
122
110
362
4
6
1 6 0.2
0.08
n.d.e
15 6 6
18
13
47 6 5
10
19
120
36
10
n.d.
9
16
b
[CoASH] was 20 mM; all other substrates and cofactors were saturating.
[NAD] was 100 mM;
c
d
all other substrates and cofactors were saturating.
Miernyk and Randall (1987).
Rubin and
e
f
Randall (1977).
n.d., Not determined.
Camp et al. (1988).
a
to be a noncompetitive inhibitor of PDC (Randall et al.,
1977), inhibited maize PDC by 36%. Ali et al. (1993) reported that the E1a-subunit of mammalian PDC contained
an essential Cys (Cys-62 in humans). Mutation of this Cys
to Ala or derivatization by sulfhydryl reagents completely
inactivated the mammalian enzyme. Because this essential
Cys is not present in prokaryotic or plastid (Johnston et al.,
1997) forms of PDC, we determined the effect of sulfhydryl
reagents on maize mtPDC. Mersalyl, p-hydroxymercuribenzoate, and N-ethylmaleimide rapidly inactivated the
maize mtPDC.
ATP-Dependent Inactivation
The 400K enzyme was almost completely inactivated in 6
min with 200 mm MgATP, but after 15 min the complex
began gradually reactivating (Fig. 6A). To determine if this
reactivation was caused by the Mg21-requiring P-PDH
phosphatase activity, the 400K enzyme was inactivated
with 200 mm ATP, the excess ATP was removed with
hexokinase plus Glc, the sample was divided into four
aliquots, and EDTA, MgCl2, or buffer was added (Fig. 6B).
EDTA prevented the gradual reactivation observed with
the control, whereas 10 mm Mg21 stimulated an 85% recovery of activity in 20 min, suggesting that the Mg21dependent reactivation is likely the result of a P-PDH
phosphatase. Incubation of the 400K enzyme with
[g-32P]ATP labeled a 43-kD protein that was recognized by
monoclonal antibodies to the maize E1a-subunit (data not
shown).
DISCUSSION
Rate-zonal density-gradient centrifugation of maize mitochondrial matrix protein yielded a distinct peak of PDC
activity with a specific activity ranging from 1.2 to 1.7 units
mg21 protein (Fig. 1A), and the predominant polypeptides
in this peak were identified as the E1b-, E1a-, E2-, and
E3-subunits. Additionally, a 110-kD Coomassie bluestained polypeptide was observed; however, this protein
did not peak with PDC peak activity (Fig. 1B) and was not
recognized by any PDC antibodies. Polypeptides of 50 and
55 kD were also observed in heavily loaded lanes; however, the 55-kD polypeptide also did not peak with PDC
peak activity. The unidentified 50-kD protein was probably
not PDH kinase or P-PDH phosphatase, since glycerolgradient-enriched mtPDC lacked these activities. Alternatively, it could have been an E3-binding protein homolog,
which is also a 50-kD protein in yeast (Behal et al., 1989).
A comparison of the apparent and calculated sizes of
plant mtPDC subunits shows that only the maize E1asubunit is identical in size to other plant E1a-subunits,
whereas the E1b- and E3-subunits are 2 to 3 kD larger
(Table III). In contrast, the maize E2-subunit (52 kD) is
much smaller than other plant E2-subunits (76 kD), which
can be explained by its variable multidomain structure.
The E2-subunit possesses a multidomain structure, with
a lipoyl domain(s) connected by flexible linkers to the
E1-/E3-binding domains followed by the catalytic domain
(Reed and Hackert, 1990; Perham, 1991). The flexible lipoyl
domains allow active-site coupling between the E1- and
E3-subunits for the following series of reactions. The E1
reductively acylates the covalently bound lipoate within
the E2 lipoyl domain. The E2-subunit catalyzes the acyltransfer step to CoA, and E3 catalyzes the reoxidation of
the dihydrolipoyl moiety using NAD1 as the electron acceptor. In addition to having a catalytic role, the inner
(second) lipoyl domain of mammalian E2 binds the kinase
and phosphatase regulatory components (Liu et al., 1995;
Chen et al., 1996).
The number of E2 lipoyl domains found in nature is
variable (Reed and Hackert, 1990). Multiple, tandemly repeated lipoyl domains have been observed in the E2subunits described previously for all organisms except bacilli and yeast (Perham, 1991). Although the tandemly
repeated lipoyl domains are functional (Allen et al., 1989),
only one is required for E2 catalytic or complex function
(Guest et al., 1985; Machado et al., 1992). The considerably
smaller size of the maize E2 can be explained if only one
lipoyl domain is present. A single lipoyl domain may be
attributable to either novel gene structure or proteolysis.
Proteolysis is unlikely, since we observed only the 52- to
53-kD band for E2 with purified mitochondria lysed di-
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1448
Thelen et al.
Plant Physiol. Vol. 116, 1998
Table III. Estimated and deduced molecular mass values of
mtPDC catalytic subunits
Values are based on SDS-PAGE analysis of isolated mitochondria
and therefore represent processed proteins. Molecular mass values
deduced from sequenced cDNA clones represent precursor proteins
(i.e. targeting peptide plus mature protein).
Molecular Mass
SDS-PAGE
Subunit
Maize
Calculated/deduced
Pea
Arabidopsis
Pea
Potato
43.5c
38.7c
n.a.
50.4h
43.2d
n.a.
n.a.
n.a.
kD
E1a
E1b
E2
E3
43
40
52 –53
62– 63
43a
38a
76a
a
57 ,60g,h
43.0b
39.2e
60.0f
n.a.i
a
b
c
Luethy et al., 1995a.
Luethy et al., 1995b.
M.H. Lued
e
thy, unpublished data.
Grof et al., 1995.
Luethy,
f
g
h
1994.
Guan et al.
Turner et al., 1992.
Bourguignon
i
et al., 1992.
n.a., Data not available.
Figure 6. A, ATP-dependent inactivation of the 400K enzyme.
Equimolar amounts of Mg21 and ATP were added to PDC preparations to the final concentrations indicated. The control did not have
any MgATP added. One-hundred-microgram samples of enzyme
were removed at various time intervals and assayed for activity.
Activity is expressed as a percentage of the control (0.16 mmol
NADH formed min21 mg21 protein) at time 0. B, The effect of Mg21
on reactivation of P-PDC. The 400K enzyme was incubated with 200
mM MgATP until the inactivation of mtPDC ceased. Free ATP was
then removed with 2.5 units of hexokinase (HK) and 2 mM Glc at 30
min. The 400K enzyme was then divided into four aliquots to which
EDTA, MgCl2, or buffer (control) was added to the final concentrations indicated.
rectly into SDS-PAGE sample buffer followed by boiling
(data not shown).
Support for the presence of a single lipoyl domain is
found in the reduced size of the maize E2, the N-terminal
amino acid sequence (which is most similar to the single E2
lipoyl domain from yeast), and its similarity to the inner
lipoyl domain of Arabidopsis and human E2. The properties of the maize E2 are consistent with previous findings,
i.e. a single lipoyl domain is sufficient for complex activity
and appears to be sufficient for binding the kinase and
phosphatase, although maybe not as tightly as with the
mammalian complex, since both the kinase and the phosphatase can be stripped away during the glycerol-gradient
purification. Molecular analysis shows that the E2 from
Arabidopsis has multiple lipoyl domains (Guan et al.,
1995), so it will be interesting to determine if this is true for
other plant species or if single lipoyl domains are characteristic of maize alone.
The pyruvate and CoA Kms for maize mtPDC are similar
to those from pea (Miernyk and Randall 1987), but unlike
the pea mtPDC the maize complex has a lower Km for
NAD1 and a higher Km for MgTPP, which may reflect
differences in the E3 and E1 components. The high Ki for
acetyl-CoA in relation to other plant PDCs may reflect a
different in vivo environment for the maize mtPDC or the
various functions of the maize mitochondria.
The maize mtPDC requires divalent cations for catalytic
activity. Of the divalent cations tested, Mg21 best satisfied
this requirement, as determined by the Vmax/Km ratio for
the three cations Mg21 (2.3), Mn21 (1.3), and Ca21 (1.0).
The Km for Mg21 was approximately 40 mm, considerably
lower than that of pea mtPDC (360 mm; Miernyk and
Randall, 1987) and pea plastid PDC (1 mm; Camp and
Randall, 1985), suggesting that the divalent cation requirement does not have regulatory significance in maize. All
plant PDCs except mtPDC from cauliflower (Randall et al.,
1977) will accept Mn21 and Ca21 as Mg21 substitutes.
The maize mtPDC is capable of regulation by reversible
phosphorylation. Increasing amounts of MgATP com-
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Maize Mitochondrial Pyruvate Dehydrogenase Complex
pletely inactivated mtPDC, although reactivation immediately ensued, indicating that PDC activity reflects the relative activities of the regulatory kinase and phosphatase.
MgATP concentrations below saturation will not entirely
inactivate mtPDC, even after extended periods. This can be
explained by contaminating ATPase activity, high P-PDH
phosphatase activity, multiple phosphorylation sites that
coordinate full inactivation, or all of the above.
In summary, we have partially purified PDC from
maize mitochondria and identified the catalytic subunits
by immunoblot analysis. The molecular masses of the
maize PDC subunits are similar to those of other plant
PDCs, the exception being the E2-subunit, which was 23
kD smaller than pea E2. Overall, the kinetic properties of
maize mtPDC were similar to those of other plant
mtPDCs, although slight differences were observed with
regard to the divalent cation and TPP requirement, as well
as the product inhibitor acetyl-CoA. The degree of similarity between maize mtPDC and C3-plant mtPDCs was
somewhat surprising considering the differences in pyruvate metabolism.
ACKNOWLEDGMENTS
The authors are grateful for discussions and critical reading by
Dr. Michael H. Luethy. We also thank Professor Thomas E. Elthon
and Dr. Gautum Sarath for the protein microsequencing performed at the Protein Core Facility, University of NebraskaLincoln.
Received September 24, 1997; accepted December 23, 1997.
Copyright Clearance Center: 0032–0889/98/116/1443/08.
LITERATURE CITED
Ali MS, Roche TE, Patel MS (1993) Identification of the essential
cysteine residue in the active site of bovine pyruvate dehydrogenase. J Biol Chem 268: 22353–22356
Allen AG, Perham RN, Allison N, Miles JS, Guest JR (1989)
Reductive acetylation of tandemly repeated lipoyl domains in
the pyruvate dehydrogenase multienzyme complex of Escherichia coli is random order. J Mol Biol 208: 623-633
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990)
Basic local alignment search tool. J Mol Biol 215: 403–410
Behal RH, Browning KS, Hall TB, Reed LJ (1989) Cloning and
nucleotide sequence of the gene for protein X from Saccharomyces
cerevisiae. Proc Natl Acad Sci USA 86: 8732–8736
Bourguignon J, Macherel D, Neuburger M, Douce R (1992) Isolation, characterization, and sequence analysis of a cDNA clone
encoding L-protein, the dihydrolipoamide dehydrogenase component of the glycine cleavage system from pea-leaf mitochondria. Eur J Biochem 204: 865–873
Bourguignon J, Merand V, Rawsthorne S, Forest E, Douce R
(1996) Glycine decarboxylase and pyruvate dehydrogenase complexes share the same dihydrolipoamide dehydrogenase in pea
leaf mitochondria: evidence from mass spectrometry and
primary-structure analysis. Biochem J 313: 229–234
Bradford MM (1976) A rapid and sensitive method for the quantitation of protein utilizing the principle of protein-dye binding.
Anal Biochem 72: 248–254
Budde RJA, Randall DD (1990) Pea leaf mitochondrial pyruvate
dehydrogenase complex is inactivated in vivo in a lightdependent manner. Proc Natl Acad Sci USA 87: 673–676
Camp PJ, Randall DD (1985) Purification and characterization of
the pea chloroplast pyruvate dehydrogenase complex. Plant
Physiol 77: 571–577
1449
Chen G, Wang L, Liu S, Chuang C, Roche TE (1996) Activated
function of the pyruvate dehydrogenase phosphatase through
Ca21-facilitated binding to the inner lipoyl domain of the dihydrolipoyl acetyltransferase. J Biol Chem 45: 28064–28070
Coppel RL, McNeilage J, Surh CD, Van de Water J, Spithill TW,
Whittingham S, Gershwin ME (1988) Primary structure of the
human M2 mitochondrial autoantigen of primary biliary cirrhosis: dihydrolipoamide acetyltransferase. Proc Natl Acad Sci USA
85: 7317–7321
Eisenthal R, Danson MJ (1992) Photometric assays. In D Rickwood, BD James, eds, Enzyme Assays: A Practical Approach.
Oxford University Press, New York, p 79
Gemel J, Randall DD (1992) Light regulation of leaf mitochondrial
pyruvate dehydrogenase complex. Plant Physiol 100: 908–914
Gopalakrishnan S, Rahmatullah M, Radke GA, PowersGreenwood S, Roche TE (1989) Role of protein X in the function
of the mammalian pyruvate dehydrogenase complex. Biochem
Biophys Res Commun 160: 715–721
Grof CPL, Winning BM, Scaysbrook TP, Hill SA, Leaver CJ
(1995) Mitochondrial pyruvate dehydrogenase: molecular cloning of the E1a subunit and expression analysis. Plant Physiol
108: 1623–1629
Guan Y, Rawsthorne S, Scofield G, Shaw P, Doonan J (1995)
Cloning and characterization of a dihydrolipoamide acetyltransferase (E2) subunit of the pyruvate dehydrogenase complex
from Arabidopsis thaliana. J Biol Chem 270: 5412–5417
Guest JR, Lewis HM, Graham LD, Packman LC, Perham RN
(1985) Genetic reconstruction and functional analysis of the
repeating lipoyl domains in the pyruvate dehydrogenase multienzyme complex of Escherichia coli. J Mol Biol 185: 743–754
Hatch MD (1987) C4 photosynthesis: a unique blend of modified
biochemistry anatomy and ultrastructure. Biochim Biophys Acta
895: 81–106
Hayes MK, Luethy MH, Elthon TE (1991) Mitochondrial malate
dehydrogenase from corn. Plant Physiol 97: 1381–1387
Johnston ML, Luethy MH, Miernyk JA, Randall DD (1997) Cloning and molecular analyses of the Arabidopsis thaliana plastid
pyruvate dehydrogenase subunits. Biochim Biophys Acta 1321:
200–206
Liu S, Baker JC, Roche TE (1995) Binding of the pyruvate dehydrogenase kinase to recombinant constructs containing the inner
lipoyl domain of the dihydrolipoyl acetyltransferase component.
J Biol Chem 2: 793–800
Luethy MH, David NR, Elthon TE, Miernyk JA, Randall DD
(1995a) Characterization of a monoclonal antibody recognizing
the E1a subunit of plant mitochondrial pyruvate dehydrogenase. J Plant Physiol 145: 443–449
Luethy MH, Miernyk JA, David NR, Randall DD (1996) Plant
pyruvate dehydrogenase complexes. In MS Patel, TE Roche, RA
Harris, eds, Alpha-Keto Acid Dehydrogenase Complexes.
Birkhauser Verlag, Basel, Switzerland, pp 71–92
Luethy MH, Miernyk JA, Randall DD (1994) The nucleotide and
deduced amino acid sequences of a cDNA encoding the E1bsubunit of the Arabidopsis thaliana mitochondrial pyruvate dehydrogenase complex. Biochim Biophys Acta 1187: 95–98
Luethy MH, Miernyk JA, Randall DD (1995b) The mitochondrial
pyruvate dehydrogenase complex: nucleotide and deduced
amino-acid sequences of a cDNA encoding the Arabidopsis thaliana E1a subunit. Gene 164: 251–254
Machado RS, Clark DP, Guest JR (1992) Construction and properties of pyruvate dehydrogenase complexes with up to nine
lipoyl domains per lipoate acetyltransferase chain. FEMS Microbiol Lett 100: 243–248
Miernyk JA, Randall DD (1987) Some kinetic properties of the pea
mitochondrial pyruvate dehydrogenase complex. Plant Physiol
83: 306–310
Niu XD, Browning KS, Behal RH, Reed LJ (1988) Cloning and
nucleotide sequence of the gene for dihydrolipoamide acetyltransferase from Saccharomyces cerevisiae. Proc Natl Acad Sci
USA 85: 7546–7550
Patel MS, Roche TE (1990) Molecular biology and biochemistry of
pyruvate dehydrogenase complexes. FASEB J 4: 3224–3233
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 1998 American Society of Plant Biologists. All rights reserved.
1450
Thelen et al.
Pawelczyk T, Easom RA, Olson MS (1992) Effect of ionic strength
and pH on the activity of pyruvate dehydrogenase complex
from pig kidney cortex. Arch Biochem Biophys 294: 44–49
Perham RN (1991) Domains, motifs, and linkers in 2-oxo acid
dehydrogenase multienzyme complexes: a paradigm in the design of a multifunctional protein. Biochemistry 30: 8501–8512
Randall DD, Miernyk JA, David NR, Gemel J, Luethy MH (1996)
Regulation of leaf mitochondrial pyruvate dehydrogenase complex activity by reversible phosphorylation. In PR Shewry, NG
Halford, eds, Protein Phosphorylation in Plants. Clarendon
Press, Oxford, UK, pp 87–103
Randall DD, Rubin PM, Fenko M (1977) Plant pyruvate dehydrogenase complex purification, characterization and regulation
by metabolites and phosphorylation. Biochim Biophys Acta 485:
336–349
Randall DD, Williams M, Rapp BJ (1981) Phosphorylationdephosphorylation of pyruvate dehydrogenase complex from
pea leaf mitochondria. Arch Biochem Biophys 207: 437–444
Rapp BJ, Miernyk JA, Randall DD (1987) Pyruvate dehydrogenase complexes from Ricinus communis endosperm. J Plant
Physiol 127: 293–306
Reed LJ, Hackert ML (1990) Structure-function relationships in
dihydrolipoamide acyltransferases. J Biol Chem 265: 8971–8974
Rubin PM, Randall DD (1977) Purification and characterization of
pyruvate dehydrogenase complex from broccoli floral buds.
Arch Biochem Biophys 178: 342–349
Plant Physiol. Vol. 116, 1998
Schuller KA, Gemel J, Randall DD (1993) Monovalent cation
activation of plant pyruvate dehydrogenase kinase. Plant
Physiol 102: 139–143
Schuller KA, Randall DD (1990) Mechanism of pyruvate inhibition of plant pyruvate dehydrogenase kinase and synergism
with ADP. Arch Biochem Biophys 278: 211–216
Smith DE, Fisher PA (1984) Identification, developmental regulation, and response to heat shock of two antigenically related
forms of a major nuclear envelope protein in Drosophila embryos: application of an improved method for affinity purification of antibodies using polypeptides immobilized on nitrocellulose blots. J Cell Biol 99: 20–28
Stoops JK, Cheng RH, Yazdi MA, Maeng CY, Schroeter JP,
Klueppelberg U, Kolodziej SJ, Baker TS, Reed LJ (1997) On
the unique structural organization of the Saccharomyces cerevisiae pyruvate dehydrogenase complex. J Biol Chem 272: 5757–
5764
Taylor AE, Cogdell RJ, Lindsay JG (1992) Immunological comparison of the pyruvate dehydrogenase complexes from pea
mitochondria and chloroplasts. Planta 188: 225–231
Turner SR, Ireland R, Rawsthorne S (1992) Purification and primary amino acid sequence of the L subunit of glycine decarboxylase. J Biol Chem 267: 7745–7750
Williams M, Randall DD (1979) Pyruvate dehydrogenase complex from chloroplasts of Pisum sativum L. Plant Physiol 64:
1099–1103
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 1998 American Society of Plant Biologists. All rights reserved.