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(Received for publication, November 6, 1995; and in revised form, December 21, 1995) From the
A novel dihydrolipoyl dehydrogenase-binding protein (E3BP) which
lacks an amino-terminal lipoyl domain, p45, has been identified in the
pyruvate dehydrogenase complex (PDC) of the adult parasitic nematode, Ascaris suum. Sequence at the amino terminus of p45 exhibited
significant similarity with internal E3-binding domains of
dihydrolipoyl transacetylase (E2) and E3BP. Dissociation and resolution
of a pyruvate dehydrogenase-depleted adult A. suum PDC in
guanidine hydrochloride resulted in two E3-depleted E2 core
preparations which were either enriched or substantially depleted of
p45. Following reconstitution, the p45-enriched E2 core exhibited
enhanced E3 binding, whereas, the p45-depleted E2 core exhibited
dramatically reduced E3 binding. Reconstitution of either the bovine
kidney or A. suum PDCs with the A. suum E3 suggested
that the ascarid E3 was more sensitive to NADH inhibition when bound to
the bovine kidney core. The expression of p45 was developmentally
regulated and p45 was most abundant in anaerobic muscle. In contrast,
E3s isolated from anaerobic muscle or aerobic second-stage larvae were
identical. These results suggest that during the transition to
anaerobic metabolism, E3 remains unchanged, but it appears that a novel
E3BP, p45, is expressed which may help to maintain the activity of the
PDC in the face of the elevated intramitochondrial NADH/NAD The pyruvate dehydrogenase complex (PDC) ( The subunit composition of the adult ascarid muscle PDC differs
significantly from the PDCs isolated from other eukaryotic organisms (7, 9, 10) . In both yeast and mammalian
PDCs, incubation of the complex with
[2- In the present study, we report that
sequence near the amino terminus of p45 exhibits significant similarity
to the putative E3-binding domains of E2 and E3BP and demonstrate that
E3 binding to the E2 core is dependent on the presence of p45. In
addition, we show that ascarid E3 bound to the bovine kidney core
(containing E3BP) appears to be more sensitive to NADH inhibition than
when it is bound to the ascarid core (containing p45). Therefore, it
appears that during the switch from aerobic to anaerobic energy
metabolism in ascarid development, E3 remains unchanged, but a novel
anaerobic E3BP, p45, is expressed to maintain PDC activity in the face
of elevated NADH/NAD
Figure 1:
Resolution of A. suum E1-depleted PDC with guanidine hydrochloride. E1-depleted A.
suum PDC (0.6 mg) was incubated in 50 mM MOPS, 1 M GdnHCl, 1 mM EDTA, 3 mM dithiothreitol, and 1
mg/ml Pluronic
For immunoblotting,
samples were transferred to nitrocellulose (Schleicher and Schuell)
overnight at 40 V in 25 mM Tris-HCl, 192 mM glycine,
and 20% (v/v) methanol. To detect bands of interest, each specific
antiserum was used in conjunction with the Promega kit,
Protoblot
Figure 2:
Binding of A. suum peripheral
components (E1 and E3) by two E2 core populations. A. suum p45-enriched E2 core (Peak 1) was reconstituted with excess A.
suum E3, and p45-depleted A. suum E2 core (Peak 2) was
reconstituted with excess A. suum E1 and E3 as described under
``Experimental Procedures.'' Samples were separated by
SDS-PAGE on 10% gels and stained with Coomassie Brilliant Blue. Lane 1, adult A. suum PDC (12 µg); Lane
2, E1-depleted A. suum PDC (10 µg); Lane 3, p45-enriched E2 core (Peak 1) (
Figure 3:
Association of p45 with the core of the
complex after selective proteolysis. E1-depleted A. suum PDC
was treated with V8 protease and the proteolytic products were
separated as described under ``Experimental Procedures.''
Samples (9 µg) were separated by SDS-PAGE on 12% gels and stained
with Coomassie Brilliant Blue. The identity of E2 domain fragments was
determined by immunoblotting and amino-terminal sequencing. Lane
1, E1-depleted PDC; Lane 2, E1-depleted PDC following
proteolysis; Lane 3, pellet; Lane 4,
supernatant.
Figure 4:
NADH sensitivity of A. suum,
bovine kidney, and hybrid pyruvate dehydrogenase complexes. Hybrid
complexes were constructed as described under ``Experimental
Procedures.'' A, PDC activity is expressed as a
percentage of the activity measured in the absence of NADH using a
fixed NAD
Figure 5:
Immunoblot of A. suum larval
homogenates with antisera against the adult A. suum E2, p45,
E1
Perhaps surprisingly, significant p45 staining
was observed in homogenates of third-stage larvae (L3), even though
they are aerobic and cyanide-sensitive. However, other key enzymes of
anaerobic metabolism, such as the 2-methyl branched-chain enoyl-CoA
reductase also have been identified in this transitional larval stage.
Their expression may represent an adaptation to the switch to
anaerobiosis that accompanies the impending molt to the
fourth-stage(30) . Taken together, these results suggest that
p45 is found predominantly in anaerobic muscle. In addition, antisera
against the adult muscle E1 To determine if
E3s also were different in aerobic and anaerobic stages, E3 was
purified to homogeneity from aerobic L2 (Table 2). E3 from L2
behaved similarly to the E3 from adult muscle during purification and
had identical kinetic properties and subunit molecular mass (55 kDa,
data not shown). More important, amino-terminal sequence analysis of
both E3s indicated that the first 53 amino acids were identical,
suggesting that stage-specific isoforms of E3 were not present in the A. suum PDC (Table 3). Therefore, it appears that during
the switch from aerobic to anaerobic energy metabolism in ascarid
development, E3 remains unchanged, but a novel anaerobic E3BP, p45, is
expressed to maintain PDC activity in the face of elevated
NADH/NAD
A. suum undergoes a marked aerobic/anaerobic
transition during its development and energy metabolism in adult muscle
mitochondria is anaerobic and characterized by elevated
NADH/NAD Although the function of a novel E3BP in anaerobic ascarid
mitochondria is still not completely clear, it appears that the binding
of E3 to p45 may decrease the sensitivity of the complex to NADH
inhibition. When a hybrid complex was constructed using the bovine
kidney core, bovine kidney E1, and the ascarid E3, the hybrid complex
had a significantly higher apparent K The function of E3BP has been elucidated most
directly in the S. cerevisiae PDC. Disruption of the gene
encoding E3BP yields a catalytically inactive complex with a properly
assembled E2 core and attached E1, but no E3, and suggests that while
E3BP is required for E3 binding, it is not an integral part of the core
or required for assembly(36) . More important, deletion of most
of the lipoyl domain of E3BP had no effect on PDC activity, suggesting
that the lipoyl domain was not essential for E3 binding or E3BP
function(36) . While E3BP has a domain structure similar to E2,
it has no catalytic activity, and lacks the HXXKG sequence
near the carboxyl terminus proposed as part of the active site of all
dihydrolipoamide acyltransferases (37) . However, after the
removal of the lipoyl domains of E2 by direct proteolysis or
site-directed mutagenesis, it does appear that the lipoyl domain of
E3BP can support the overall reaction of the complex, albeit at a
greatly reduced rate(38, 39) . Indeed, the final
specific activity of the PDC purified from A. suum muscle is
significantly lower than that reported for PDCs from either yeast or
mammals(7) . Whether this reduced specific activity is related
to the absence of a lipoyl domain on E3BP is not clear. Recently, an
E3BP in the PDC of the insect trypanosomatid, Crithidia
fasciculata, has been identified which appears to contain, not
one, but multiple lipoyl domains(40) . A similar situation has
been described previously for E2s, where 1, 2, or 3 lipoyl domains have
been identified, depending on the species, and there appears to be no
physiological significance to the number of lipoyl domains in
E2(41) . For example, site-directed mutagenesis of the three
lipoyl domains of the E. coli E2 suggests that the
inactivation of two of the three lipoyl domains does not affect
catalysis(42) . Whether more subtle regulatory properties are
affected by these alterations has not been determined.
Volume 271,
Number 10,
Issue of March 8, 1996 pp. 5451-5457
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
ratios associated with anaerobiosis.
)plays a
key role in the unique mitochondrial metabolism of the parasitic
nematode, Ascaris suum(1, 2) . During larval
development, A. suum exhibits a marked aerobic-anaerobic
transition in energy metabolism. Early larval stages are aerobic, and
the PDC functions to supply acetyl-CoA for tricarboxylic acid cycle
oxidation(3) . In contrast, energy metabolism in adult ascarid
muscle is anaerobic. Adult muscle mitochondria lack both a functional
tricarboxylic acid cycle and cytochrome oxidase activity and use
unsaturated organic acids, such as fumarate and 2-methyl branched-chain
enoyl-CoAs, instead of oxygen, as terminal electron
acceptors(4, 5, 6) . The PDC is overexpressed
in adult muscle and is designed to function under the elevated
NADH/NAD and acyl-CoA/CoA ratios present in the
microaerobic environment of the host gut(7, 8) .
C]pyruvate in the absence of CoA results in
the acetylation of two proteins, dihydrolipoyl transacetylase (E2) and
dihydrolipoyl dehydrogenase (E3)-binding protein
(BP)(11, 12) . E2 and E3BP are present in a ratio of
about 5:1, and both contain similar multidomain structures consisting
of an amino-terminal lipoyl domain, a subunit binding domain, and an
inner domain(13, 14, 15) . E2 preferentially
binds pyruvate dehydrogenase (E1) and exhibits transacetylase activity,
whereas E3BP contains a high-affinity binding site for E3(16) .
In contrast, incubation of the ascarid complex under similar conditions
results in the acetylation of only E2 and no protein corresponding to
E3BP has been identified(10) . In addition, the PDC from A.
suum and other closely related nematodes contains subunits of 43
kDa (p43) and 45 kDa (p45) of unknown function, which do not appear to
be present in PDCs from other organisms(17) . Whether the
E3-binding site in the ascarid PDC resides in E2, as has been observed
for PDCs from prokaryotes, or in one of these novel proteins has not
been determined(18) . ratios.
Materials
A. suum were obtained from Routh Packing (Sandusky,
OH). PDC (3.5 µmol of NADH min (mg of
protein)
) was isolated from frozen A. suum muscle strips as described previously(19) . A. suum E3 was prepared as described previously(20) . A. suum larval stages were prepared as described previously(21) .
Bovine kidney PDC, E2
E3BP core, and E1 were the generous gift of
Dr. Thomas E. Roche (Kansas State University, Manhattan, KS). All
chromatographic matrices were purchased from Pharmacia Biotech
(Piscataway, NJ), and all electrophoresis reagents were from Bio-Rad.
All other chemicals and reagents were of the highest grade available
and purchased from Sigma.Methods
Sequential Dissociation and Resolution of A. suum
PDC
A. suum E1-depleted PDC and E1 were prepared as
described previously (10) with the following modification:
dissociation was performed in 50 mM MOPS (pH 7.4), 2 M NaCl, 1 mM MgCl
, 0.1 mM EDTA, 3
mM dithiothreitol, and 0.8 mg ml Pluronic
F68 (BASF Corp.). After incubation at
room temperature for 90 min, the sample was applied to a Sephacryl
S-400 column (2.6
90 cm) equilibrated with the same buffer. The
first peak contained E1-depleted PDC and was concentrated by
centrifugation at 155,000 g for 3 h. The pellet was
resuspended in 50 mM MOPS (pH 7.4), 1 mM MgCl, 1 mM EDTA, 0.6 mg ml Pluronic
F68, and 3 mM dithiothreitol
(Buffer A). E1-depleted PDC (0.5-0.8 mg) was incubated for 60 min
at room temperature in 50 mM MOPS (pH 7.4), 1 M GdnHCl (Pierce), 1 mg ml
Pluronic
F68, 1 mM EDTA, and 3 mM dithiothreitol (final
volume 250 µl), followed immediately by fast protein liquid
chromatography on a Superose 12 HR10/30 column equilibrated in the same
buffer. Peak fractions were pooled. After removal of GdnHCl by
dialysis, Peak 1 was centrifuged at 155,000
g for 4 h.
The pellet (Peak 1, see Fig. 1) was insoluble using a variety of
solubilization techniques including detergents, heparin, and
NH
Cl. Therefore, following centrifugation the pellet was
resuspended directly in Laemmli sample buffer for SDS-polyacrylamide
gel electrophoresis (PAGE). Peaks 2 and 3 were desalted and
concentrated in a Centricon 30 (Amicon Inc.) with
several changes of Buffer A. Protein was determined according to
Bradford using bovine serum albumin as a standard(22) .
F68 (pH 7.4) as described under
``Experimental Procedures.'' The sample (0.2 ml) was applied
to a Superose 12 column equilibrated in the same buffer. The flow rate
was 0.4 ml/min and 0.4-ml fractions were collected. Inset,
Coomassie Brilliant Blue-stained 10% SDS-polyacrylamide gel of the
pooled peak fractions; *, adult A. suum PDC (12 µg); SC, E1-depleted A. suum PDC (10 µg); 1,
Peak 1, fractions 18-20 (
8 µg), (p45-enriched E2 core); 2, Peak 2, fractions 23-27 (8 µg) (p45-depleted A. suum E2 core); and 3, Peak 3, fractions
29-32, (4 µg). Molecular mass markers of 64, 50, and 36 kDa
are indicated at the right.
Reconstitution and Binding of Resolved Components
following Guanidine Hydrochloride Dissociation
After resolution
and the removal of 1 M GdnHCl, Peak 2 (100 µg) was
incubated with excess A. suum E1 (200 µg) and E3 (100
µg) for 30 min at room temperature in Buffer A (final volume of 2.5
ml). PDC activity of the reconstituted sample was measured
spectrophotometrically as described previously(7) . The
reconstituted sample was layered over 4 ml of 20% (w/v) sucrose in
Buffer A. After centrifugation at 155,000 g for 5 h,
the pellet was resuspended in Buffer A and the supernatant was
concentrated by Centricon 30 with several changes of
Buffer A. Since Peak 1 was insoluble after removal of GdnHCl, Peak 1
(approximately 150 µg) was dialyzed against 50 mM MOPS (pH
7.4), 1 mM EDTA, and 3 mM dithiothreitol (Buffer B)
containing 0.5 M GdnHCl. After 2 h, E3 (100 µg) was added
and the sample was dialyzed against Buffer B containing 0.1 M GdnHCl for an additional 3 h. After a final dialysis against
Buffer B containing 0.02 M GdnHCl for 6 h, the sample was
layered over 4.0 ml of 20% (w/v) sucrose in Buffer A and centrifuged at
155,000
g for 5 h. This pellet was resuspended
directly in Laemmli sample buffer.V8 Protease Treatment
E1-depleted PDC (500 µg)
was incubated in 15 mM Tris-HCl (pH 7.4), 3 mM MgCl, 1 mM dithiothreitol, and 50 µg of
V8 protease (final volume of 0.5 ml). After incubation at room
temperature for 60 min, the reaction was stopped with the addition of
3,4-dichloroisocoumarin (final concentration of 0.2 mM), and
400 µl of the stopped reaction was layered over 2 ml of 15% (w/v)
sucrose in 50 mM MOPS (pH 7.4), 1 mM dithiothreitol,
and 0.1 mM EDTA. After centrifugation at 155,000 g for 3 h, the pellet was resuspended in 50 mM MOPS (pH
7.4), 1 mM dithiothreitol, and 0.1 mM EDTA (Buffer C)
and the supernatant was concentrated in a Centricon 30
with several changes of Buffer C.
NADH Sensitivity of Native and Hybrid PDCs
A.
suum PDC activity was measured spectrophotometrically as described
previously(7) . The reaction mixture contained 50 mM potassium phosphate buffer (pH 7.4), 1 mM MgCl, 0.2 mM NAD, 2 mM dithiothreitol, 0.1 mM thiamine pyrophosphate, 0.1 mM CoA, and 4 mM pyruvate in a final volume of 1 ml. Bovine
kidney PDC activity was measured spectrophotometrically as described
previously(23) . The reaction mixture contained 50 mM potassium phosphate (pH 8.0), 1 mM MgCl
, 2.5
mM NAD, 2.6 mM dithiothreitol, 0.2
mM thiamine pyrophosphate, 0.13 mM CoA, and 2 mM pyruvate in a final volume of 1 ml. A. suum E3 was
prepared as described previously(20) . The bovine kidney-A.
suum hybrid complexes were prepared as follows: bovine kidney
E2
E3BP (2 µg) and bovine kidney E1 (4 µg) were
preincubated in 50 mM potassium phosphate (pH 8.0), 1 mM MgCl, and 3 mM dithiothreitol at room
temperature for 5 min. A. suum E3 (1 µg) was then added
and the preparation was incubated at room temperature for an additional
15 min. PDC activity of the hybrid complex was measured using the
conditions described for the bovine kidney PDC(23) .
Sensitivity of the different complexes to NADH was determined using
various concentrations of NAD and NADH (constant
nicotinamide pool of 200 µM) in the PDC activity assay, or
a fixed NAD
concentration (either 10 or 20
µM) with various concentrations of NADH in the PDC
activity assay.
Preparation of A. suum Larval Homogenates
A.
suum unembryonated ``eggs'' (UE), first-stage (L1),
second-stage (L2), or third-stage larvae (L3) were resuspended (1 g of
larvae/4 ml of buffer) in 20 mM MOPS (pH 7.2), 2 mM EDTA, 2 mM EGTA, and a protease inhibitor mixture
consisting of: 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 2.1 µM leupeptin, 3 µM aprotinin, 1 µM soybean trypsin inhibitor, 66
µM antipain, 33 µM chymostatin, 152
µM -amino caproic acid, and 29 µM pepstatin A. Homogenates were prepared by three passes through a
French pressure cell at 20,000 p.s.i. Triton X-100 then was added to a
final concentration of 1% (w/v). After 30 min on ice, samples were
centrifuged for 30 min at 10,000
g and the
supernatants frozen in liquid nitrogen. Samples were concentrated by
ultrafiltration and centrifuged for 15 min at 9,000 g to remove insoluble material.Purification of E3 from Second-stage Larvae
All
procedures were performed at 4 °C unless otherwise stated. L2s (50
ml) were washed with 20 mM potassium phosphate (pH 7.4) and
resuspended in 20 mM potassium phosphate (pH 7.4), 2 mM EDTA, and protease inhibitor mixture, and passed through a French
pressure cell at 20,000 p.s.i. three times. The homogenate was
centrifuged at 1,000 g for 10 min, and the resulting
supernatant was then centrifuged at 10,000 g for 30
min. The pellet was discarded and the supernatant was centrifuged for 3
h at 155,000 g. The resulting pellet was resuspended
in 20 mM potassium phosphate (pH 7.4), 1 mM EDTA, 1
mM EGTA, and protease inhibitor mixture, incubated in a 70
°C water bath for 10 min, and immediately cooled on ice for 15 min.
Following an initial clarification at 9,600 g for 15
min, the supernatant was centrifuged again for 3 h at 155,000 g. The pellet was discarded and the supernatant was
concentrated using Centricon 10 with the buffer
exchanged to 20 mM Tris-HCl (pH 7.2), 1 mM EDTA, and
1 mM EGTA (Buffer D). The concentrated sample was applied to a
DEAE-Sepharose column (1
7 cm) equilibrated with Buffer D.
After washing with 15 ml of Buffer D, activity was eluted with a
step-gradient comprised of Buffer D containing 75, 100, 150, and 200
mM NaCl, respectively. Activity eluted with 100 and 150 mM NaCl. Fractions with the highest specific activity were combined
and applied directly to a hydroxylapatite column equilibrated with
Buffer D. Activity eluted with 300 and 400 mM potassium
phosphate (pH 7.2) was desalted and concentrated to 1 mg
ml and stored at -70 °C. The purified E3
migrated as a single band with an apparent molecular mass of 55 kDa
during SDS-PAGE.
Gel Electrophoresis and Immunoblotting
SDS-PAGE
was conducted according to Laemmli (24) and proteins were
visualized with Coomassie Blue R-250 staining. Western blot AP system (Promega Corp.), using
goat anti-rabbit IgG secondary antibody conjugated to alkaline
phosphatase. The procedure was performed as described by the
manufacturer. Affinity purified antisera against p45, E1
, and
E1
were prepared as described previously (10, 25, 26) . E2 antiserum was
affinity-purified in a manner previously
described(25, 27) .Determination of Amino-terminal Sequence
Samples
were separated by SDS-PAGE on a 12% gel, and transferred to
Problott membranes (Applied Biosystems) in 100 mM CAPS (pH 11), and 10% (v/v) methanol. After transfer for 90 min,
the membranes were stained with Coomassie Blue R-250 as described by
the manufacturer. Individual p45 bands were excised and stored at
-20 °C. Sequencing was performed by Andrew Brauer of ARIAD
Pharmaceuticals Inc. (Cambridge, MA), and William Burkhart of Glaxo
Wellcome Inc. (Durham, NC).
Identification of a Putative E3-binding Domain in
p45
As described previously, incubation of the adult A. suum PDC with [2-C]pyruvate, followed by
SDS-PAGE and autoradiography yields only a single acetylated subunit,
suggesting that the lipoyl-containing E3BP observed in other eukaryotic
PDCs was not present in the adult ascarid complex(10) .
However, analysis of one novel subunit of the ascarid complex, p45, has
indicated that its amino-terminal sequence is similar to that of the
internal E3-binding domains of E2, E3BP, and succinyltransferase from a
variety of other organisms (Table 1). While the sequence identity
is limited, the conserved hydrophobic residues predicted to be involved
in intrahelical contacts within the hydrophobic domain and the charged
residues predicted to be involved in E3 binding are predominantly
conserved(28) . In addition, Pro at position 14 in the A.
suum sequence also is conserved except in the E3BP sequence from Saccharomyces cerevisiae. However, the yeast E3BP sequence
does contain a Pro upstream at position 142. It has previously been
suggested that this Pro represents the amino-terminal boundary of the
binding domain(28) . In contrast, the results of the present
study indicate that the p45 of the adult A. suum muscle PDC is
an E3-binding protein with a novel domain structure.
Dissociation of E3 from the A. suum E2 Core and the
Generation of two Distinct E2 Core Populations
Techniques which
dissociate both E1 and E3 from the mammalian PDC result in the
dissociation of only E1 from the ascarid complex(10) . To
generate an E2 core free of E1 and E3, E1-depleted PDC prepared by
treatment with 2 M NaCl was incubated with 1 M GdnHCl
followed by fast protein liquid chromatography on Superose 12. This
procedure separated the E1-depleted PDC into three fractions and
generated two E2 core populations (Fig. 1). Peak 1, which eluted
with the void volume, contained E2 and was enriched in p43 and p45.
After the removal of GdnHCl, this fraction was insoluble. In contrast,
Peak 2 contained E2 and trace amounts of both E3 and p45, and remained
soluble after the removal of GdnHCl. Peak 3 only contained E3. The
selective removal of E1 and E3 by different reagents suggests that E1
and E3 have specific, but different, interactions with the E2 core. The
GdnHCl concentration used in this study was critical. For example,
treatment of the E1-depleted PDC with 0.7 M, instead of 1.0 M GdnHCl, released only E3 and had no observable effect on the
ratio of E2 to p45 in the core (data not shown).Removal of p45 from the Core Decreases the Binding of E3,
but Not E1
Incubation of the soluble, p45-depleted E2 core (Peak
2 from the separation in GdnHCl described above; Fig. 2, lane 6) with both E1 and E3 restored a portion of the PDC
activity (about 20%, 1.1-1.5 µmol min (mg
protein)
). Sedimentation of the reconstituted
complex revealed that E1 binding appeared to be unaffected (Fig. 2, lane 1), while E3 binding was significantly
diminished (Fig. 2, lane 7) and almost all of the E3
remained in the supernatant fraction (Fig. 2, lane 8).
In contrast, incubation of the p45-enriched E2 core (Peak 1 from the
separation in GdnHCl described above; Fig. 2, lane 3)
with excess E3, followed by dialysis to remove GdnHCl, revealed that E3
binding to the core was enhanced (Fig. 2, lane 4).
These results suggest that p45 plays a role in E3 binding.
8 µg); Lane 4, reconstituted Peak 1 pellet (
12 µg); Lane 5,
reconstituted Peak 1 supernatant (4 µg); Lane 6,
p45-depleted A. suum core (Peak 2) (8 µg); Lane
7, reconstituted Peak 2 pellet (10 µg); and Lane 8,
reconstituted Peak 2 supernatant (5
µg).
p45 Remains Intact and Associated with the Core After the
Removal of the Lipoyl Domains of E2 by Selective Proteolysis
The
E1-depleted PDC was incubated with V8 protease and pelleted at 155,000
g for 3 h (Fig. 3). E2 was sensitive to
proteolysis, while p45 and E3 were resistant. Analysis of the
proteolytic products by amino-terminal sequencing and immunoblotting
with affinity purified polyclonal antisera against either E2 or p45
revealed that E2 was digested to a mixture of E2
(VAPPARVGVAATMAGPVXXGGFIDIPVSENR), E2 (APPNYHKP),
and two fragments of E2
(PDLPEHKKIPLPALSPTM), while the
breakdown of p45 was minimal (data not shown). E2,
E2, and p45 sedimented with the pellet, while E2
was found in the supernatant fraction. These results confirm the
tight association of p45 with the E2 oligomer. In contrast, E3 was
present in both the pellet and supernatant fractions after proteolysis.
These results suggest that in addition to p45, E2 also may play a role
in the binding of E3 to the core of the ascarid complex.
NADH Inhibition of Native and Hybrid Complexes
To
determine if p45 plays a role in the decreased NADH sensitivity of the
adult A. suum muscle PDC, the NADH sensitivities of the native
bovine kidney and adult A. suum PDCs were compared with that
of a hybrid complex constructed using the bovine kidney E2E3BP
core and E1, and the A. suum E3 (Fig. 4). The native A. suum PDC exhibited an apparent K
for
NAD of 12.0 ± 0.7 µM (µM ± S.E., n = 6) which was significantly
lower than that exhibited by either the bovine kidney (59.4 ±
1.5 µM, µM ± S.E., n =
4) or hybrid complex containing the ascarid E3 (54.2 ± 1.6
µM, µM ± S.E., n = 4).
As reported previously and confirmed in the present study, the adult A. suum muscle PDC was less sensitive to NADH inhibition than
the bovine kidney PDC, when assayed at either a fixed NAD
or total nicotinamide nucleotide pool (Fig. 4). In fact,
the A. suum PDC appears to be the most insensitive to NADH
inhibition of all PDCs studied thus far(29) . Most important,
the hybrid complex constructed with bovine kidney E2
E3BP and E1,
and ascarid E3 was more sensitive to NADH inhibition than the native A. suum PDC (Fig. 4). An NADH/NAD ratio of about 2 was required for 50% inhibition of the native A. suum PDC, while a ratio of about 1 was required for either
the bovine kidney or hybrid complexes (Fig. 4). These results
suggest that the binding of E3 to p45 may decrease the sensitivity of
the complex to NADH inhibition. It should be noted that the assay
conditions for the different PDCs were selected to maintain the
activity of other components such as the rate-limiting E1 and maximize
overall PDC activity. Therefore, the A. suum PDC was assayed
at pH 7.4 and the bovine kidney and hybrid complexes at pH 8.0.
Importantly, changing the pH of the assay buffer from 7.4 to 8.0 had
only a negligible effect on the apparent K
for
NAD and NADH inhibition for the native ascarid PDC,
suggesting that the differences in E3 inhibition did not result from
the different assay conditions.
concentration with varying NADH
concentrations. B, PDC activity is expressed as a percentage
of the activity measured in the absence of NADH using conditions of a
fixed nicotinamide pool (200 µM). Closed circles, A. suum PDC; squares, bovine kidney PDC; and open
circles, bovine kidney E2
E3BPE1/A. suum E3
hybrid complex.
Regulation of the PDC during Ascarid Larval
Development
The role of the PDC during A. suum larval
development changes dramatically and stage-specific, aerobic and
anaerobic, isozymes of many mitochondrial enzymes have been identified
previously(25, 30, 31) . To examine the role
of p45 during this aerobic/anaerobic transition, ascarid larval
homogenates were immunoblotted with affinity purified polyclonal
antisera against p45 and other subunits of the adult A. suum PDC (Fig. 5). All ascarid larval stages contained PDC, as
evidenced by the specific staining with E2 antisera. However, as
predicted, p45 appeared to be much less abundant or absent in the
aerobic early larval stages (UE, L1, and L2). Previous work reported
that p45 also is absent from the PDC of adult ovaries and
testis(10) .
, and E1. Larval homogenates and the affinity purified
antisera were prepared as described under ``Experimental
Procedures.'' E2 and p45 antisera were used at dilutions of 1:5000
and 1:2000, respectively. E1 and E1 antisera were both used
at dilutions of 1:2000. UE, unembryonated eggs (150 µg); L1 (150 µg); L2 (150 µg); L3 (80
µg); and M, adult muscle PDC (1 µg). *, rabbit IgG: L3
are recovered from rabbit lungs and homogenates often contain rabbit
IgG.
and E1 recognize proteins of
different mobilities in the aerobic larval stages. This confirms the
presence of stage-specific E1 isozymes and further suggests that
stage-specific forms of E1 also may be present. ratios observed in adult body wall muscle.
ratios(3) . The results of the
present study suggest that E3 is identical in both aerobic and
anaerobic stages, but that the PDC from anaerobic mitochondria has a
novel E3BP, p45. In contrast to E3BPs identified in either yeast or
mammalian PDCs, p45 is not acetylated during incubation in
[2-
C]pyruvate and does not contain the highly
conserved amino-terminal PS/ALSPTM sequence characteristic of lipoyl
domains(10, 32) . In contrast, the amino terminus of
p45 does contain sequence which exhibits significant similarity to
E3-binding domains found internally in E2s, E3BPs, and
succinyltransferases isolated from a number of organisms. However, it
should be noted that the E3-binding domain of p45 also exhibits
significant differences from these putative E3-binding domains.
Supporting the proposed role of p45 as an E3-binding protein is the
observation that E2 core substantially depleted of p45 still retains
its ability to fully bind E1 and function catalytically, but does not
bind additional E3, while E2 core enriched with p45 binds additional
E3. Whether the PDC from early larval stages contains an E3BP similar
to that observed in yeast and mammals must await the purification of
the PDC from these stages. Unfortunately, purification is complicated
by the limited amounts of available tissue and the high protease
activity found in the hatching fluids of those early larval stages. for
NAD and was more sensitive to NADH inhibition than the
native ascarid complex. PDCs exhibit a wide range of sensitivities to
NADH inhibition. For example, PDCs from facultative anaerobes, such as Escherichia coli, are very sensitive to NADH inhibition. As
NADH accumulates during anaerobiosis, PDC activity is inhibited, which
in turn, diverts pyruvate from the TCA cycle to fermentative
pathways(33) . In contrast, PDCs from anaerobes, such as Enterococcus faecalis, are much less sensitive to NADH
inhibition(29) . PDCs from higher eukaryotes also are less
sensitive to NADH inhibition. In these organisms, PDC activity is
regulated primarily by covalent modification and not end product
inhibition. In addition, elevated NADH/NAD
levels have
a marked stimulatory effect on pyruvate dehydrogenase kinase activity
which catalyzes the phosphorylation and inactivation of the
complex(34, 35) . The PDC from adult A. suum muscle and other gut dwelling parasitic helminths is the most
insensitive to NADH inhibition, and pyruvate dehydrogenase kinase
activity in these complexes is also less sensitive to activation by
NADH(7, 17) . Interestingly, when the amino acid
sequence of the conserved redox active disulfide site of E3s from
anaerobic parasitic helminths is compared to that of other E3s, a
consistent sequence difference is observed. Phenylalanine is
substituted for leucine at position 40 and a proline is located at
position 38. These sequence alterations are conserved in E3s from
anaerobic helminths representing two distinct helminth phyla, but not
in the E3 from the aerobic free-living nematode, Caenorhabditis
elegans. The significance of this sequence difference remains to
be determined.
), catalytic domain of E2;
E2, catalytic domain plus subunit-binding domain of E2;
E2
, lipoyl domain of E2; E3, dihydrolipoyl dehydrogenase;
E3BP, E3-binding protein (protein X); p45, 45-kDa component of A.
suum PDC; p43, 43-kDa component of A. suum PDC; L1,
first-stage larvae; L2, second-stage larvae; L3, third-stage larvae;
GdnHCl, guanidine hydrochloride; MOPS,
3-(N-morpholino)propanesulfonic acid; PAGE, polyacrylamide gel
electrophoresis; CAPS, 3-(cyclohexylamino)-1-propanesulfonic acid.
We thank the personnel at Routh Packing in Sandusky,
OH, for their permission to collect ascarids at their facility. We
express our appreciation also to Emilio Duran for the affinity purified
antisera.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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