ANALYTICAL

BIOCHEMISTRY
Analytical Biochemistry 356 (2006) 44–50
www.elsevier.com/locate/yabio

Assay of the pyruvate dehydrogenase complex by coupling

with recombinant chicken liver arylamine N-acetyltransferase
Nam Ho Jeoung, Paresh C. Sanghani, Lanmin Zhai, Robert A. Harris *

Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA

Received 13 January 2006

Available online 30 June 2006

Abstract

The activity of the pyruvate dehydrogenase complex has long been determined in some laboratories by coupling the production of
acetyl-coenzyme A (acetyl-CoA) to the acetylation of 4-aminoazobenzene-4 0 -sulfonic acid by arylamine N-acetyltransferase. The assay
has some advantages, but its use has been limited by the need for large amounts of arylamine N-acetyltransferase. Here we report pro-
duction of recombinant chicken liver arylamine N-acetyltransferase and optimization of its use in miniaturized assays for the pyruvate
dehydrogenase complex and its kinase.
 2006 Elsevier Inc. All rights reserved.

Keywords: Pyruvate dehydrogenase complex; Pyruvate dehydrogenase kinase; Pyruvate dehydrogenase phosphatase; Arylamine N-acetyltransferase;
4-Aminoazobenzene-4 0 -sulfonic acid; Acetyl-CoA

The pyruvate dehydrogenase complex (PDC)1 catalyz-
es the oxidative decarboxylation of pyruvate with the
production of acetyl-coenzyme A (acetyl-CoA), NADH,
and CO2. PDC is a regulatory enzyme, subject to inacti-
vation by pyruvate dehydrogenase kinase (PDK) and
activation by pyruvate dehydrogenase phosphatase
(PDP). Because of this mode of regulation and the stra-
tegic location of the complex in the catabolic pathway
for glucose, the activity of the complex in different phys-
*
Corresponding author. Fax: +1 317 278 9739.
E-mail address: raharris@iupui.edu (R.A. Harris).
1
Abbreviations used: PDC, pyruvate dehydrogenase complex; acetyl-
CoA, acetyl-coenzyme A; PDK, pyruvate dehydrogenase kinase; PDP,
pyruvate dehydrogenase phosphatase; AABS, 4-aminoazobenzene-4 0 -sul-
fonic acid; AAT, arylamine N-acetyltransferase; ATP, adenosine triphos-
phate; EST, expressed sequence tag; EDTA, ethylenediaminetetraacetic
acid; BSA, bovine serum albumin; DTT, dithiothreitol; TPCK, tosylphe-
nylalanylchloromethane; ORF, open reading frame; LB, Luria–Bertani;
IPTG, isopropyl-b-D-thiogalactopyranoside; SDS–PAGE, sodiumdodecyl
sulfate–polyacrylamide gel electrophoresis; PDCa, actual PDC activity;
PDCt, total PDC activity; EGTA, ethylene-bis(oxyethylenenitrilo)tetra-
acetic acid; PCR, polymerase chain reaction; CAT, carnitine
acetyltransferase.
iological states has long been of interest. Several spectro-
photometric and radiochemical methods have been
described for measuring the activity of the complex and
its individual components. The absorbance of NADH
at 340 nm [1] can be used for the purified complex but
not with crude tissue extracts because NADH produced
by the complex is oxidized by lactate dehydrogenase.
Radiochemical assays based on the generation of labeled
CO2 [2], citrate [3], and acetyl-carnitine [4] are used in
some laboratories. Spectrophotometric assays that can
be used with crude tissue extracts include reduction of
tetrazolium [5], acetylation of 4-aminoazobenzene-4 0 -sul-
fonic acid (AABS) by arylamine N-acetyltransferase
(AAT) [6], and reduction of NAD+ following immuno-
capture of the PDC from tissue extracts [7]. A fluorimet-
ric assay based on acetylation of 2-aminoanthracene by
AAT has also been described [8].
Much of our current understanding of the physiological
role of PDC was defined in Randle’s laboratory with the
assay that couples the generation of acetyl-CoA by PDC
(reaction 1) with the acetylation of AABS by AAT
(reaction 2) [6]:

0003-2697/$ - see front matter  2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.ab.2006.06.017
 Assay of the pyruvate dehydrogenase complex / N.H. Jeoung et al. / Anal. Biochem. 356 (2006) 44–50
Pyruvate þ NADþ þ CoAASH
PDC þ
! AcetylACoA þ NADH þ H þ CO2
AcetylACoA þ AABS
AAT
! CoAASH þ acetylAAABS
Loss of the absorption of light by AABS at 460 nm oc-
curs on acetylation. In spite of inherent problems, this
assay has long been the one favored by several laborato-
ries [6,8–10], including our laboratory [11,12]. It gives a
continuous readout of the absorbance change, which
facilitates assessment of the linearity of the reaction.
There is no interference by lactate dehydrogenase or
any other known enzyme activity. The use of radioiso-
topes is avoided. In addition, it provides the most conve-
nient assay for determining the first-order rate constant
for adenosine triphosphate (ATP)-dependent loss of
PDC activity, the most frequently used measure of
PDK activity. In spite of these good things about the as-
say, a frustrating problem exists that has prevented wide
acceptance of the assay as the one of choice by many
laboratories. AAT, the coupling enzyme required to trap
the acetyl group of acetyl-CoA produced by PDC activ-
ity, is not readily available. Most laboratories that use
the assay deal with this problem by partially purifying
AAT from pigeon liver or chicken liver [9,13–15]. Pigeon
liver is the best source of the enzyme because AAT activ-
ity in pigeon liver is at least eight times greater than the
activity present in chicken liver and the specific activity
of the pigeon enzyme may also be higher than that of
the chicken enzyme [9,15]. However, there are barriers
to the purification of the enzyme from pigeon liver. Feral
pigeons are considered to be a health hazard for labora-
tory workers [16,17]. Most large cities require a permit to
dispose of feral pigeons. Permits held by pest control
companies usually prohibit transfer of dead or live ani-
mals to a third party. Domesticated pigeons can be pur-
chased from breeders but are prohibitively expensive in
the number required for large-scale preparations of
AAT. Acetone powder of pigeon liver can be purchased
from commercial suppliers but has not proven to be a
reliable source of the enzyme in our hands. Beyond these
issues, purification of the enzyme from fresh or frozen
liver is tedious, the yield is poor, and the specific activity
often is disappointing. Partially purified pigeon liver
AAT is commercially available, but the purity and specif-
ic activity are variable and our laboratory has found it
to be prohibitively expensive.
The need for a large, reliable, and inexpensive supply of
AAT made it desirable to produce the recombinant protein
for the assay of PDC. Our first choice would have been the
pigeon enzyme, but no DNA sequence information is avail-
able for the pigeon AAT gene. The cDNA sequence for the
chicken liver enzyme was published previously [18], and
chicken liver expressed sequence tags (ESTs) are available
from MRC Geneservice (Cambridge, UK).
45
Materials and methods
ð1Þ Reagents
AABS (technical grade, cat. No. 11965), also called p-(p-
ð2Þ aminophenylazo)benzene sulfonic acid, (4-anilinoazo)ben-
zene-4 0 -sulfonic acid, and p-aminophenylazobenzene
sulfonic acid, was purchased from Lancaster Synthesis
(Windham, NH, USA). Pyruvate dehydrogenase (cat.
No. P-5194) and creatine phosphokinase (cat. No.
C-3755) were obtained from Sigma Chemical (St. Louis,
MO, USA). Recombinant PDP1 was expressed in
Escherichia coli and purified as described previously [19].
Restriction enzymes were obtained from Promega
(Madison, WI, USA). All other chemicals were purchased
from Sigma Chemical.
Animals and tissue extraction
Ten-week-old male C57Bl/6J mice were obtained from
Jackson Laboratory (Bar Harbor, ME, USA). Fed mice
had continuous access to a mouse chow diet prior to sacri-
fice. Starved mice were fasted for 24 h in cages fitted with
wire mesh bottoms to minimize coprophagy. Mice were
anesthetized with an intraperitoneal injection of Nembutal
(60 mg/kg body weight). Hearts were removed within 10 s
of opening the thoracic cavity, freeze-clamped at the tem-
perature of liquid nitrogen, and stored 80 C until used
for assays.
Frozen hearts were pulverized with a mortar and pes-
tle under liquid nitrogen. A portion of the powder was
weighed on an analytical balance; suspended in 5 vol-
umes (w/v) of extraction buffer containing 30 mM
Hepes–KOH (pH 7.5), 3% (v/v) Triton X-100, 2 mM eth-
ylenediaminetetraacetic acid (EDTA), 2% (v/v) bovine
serum albumin (BSA), 5 mM dithiothreitol (DTT),
10 lM tosylphenylalanylchloromethane (TPCK), 10 lg/
ml trypsin inhibitor, and 1 lM leupeptin; and homoge-
nized with a motor-driven Teflon homogenizer (Poly-
science, Niles, IL, USA). The supernatant obtained
from centrifugation of the homogenate at 10,000g for
10 min at 4 C was used immediately for the assay of
PDC activity.
Expression and purification of chicken AAT
Three EST clones for chicken liver AAT (clone IDs
ChEST912L18, ChEST254H16, and ChEST46F8) were
obtained from MRC Geneservice. The open reading
frames (ORFs) in each of the three ESTs were complete
and matched the published sequence (Swiss–Prot entry
P12275) except that each of them encoded nine addition-
al amino acids at the carboxy-terminus past the
published sequence. The entire ORF containing the nine
additional amino acids was directly subcloned into the
pET-100 (Invitrogen, Carlsbad, CA, USA) expression
vector using the directional cloning strategy described
46 Assay of the pyruvate dehydrogenase complex / N.H. Jeoung et al. / Anal. Biochem. 356 (2006) 44–50
in the pET-100 cloning manual. For expression, 50 ml of
starter culture of Luria–Bertani (LB) broth culture con-
taining 50 lg/ml of carbenicillin was inoculated with
1 ml of E. coli BL21 cell stock that had been trans-
formed with the expression vector. After approximately
3 h, the starter culture was inoculated into 10 L of LB
broth containing 50 lg/ml of carbenicillin. When the cul-
ture density reached A595 of 0.5, 1 mM isopropyl-b-D-
thiogalactopyranoside (IPTG) was added to induce the
expression of the enzyme. The cells were grown for an
additional 8–10 h at 20 C before harvesting them with
a Millipore Pellicon concentrator fitted with a 0.45-lm
filter. The concentrated cells were pelleted at 4500g for
20 min and lysed using a Thermospectronic French Press
in a lysis buffer (50 mM Tris–HCl [pH 8.0] containing
0.3 M NaCl, 1 lg/ml leupeptin, 2 mM benzamidine, and
10 mM imidazole). The lysate was spun for 1 h at
40,000g, and the supernatant was incubated with 15 ml
of Ni–NTA resin for 3 h at 4 C. The Ni–NTA resin
was packed into a column and eluted with 0–0.2 M imid-
azole containing lysis buffer in step gradients. The
enzyme-containing fractions were pooled and concentrat-
ed before changing the buffer into 10 mM Tris–HCl (pH
7.9) containing 1 mM DTT and 1 mM EDTA. The
enzyme was stored at 20 C in 40% glycerol. Proteins
of E. coli extracts and purified recombinant chicken
AAT were separated by sodium dodecyl sulfate–poly-
acrylamide gel electrophoresis (SDS–PAGE) according
to Laemmli [20] and were stained with Coomassie blue
R350.
Measurement of enzyme activities
AAT activity was measured by the decrease of the
absorbance of AABS at 460 nm with a Cary 50 Bio spec-
trophotometer (Varian Analytical Instruments, Walnut
Creek, CA, USA). The assay was carried out with 50
mM potassium phosphate buffer (pH 7.8) containing
5 mM b-mercaptoethanol, 1 mM EDTA, 0.2 mM acetyl-
CoA, and 0.3 mM AABS at 30 C [9].
Activity of purified PDC was measured spectrophoto-
metrically at 30 C by the rate of NAD+ reduction in a
cocktail containing 100 mM Tris–HCl (pH 7.8), 0.5 mM
EDTA, 1 mM MgCl2, 1 mM thiamine pyrophosphate,
5 mM b-mercaptoethanol, 0.1 mM NAD+, and 0.1 mM
CoA. The reaction was initiated with the addition of
0.8 mM pyruvate (final concentration).
To conserve reagents and the amount of tissue or cell
extract required, measurements of actual PDC activity
(PDCa), total PDC activity (PDCt), and PDK activity
by the AAT coupled assay were carried out in 96-well
microplates with a SpectraMax 190 microplate reader
(Molecular Devices, Sunnyvale, CA, USA). PDCa
designates the activity of PDC prior to activation by
its phosphatase, that is, the actual activity of the com-
plex as it existed in the tissue prior to extraction. PDCt
designates the activity of the complex after complete
activation by dephosphorylation with its phosphatase,
that is, the maximum activity of the complex. PDK
activity refers to the rate of ATP-dependent inactivation
of PDC by pyruvate dehydrogenase kinases present in
the tissue extract.
For the determination of PDCa activity, an aliquot of
tissue extract (50 ll) was mixed with an equal volume of
resuspension buffer (60 mM Hepes–KOH [pH 7.5], 2% [v/
v] Triton X-100, 0.4 mM EDTA, 4% [v/v] BSA, 2 lM leu-
peptin, and 5 mM DTT containing 20 mM dichloroacetate
and 100 mM KF). For the determination of PDCt activity,
an aliquot of tissue extract (100 ll) was mixed with an
equal volume of activation buffer (60 mM Hepes–KOH
[pH 7.5], 2% [v/v] Triton X-100, 0.4 mM EDTA, 4% [v/v]
BSA, 2 lM leupeptin, and 5 mM DTT containing 40 mM
MgCl2, 2 mM CaCl2, and 1.5 lg/sample of recombinant
PDP1 [19]). Complete activation of PDC by dephosphoryl-
ation was achieved by incubation of this mixture at 30 C
for 10–15 min.
PDC activity was determined at 30 C in 96-well
microplates in a total volume of 200 ll. An aliquot
(3 ll) of tissue extracts prepared for PDCt or PDCa
determination was added to 193 ll of reaction cocktail
solution that was 100 mM Tris–HCl (pH 7.8), 0.5 mM
EDTA, 1 mM MgCl2, 1 mM thiamine pyrophosphate,
5 mM b-mercaptoethanol, 0.5 mM NAD+, 0.1 mM
CoA, 0.1 mM AABS, and 20 mU in recombinant
AAT. The reaction was started with the addition of
4 ll of 40 mM pyruvate (final concentration of
0.8 mM). PDC activity was calculated from the linear
rate of change in absorbance between 6 and 8 min of
incubation. Calculations were based on the extinction
coefficient of 7.11 · 106 cm2/mol for AABS. Values were
multiplied by 0.788 to correct for the reduced length of
the light path in the microplate reader. One unit of
PDC activity corresponds to the acetylation of 1 lmol
AABS/min at 30 C.
For PDK assay, an aliquot of the extract prepared
for the determination of PDCt was made 1 mM in eth-
ylene-bis(oxyethylenenitrilo)tetraacetic acid (EGTA) and
50 mM in KF to inhibit PDP1 activity. An aliquot of
the extract (30 ll) was mixed with 30 ll of a kinase
buffer containing an ATP-regenerating system (50 mM
potassium phosphate [pH 7.8], 1 mM MgCl2, 20 mM
KF, 10 mM phosphocreatine, and 5 U/ml creatine phos-
phokinase) and preincubated at 30 C for 2 min. The
reaction was started with the addition of ATP to a final
concentration of 0.5 mM. Aliquots of the incubation
mixture (6 ll) were removed at 0, 30, 60, and 120 s
for the measurement of PDC activity by the miniatur-
ized AAT coupled assay.
Statistical analysis
Results are presented as means ± SEM. Statistical anal-
ysis was performed by Student’s t test. P < 0.05 was consid-
ered to be statistically significant.
 Assay of the pyruvate dehydrogenase complex / N.H. Jeoung et al. / Anal. Biochem. 356 (2006) 44–50
Results
Expression and purification of recombinant chicken liver
AAT
Three EST clones (ChEST912L18, ChEST254H16, and
ChEST46F8) with ORFs encoding chicken liver AAT were
sequenced. All three encoded the identical ORF of 891 bp.
The deduced sequence of the first 287 amino acids of the
protein was identical to the sequence published previously
for chicken liver AAT [18] (GenBank Accession No.
NM_205526). However, the ORF encodes an additional
nine amino acids at the carboxy-terminal end of the protein
(Fig. 1). Therefore, the published chicken AAT sequence
has 287 amino acids [18], whereas the clones sequenced in
the current study encode a protein that is nine amino acids
longer. One base difference in codon 288 accounts for the
difference between the sequence published previously and
the sequence found with ESTs encoding the enzyme in this
study (Fig. 1).
The coding sequence of chicken liver AAT was amplified
by polymerase chain reaction (PCR) and inserted into pET-
100. Recombinant His-tagged AAT was expressed in E. coli
and purified in one step by chromatography on a Ni–NTA
resin column (Fig. 2). The specific activity of AAT in crude
E. coli extracts was 21 ± 1 nmol min1 mg1 of protein.
The protein was estimated to be 97% pure based on Coomas-
sie blue R350 staining (Fig. 2) with a specific activity of
223 ± 10 nmol min1 mg1 of protein. Yields of purified
enzyme in several preparations from 10-L cultures have ran-
ged from 120 to 180 mg. The molecular weight of the recom-
binant His-tagged enzyme estimated from mobility on SDS–
PAGE is approximately 35 kDa, which (as expected) is
slightly larger than the molecular weight calculated from
the amino acid composition of the non-His-tagged protein
(33,877 kDa). Km values for AABS and acetyl-CoA were
found to be 32 ± 5 (Fig. 3A) and 23 ± 5 lM (Fig. 3B),
respectively. No change in enzyme activity occurred during
1 month of storage in 40% glycerol at 20 C.
Determination of the optimal amount of the AAT for the
coupled enzyme assay for PDC
The amount of AAT required for measuring PDC activ-
ity with the coupled assay was determined empirically by
titrating a known activity of purified PDC with increasing
Codon #
288 289 290 291 292 293 294 295 296 297
Chicken AAT
(NM_205526)
TAG CCA GGT AAT CTG GTG GAT ACC ATA TAA
Stop
ChEST912L18 TTG CCA CCT AAT CTG GTG GAT ACC ATA TAA
L P P N L V D T I Stop
Fig. 1. Comparison of the DNA sequences and amino acid sequences at
the C terminus of chicken liver AAT (GenBank Accession No.
NM_205526 [18]) and EST clone ChEST912L18. The numbers above
the nucleotide sequence indicate codon numbers for the DNA sequences.
47
M 1 2
75 kDa
51 kDa
36 kDa
25 kDa
Fig. 2. SDS–PAGE of recombinant chicken liver AAT. Recombinant
AAT was expressed in E. coli BL21 and purified as described in Materials
and methods. Samples of E. coli lysates (10 lg of protein, lane 1) and
purified AAT (10 lg of protein, lane 2) were separated on a 10% SDS–
PAGE gel. Size marker proteins are shown in lane M. Molecular weights
of the marker proteins are indicated on the left side of the gel.
amounts of AAT (Fig. 4). Assays were carried out with a
recording spectrophotometer in 1.0 ml of assay cocktail
containing 8.4 ± 0.1 mU of PDC activity (measured by
increased absorbance at 340 nm produced by the reduction
of NAD+ to NADH). Increasing the amount of AAT in
the coupled assay increased the rate of the AABS acetyla-
tion until it reached (within experimental error) the same
value for PDC activity (8.7 ± 0.3 mU) measured by the
reduction of NAD+ to NADH. As expected from our pre-
vious experience with native pigeon and chicken AAT, a
large amount of recombinant AAT (P10 units of AAT
activity per unit of PDC activity) needed to be added to
achieve maximum PDC activity. A lag corresponding to
the time required to achieve a linear rate after initiation
of the reaction was reduced from nearly 4 min at the lowest
amount of AAT added to the assay (45 ng/ll) to less than
1 min at the highest level of AAT added to the assay
(450 ng/ll).
Linearity of the PDC assay with amount of PDC
The activity of PDC increased linearly with the amount
of PDC in the assay (Fig. 5).
Application of the assay with recombinant AAT to the
measurement of PDC and PDK activities in tissue extracts
Complete activation of PDC for the measurement of
PDCt requires complete dephosphorylation of PDC by
the action of a phosphoprotein phosphatase. With crude
extracts of some tissues, this can be achieved by simply
adding Mg2+ and Ca2+ to activate endogenous phospha-
tases [21,22]. To decrease the incubation time required
48 Assay of the pyruvate dehydrogenase complex / N.H. Jeoung et al. / Anal. Biochem. 356 (2006) 44–50

A 0.15 B
0.2

Activity (μmol/min/mg)

Activity (μmol/min/mg)
0.15
0.1

0.1

0.05
0.05

0 0
0 50 100 150 200 0 50 100 150 200
[AABS] (μM) [Acetyl-CoA] (μM)

Fig. 3. Substrate saturation curves for acetyl-CoA and AABS with recombinant chicken AAT. (A) The acetyl-CoA concentration was fixed at 0.2 mM,
and the concentration of AABS was varied from 0 to 0.2 mM. (B) The AABS concentration was fixed at 0.2 mM, and the concentration of acetyl-CoA was
varied from 0 to 0.2 mM.

10 4 for complete activation of PDC, we routinely add recombi-

PDC activity (nmol/min)

nant PDP1 to the PDC activation cocktail (Fig. 6). With

8
Lag time (min)

3 added PDP1, complete activation of PDC present in crude

6 tissue extracts usually can be achieved within 5 min. PDC
4
2
0 0
0 90
Fig. 4. Determination of the optimal amount of AAT needed for
maximum PDC activity by the coupled assay. Purified PDC
(8.4 ± 0.1 mU determined spectrophotometrically by reduction of
NAD+ to NADH) was used as the source of enzyme. The amount of
recombinant AAT was varied from 45 to 450 ng of protein/ll. Lag time
refers to the amount of time required to achieve a linear rate after
initiation of the reaction by the addition of 0.8 mM pyruvate. PDC
activity was determined from the linear rate achieved after the lag. The
values for activities and lag times correspond to means ± SEM obtained
from triplicate determinations.
10.0
PDC activity (mU/min)
2 activity is stable to longer incubations at 30 C (Fig. 6),
but we prefer to keep the incubations short to minimize
1 possible loss of PDC and PDK activities by proteolytic
activity that might not be completely suppressed in crude
tissue extracts.
180 270 360 450 To illustrate the use of the recombinant AAT-based
AAT (ng/μl) assay, PDCt and PDCa activities were measured with the
assay in crude heart extracts prepared from fed mice and
mice starved for 24 h (Table 1). Total PDC activities mea-
sured for mouse heart ( 15 lmol/min/g wet weight) are
significantly higher than the highest values (8.4 lmol/
min/g wet weight) reported for rat heart [9,23] and mouse
heart [10]. Indeed, to our knowledge, these are the highest
values recorded for any tissue of any animal. As expected,
actual activities are much lower, with only 13% of the
enzyme active in the fed heart and less than 1% of the
enzyme active in the starved heart. As expected from
previous work with rats, starvation induced a very large

8.0 14
PDC activity (μmol/min/g)

12
6.0
10
4.0 8

6
2.0
4
0.0 2
0 2 4 6 8 10 12 14
0
Protein amount (μg) 0 5 10 15 20 25 30
Incubation time (min)
Fig. 5. Linearity of the assay for PDC with the amount of PDC. Purified
PDC was used as the source of enzyme. Reactions were carried out with Fig. 6. Activation of PDC by PDP1. Mouse heart extract was incubated
the 96-well plate method described in Materials and methods. PDC was with recombinant PDP1 for the indicated period of time as described in
added in the indicated amounts with 90 lg of AAT per 200 ll of reaction Materials and methods. PDC activity was measured at the indicated time
mixture. Activity correlated directly with the amount of PDC points with the AAT coupled assay described in Materials and methods.
(R2 = 0.996). The values represent means ± SEM obtained from triplicate The values represent means ± SEM obtained from triplicate
determinations. determinations.
 Assay of the pyruvate dehydrogenase complex / N.H. Jeoung et al. / Anal. Biochem. 356 (2006) 44–50
Table 1
Activity of PDC and its kinase in mouse heart
Nutritional PDC activity
state of mice
Actual PDC (lmol/min/g) Total PDC (lmol/min/g)
Fed 1.88 ± 0.62c 14.6 ± 1.0
Starvedd 0.12 ± 0.03 16.1 ± 0.1
a
(Actual PDC activity/Total PDC activity) · 100.
b
Values are reported as first-order rate constants for ATP-mediated inactivation of the PDC complex.
c
All values are means ± SEM for independent measurements with hearts from four animals.
d
Mice were starved for 24 h prior to being sacrificed.
increase (fivefold) in PDK activity in the heart of the mouse
(Table 1).
Discussion
Many investigators have used pigeon liver AAT enzyme
to assay the PDC activity [6,8,10,24]. However, purification
of AAT from pigeon liver is a time-consuming and tedious
process. The specific activity of the AAT prepared from fresh
pigeon liver is higher than that prepared from commercially
available acetone powder from pigeon liver [9,15], but fresh
pigeon liver is difficult to obtain and acetone powder from
pigeon liver is expensive and not a reliable source for purifi-
cation of the enzyme. Chicken liver AAT can be used for the
assay in place of the pigeon enzyme [9,15]. The specific activ-
ity of chicken liver AAT is significantly lower than that of
pigeon liver AAT but higher than the enzyme of other ani-
mals that have been examined [15]. Chicken liver is abun-
dantly available, but considerable time is still required for
preparation of the enzyme, the yield is relatively poor, and
the purity often remains poor after several column chroma-
tography steps. For these reasons, a chicken liver AAT
cDNA was cloned in this study and the recombinant protein
was expressed in E. coli and purified to near homogeneity.
Approximately 150 mg of nearly pure AAT can be obtained
from a 10-L culture with a single purification step. The
recombinant protein has a high specific activity
(223 ± 10 nmol min1 mg1) compared with purified AATs
from pigeon liver (7.7 ± 0.5 nmol min1 mg1) and chicken
liver (1.0 ± 0.2 nmol min1 mg1) [15]. Recombinant chick-
en liver AAT was expressed previously in Chinese hamster
ovary cells by Ohsako and co-workers [18]. They demon-
strated N-acetyltransferase activity in crude cell extracts,
but the enzyme was not purified and characterized.
Previous measurements of total PDC activity in mouse
heart have reported values in the range of 6 U/g of wet
weight [10,25]. We found a significantly higher value
(16 U/g wet weight) in this study. This may reflect the
use of different strains of mice or perhaps a limitation
in the amount of AAT coupling enzyme used in previous
studies. A large amount of AAT must be used to maxi-
mize PDC activity and minimize the lag. PDC activities
reported in the literature may, in some cases, be underes-
timated because the effort required for its purification or
the expense associated with its purchase resulted in the
49
PDC activity state (%)a PDC kinase activity (min1)b
13.0 ± 4.4 0.74 ± 0.23
0.7 ± 0.1 3.70 ± 0.32
use of less than optimal amounts of coupling enzyme
in the assay. We found that the activity of AAT needs
to exceed the activity of PDC by a factor of at least
10 for maximal PDC activity. This agrees well with the
recommendation by Bergmeyer [26] for a large excess
of indicator reaction in coupled enzymatic reactions.
The amount of AAT required has been a major hurdle
for the use of the AAT assay. The availability of the
recombinant enzyme solves this problem because it is rel-
atively inexpensive to prepare and is stable to storage
and during the assay.
As pointed out by others [8], AAT could be used for the
assay of the activities of acetyl-CoA synthetase, acetyl-CoA
carnitine acetyltransferase (CAT), and citrate cleavage
enzyme in crude tissue extracts. It could also be readily
used for quantification of acetyl-carnitine and acetyl-CoA
in acid extracts of tissues. Because recombinant AAT can
be readily prepared in a much purer form than can the
native enzyme from chicken or pigeon, recombinant AAT
should prove to be even more useful for these assays.
In summary, large amounts of nearly pure AAT can
now be produced as the recombinant protein. A miniatur-
ized assay based on the use of recombinant AAT has been
developed for the measurement of the activities of PDC
and its kinase in mammalian tissues. The method saves
on reagents and the amount of tissue extract required
and can be used for the simultaneous assay of large num-
bers of samples.
Acknowledgments
This work was supported by grants to R.A.H. from the
American Diabetes Association and the U.S. Public Health
Service (DK47844) and by a postdoctoral fellowship award
(N.H.J.) from the Midwest affiliation of the American
Heart Association. We thank the Indiana Genomics Initia-
tive (INGEN) Protein Expression Core of Indiana Univer-
sity (supported in part by Lilly Endowment) for expression
and purification of AAT and PDP1.
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