From the Tohoku University Gene Research Center,
Sendai 981-8555, the § Yokohama Research Center,
Mitsubishi-Tokyo Pharmaceuticals Inc., Yokohama, 227-0033, and the
¶ Department of Structural Biology, Biomolecular Engineering
Research Institute, Suita 565-0874, Japan
Received for publication, September 26, 2000, and in revised form, December 21, 2000
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ABSTRACT |
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Using peptide sequences derived from
bovine cardiac acetyl-CoA synthetase (AceCS), we isolated and
characterized cDNAs for a bovine and murine cardiac enzyme
designated AceCS2. We also isolated a murine cDNA encoding a
hepatic type enzyme, designated AceCS1, identical to one reported
recently (Luong, A., Hannah, V. C., Brown, M. S., and
Goldstein, J. L. (2000) J. Biol. Chem. 275, 26458-26466). Murine AceCS1 and AceCS2 were purified to homogeneity and characterized. Among C2-C5 short and medium chain fatty acids, both enzymes preferentially utilize acetate with similar affinity. The
AceCS2 transcripts are expressed in a wide range of tissues, with the
highest levels in heart, and are apparently absent from the liver. The
levels of AceCS2 mRNA in skeletal muscle were increased markedly
under ketogenic conditions. Subcellular fractionation revealed that
AceCS2 is a mitochondrial matrix enzyme. [14C]Acetate
incorporation indicated that acetyl-CoAs produced by AceCS2 are
utilized mainly for oxidation.
Acetyl-CoA synthetase
(AceCS,1 EC 6.2.1.1)
catalyzes the ligation of acetate with CoA to produce acetyl-CoA, an
essential molecule utilized in various metabolic pathways including
fatty acid and cholesterol synthesis and the tricarboxylic acid
cycle (for review, see Ref. 1). In ruminant animals, AceCS plays a key
role in the catabolism of acetate produced by microorganisms in the
rumen. In nonruminant mammals, acetate production occurs in the liver
from ethanol by alcohol dehydrogenase and acetaldehyde dehydrogenase
and from acetyl-CoA by acetyl-CoA hydrolase (2). Furthermore, AceCS is
postulated to play a key role in the recycling of acetate released by
acetylcholine esterase for the formation and release of acetylcholine
in cholinergic nerve terminals (3, 4).
AceCS from various microorganisms has revealed that the enzyme
belongs to the firefly luciferase superfamily (5, 6), which includes
mammalian long chain acyl-CoA synthetases, ACS1-ACS5 (7-11),
bacterial antibiotic synthetases, 4-coumarate:CoA ligases, 4-chlorobenzoate: CoA ligase, and luciferases of various origins. All
enzymes in this superfamily contain a common sequence motif of
Ser-Gly-(small hydrophilic residue)2-Gly-(any
residue)-Pro-Lys-Gly and catalyze common two-step reactions:
adenylation of substrates and subsequent thioester formation (5,
6).
To evaluate the role of AceCS, we purified AceCS from bovine
heart and cloned bovine and murine cDNAs encoding this enzyme. In
addition to the cardiac enzyme, we also obtained a cDNA for hepatic
type enzyme. The murine cDNAs encoding cardiac and hepatic types of
AceCSs were introduced into COS cells, and the resulting enzymes were
purified and characterized. Subcellular fractionation revealed that the
hepatic type enzyme (termed AceCS1) is a cytosolic enzyme, whereas the
cardiac enzyme (termed AceCS2) is located in the mitochondrial matrix.
During the preparation of this paper, Luong et al. (12)
published a molecular characterization of AceCS which is identical to
murine AceCS1. Here, we provide evidence that AceCS2 provides
acetyl-CoA that is utilized mainly for oxidation under ketogenic conditions.
Standard Methods--
Standard molecular biology and
immunochemical techniques were performed essentially as described by
Sambrook et al. (13) and Harlow and Lane (14), respectively.
cDNA clones were subcloned into pBluescript vectors and sequenced
by the dideoxy chain termination method with a Bigdye Terminator Cycle
Sequencing Ready Reaction kit (PE Biosystems) and a DNA sequencer
(model 310; PE Biosystems). Total RNA was prepared using standard
guanidinium thiocyanate lysis buffer and centrifugation over a cesium
chloride cushion. For Northern blotting, total RNA was denatured with 1 M glyoxal and 50% dimethyl sulfoxide, fractionated in a
1.5% agarose gel, and transferred to a Zeta Probe nylon membrane
(Bio-Rad). To analyze RNA in murine tissues, commercially
available Northern blots (CLONTECH) were used for
Northern blot analysis. 32P-Labeled probes were prepared by
priming with random hexanucleotides. The probes used for Northern blots
included the murine AceCS1 cDNA nucleotides 154-1318 and the
murine AceCS2 cDNA nucleotides 160-1696. Quantitative analysis was
performed with a bioimage analyzer (BAS2000, Fuji Film, Tokyo). For
immunoblotting, polyclonal antibodies against murine AceCS1 and AceCS2
(see below) were used as first antibodies. Antibody binding was
detected by chemiluminescence (ECL, Amersham Pharmacia Biotech) kit.
Peptide Sequence Analysis--
Bovine AceCS was purified
from heart as described previously (15). The purified enzyme (specific
activity 45 µmol/min/mg at 37 °C) was digested with lysyl
endopeptidase (Wako Pure Chemicals, Osaka) according to the
manufacturer's instructions. The resulting peptides were separated by
a C8 reverse phase column using a linear gradient of 0-80%
acetonitrile containing 0.1% trifluoroacetic acid and sequenced by
automated Edman degradation using a peptide sequencer (model 470 A, PE Biosystems).
cDNA Cloning--
Reverse transcription polymerase chain
reaction was performed to obtain a partial cDNA for bovine AceCS. 1 µg of poly(A) RNA prepared by oligo(dT)-LatexTM (Takara Shuzo,
Kyoto, Japan) was used to prime cDNA synthesis with Superscript
II reverse transcriptase (Life Technologies, Inc.) and random hexamers.
The resulting cDNA was then amplified with a set of degenerate
primers corresponding to the amino acid residues 289-296 and 471-478:
5'-GGN GTN GTN CA(C/T) ACN CA(A/G) GCN GG-3' and 5'-CT NCC NCC (C/T)TC
NAG NAC (A/G)TT NCC-3'. Polymerase chain reaction amplification was
carried out under the conditions recommended for Ex TaqTM
(Takara Shuzo Corp.).
Three cDNA libraries were constructed using poly(A) RNA from bovine
and murine hearts, and murine livers using an Okayama-Berg vector (16).
Bovine and murine cardiac cDNA libraries were screened with partial
cDNA for the bovine enzyme. We also screened a murine hepatic
cDNA library under reduced conditions. After screening of
~105 clones from each library, we obtained several
clones. Representative clones encoding near full-length cDNAs were
further characterized.
Cell Culture and DNA Transfection--
COS-7 cells were grown in
monolayer culture in Dulbecco's modified Eagle's medium supplemented
with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml
streptomycin. Cells were transfected with 10 µg of plasmid DNA by the
DEAE-dextran procedure (17). 3e days after DNA transfection, the cells
were harvested for purification and [14C]acetate
incorporation (see below).
Assay of AceCS Activity--
AceCS activity was determined at
37 °C by either an isotopic or by a spectrophotometric method, which
is based on the formation of AMP using adenylate kinase, pyruvate
kinase, and lactate dehydrogenase (18). The latter was used only for
the purified enzyme. The standard reaction mixture for the isotopic
method contained 100 mM Tris-HCl, pH 8.5, 10 mM
MgCl2, 10 mM ATP, 1 mM CoA, and 10 mM [14C]acetate (940 dpm/nmol) in a total
volume of 0.2 ml. After 1 min of preincubation at 37 °C, the
reactions were initiated by the addition of enzyme solution. After a
30-min incubation, the reaction was terminated by adding 50 µl of
ice-cold glacial acetic acid. The reaction product
([14C]acetyl-CoA) was isolated by spotting onto a piece
of chromatography media (ITLC-SG type, Gelman Sciences) and extensive
washing with water-saturated ether/formic acid (7:1) for measurement of radioactivity.
The standard reaction mixture for the spectrophotometric method
contained 100 mM Tris-HCl, pH 8.5, 1 mM
dithiothreitol, 15 mM MgCl2, 10 mM
ATP, 0.25 mM potassium phosphoenolpyruvate, 1 mM acetate, 0.3 mM NADH, 80 units of adenylate
kinase (Roche Molecular Biochemicals), 17 units of lactate
dehydrogenase (Roche Molecular Biochemicals) and 6 units of pyruvate
kinase (Roche Molecular Biochemicals) in a total volume of 1 ml. After
a 1-min preincubation at 37 °C, the reactions were started by adding
24 µl of 25 mM CoA. The oxidation of NADH was measured at
340 nm on a recording spectrophotometer. The formation of 1 mol of AMP
corresponds to the oxidation of 2 mol of NADH.
All assays were carried out within the range where the reaction
proceeded linearly with time, and the initial rate of reaction was
proportional to the amount of enzyme added. The protein content was
determined as described (19).
Purification of AceCS1 and AceCS2--
All purification steps
were carried out at 0-4 °C, and the results are summarized in Table
I.
COS-7 cells (80 dishes, 100-mm diameter) transfected with either murine
AceCS1 or AceCS2 were suspended in 30 ml of 20 mM Tris-HCl,
pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 mM
phenylmethylsulfonyl fluoride and disrupted by sonication. The cell
extracts were centrifuged at 230, 000 × g for 1 h, and the supernatant was used for purification.
For purification of AceCS1, the following purification steps were
carried out. Protein in the 230, 000 × g supernatant
was precipitated with (NH4)2SO4 at
60% saturation and centrifugation at 16, 000 × g for
15 min. The resulting precipitate was suspended in buffer A (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, and 10% glycerol (w/v)), dialyzed
against buffer A, and applied to a DEAE-Sepharose FF (Amersham
Pharmacia Biotech) column (1.5 × 5 cm) equilibrated with buffer
A. After washing with buffer A, the column was eluted with an
increasing gradient of 0-0.25 M KCl containing buffer A. Active fractions were combined, and (NH4)2SO4 was added to give 30%
saturation and then applied to a phenyl-Sepharose FF (Amersham
Pharmacia Biotech) column (1.5 × 5 cm) equilibrated with buffer A
containing 30% (NH4)2SO4. After washing with buffer A containing 30%
(NH4)2SO4, the column was eluted
with a decreasing gradient of 30-0%
(NH4)2SO4 containing buffer A. Active fractions were collected and dialyzed against buffer A and then
applied to a blue Sepharose CL-6B (Amersham Pharmacia Biotech) column
(1.0 × 3 cm) equilibrated with buffer A. The column was washed
with buffer A and eluted with an increasing gradient of 0-1
M KCl containing buffer A. The fractions exhibiting enzyme
activities were combined, concentrated by ultrafiltration with Amicon
Centriplus 10 (Amicon Inc.), and then applied to a Sephacryl S-200 HR
column (1.6 × 60 cm). The column was eluted with 150 ml of buffer
A containing 150 mM KCl. Active fractions were collected,
dialyzed against buffer A, and stored at
For purification of AceCS2, a similar procedure was followed except
DEAE-Sepharose FF and Sephacryl S-200 were replaced by Q-Sepharose FF
(Amersham Pharmacia Biotech). After dialyzing against buffer A, the
enzyme solution was applied to a Q-Sepharose FF column (1.5 × 2 cm) equilibrated with buffer A. The column was washed with buffer A and
eluted with an increasing gradient of 0-0.25 M KCl
containing buffer A. The purified enzyme from the second Q-Sepharose FF
step was dialyzed against buffer A and stored at Antibodies--
To obtain an anti-murine AceCS1, a glutathione
S-transferase fusion protein with murine AceCS1 was used to
immunize rabbits. To produce the glutathione S-transferase
fusion protein, a cDNA fragment encoding amino acids 456-600 of
murine AceCS1 was ligated in-frame to a pGEX-4T bacterial expression
vector (Amersham Pharmacia Biotech). The fusion protein was induced in
Escherichia coli DH5
For the production of an anti-murine AceCS2, a 15-residue peptide
corresponding to the C terminus of murine AceCS2 (CSAFQKYEEQRAATN) was
synthesized by Nippon Gene Research Laboratories (Sendai, Japan). The
amino acid composition and sequence were confirmed by the supplier. The
peptide was coupled to keyhole limpet hemocyanin and injected into New
Zealand White rabbits as described (20). IgG fractions were prepared by
affinity chromatography on protein A-Sepharose (Amersham Pharmacia Biotech).
Subcellular Fractionation--
Mouse kidneys (8 g) were
suspended in 30 ml of buffer B (10 mM Tris-HCl, pH 7.5, 0.25 M sucrose, and 0.2 mM EDTA) and
homogenized with a glass Dounce homogenizer. The homogenate was
centrifuged (1, 000 × g, 10 min) to remove cell debris
and nuclear pellets. A heavy mitochondrial fraction was prepared from
the 1,000 × g supernatant by centrifugation at
3,300 × g for 10 min and then washed twice with buffer
A. The 3, 300 × g supernatant was centrifuged further
at 12, 500 × g for 20 min to obtain a crude
mitochondrial fraction containing a light mitochondria fraction,
peroxisomes, and lysosomes. The 12,500 × g supernatant
was then subjected to ultracentrifugation (120,000 × g
for 1 h) to yield a microsomal and a cytosolic fraction. The crude
mitochondrial fraction was suspended in 20 ml of buffer B and
fractionated on a discontinuous sucrose density gradient
ultracentrifugation to obtain a light mitochondrial and a peroxisomal
fraction (21). The pellet of each spin was suspended in a volume of
buffer B equal to the volume of the supernatant from the same spin.
Experimental Animals--
Male C57BL/6J mice (age 10 weeks,
individually caged) were purchased from Clea Japan (Tokyo) and housed
at the animal laboratory of Tohoku University Gene Research Center
under protocols in accord with the institutional guidelines for animal
experiments at Tohoku University. Animals had free access to the
commercial stock diet (Clea CE2) and water. Fasted mice were deprived
of food for 48 h. Zucker diabetic fatty rats (14-week-old males)
and their normal littermate males were obtained from Nippon
Experimental Animal Co.
Metabolism of [14C]Acetate in Transfected
Cells--
3 days after transfection, COS cells were scraped,
suspended with 5 ml of Hanks' balanced salt solution containing 25 mM glucose and 1 mM [14C]acetate
(940 dpm/nmol), and incubated at 37 °C in a tightly sealed flask.
After a 2-h incubation, 500 µl of 30% (w/v) trichloroacetic acid was
added immediately into the flask, and
[14C]CO2 was trapped into ethanolamine in a
counting vial connected with the flask by constant flow of
N2 gas. Incorporation of 14C in lipids was
determined by extracting the cells with chloroform-methanol (2:1).
Comparison of Amino Acid Sequences of AceCS1 and AceCS2--
In
the previous study, we purified and characterized AceCS from bovine
heart (15). To clone a cDNA encoding AceCS, we determined the
partial amino acid sequence of the bovine enzyme: the purified enzyme
was digested with lysyl endopeptidase, and several peptides were
obtained after separation on a C8 reverse phase column (data not
shown). Based on two peptide sequences (amino acid residues 286-296
and 471-478) derived from the purified enzyme, we designed a set of
degenerate primers to amplify a cDNA fragment encoding the bovine
enzyme (see "Experimental Procedures"). Reverse
transcription-polymerase chain reaction of bovine cardiac poly(A) RNA
with the degenerate primers resulted in amplification of a major
fragment of 576 base pairs encoding a partial cDNA for the bovine
enzyme. Using the amplified 576-base pair fragment as a probe, we
obtained a near full-length cDNA for bovine cardiac AceCS (Fig.
1). We also isolated a near full-length
cDNA for a murine ortholog 83.5% identical to the bovine cardiac
AceCS. In addition, a near full-length cDNA encoding an AceCS
distinct from the cardiac enzyme was obtained by screening a murine
hepatic cDNA library using reduced hybridization conditions. The
nucleotide sequence of the cDNA encoding the hepatic enzyme is
completely identical to that described by Luong et al. (12).
The hepatic and cardiac type enzymes were designated AceCS1 and AceCS2,
respectively. Although Luong et al. abbreviated the enzyme
name to ACS, we prefer to refer to it as AceCS because we have already
designated long chain acyl-CoA synthetase as ACS and characterized five
enzymes, ACS1-5 (7-11).
Fig. 1A shows the deduced primary amino acid sequences of
bovine and murine AceCS2 compared with that of murine AceCS1. There is
only 45.8% amino acid identity between murine AceCS1 and AceCS2, and
the phylogenetic tree of AceCSs of various origins indicates that
AceCS1 and AceCS2 belong to completely different groups (Fig. 1B). Bovine and murine AceCS2 consist of 675 and 682 amino
acids with calculated molecular weights of 74,309 and 74,662, respectively. Sequence analysis by the PSORT program (22) predicted
that AceCS2 contains a potential mitochondrial targeting signal at the
N terminus (Fig. 1A).
Purification of AceCS1 and AceCS2--
To compare the enzymatic
properties of the two enzymes, murine AceCS1 and AceCS2 cDNAs were
individually introduced into COS cells, and the resulting enzymes were
purified to homogeneity using the 230, 000 × g
supernatant fractions of the sonicated extracts. The overall
purification and yields of AceCS1 and AceCS2 were 9.9-fold with a yield
of 2.8% and 15-fold with a yield 2.3%, respectively. The specific
activities of the purified AceCS1 and AceCS2 were, respectively, 26.8 and 11.9 µmol/min/mg when assayed with acetate as a substrate. The
specific activity of the purified AceCS1 was 18-fold higher than that
purified by Luong et al. using an expression plasmid
containing six consecutive histidines at the N terminus and nickel
column chromatography (12).
The two purified enzymes were essentially homogenous when analyzed by
SDS-PAGE (Fig. 2). The molecular mass of
the purified AceCS1 estimated by SDS-PAGE was ~78 kDa, in good
agreement with that calculated molecular mass from the deduced amino
acid sequence of the cDNA. The purified AceCS2 exhibited a
molecular mass of 71 kDa, which agrees well with the purified enzyme
from bovine heart but is ~3.7 kDa smaller than the calculated
molecular mass deduced from the cDNA. This suggests that the
putative mitochondrial targeting signal is cleaved during the
transportation of the enzyme into the mitochondria matrix (see
below).
Enzymatic Properties--
Using the purified preparation, the
fatty acid chain length preferences of AceCS1 and AceCS2 were
determined. As shown in Fig.
3A, acetate is the most
preferred substrate among short and medium chain fatty acids with 2-5
carbon atoms. The enzymes can also utilize propionate but with lower
affinities (Fig. 3B). The calculated Km
values for acetate of both AceCS1 and AceCS2 are similar to that of
the purified histidine-tagged AceCS1 described by Luong et
al. Both AceCS1 and AceCS2 require ATP and CoA for their
activities (data not shown). When adenylate kinase was omitted from the
reaction mixture for the spectrophotometric assay, no oxidation of NADH
occurred, indicating that AMP was a reaction product (data not
shown).
Tissue Expression and Regulation--
Northern blotting of RNA
from various mouse tissues (Fig. 4)
revealed hybridization to major transcripts of 4.4 kilobases for
AceCS2, expressed in a wide range of tissues with the highest level in
heart, relatively high levels in spleen, lung, skeletal muscle, kidney,
and testis and lower levels in the brain (middle panel). No
AceCS2 transcripts were detected in the liver, in contrast to AceCS1
transcripts (12) (Fig. 4, top panel). We also analyzed AceCS1 and AceCS2 mRNAs in 3T3-L1 cells. Although marked induction of AceCS1 mRNA and protein were seen during the differentiation of
3T3-L1 cells, neither AceCS2 mRNA nor protein was detected in
undifferentiated or differentiated 3T3-L1 cells (data not shown).
Regulation of AceCS1 and AceCS2 under Ketogenic
Conditions--
Luong et al. (12) have demonstrated that
the expression of AceCS1 is regulated by sterol regulatory
element-binding proteins in parallel with fatty acid synthesis in
animal cells. Because AceCS2 is highly expressed in the heart and
skeletal muscle, we analyzed the effects of starvation. Northern
blotting was carried out to analyze the levels of mRNAs in the
heart and skeletal muscle of adult male mice because the available
antibodies were not sufficiently sensitive to detect AceCS1 and AceCS2
proteins in these tissues by immunoblotting.
After 48 h of fasting, a marked induction of AceCS2 mRNA was
seen in the heart (2-fold, p < 0.01, Fig.
5A, right panel),
and skeletal muscle (6.5-fold, p < 0.01, Fig.
5B, right panel). In contrast, the AceCS1
mRNA level in the skeletal muscle decreased by ~50%
(p < 0.05, Fig. 5B, left panel),
whereas no significant changes were seen in the heart (Fig.
5A, left panel). We also analyzed the levels of
the two AceCS mRNAs in Zucker diabetic fatty rats. As shown in Fig.
5C, the levels of AceCS2 mRNA in the skeletal muscle of
Zucker diabetic fatty rats were ~3-fold higher than those in the
normal littermates (p < 0.05, right panel), whereas no changes were found in the levels of AceCS1 mRNA
(left panel). These data indicate that the AceCS2
transcripts are induced in the heart and skeletal muscle under
ketogenic conditions.
Subcellular Localization--
To determine the subcellular
distributions of AceCS1 and AceCS2, a subcellular fractionation was
performed on mouse kidney homogenate. The tissue homogenate was
fractionated into a light and heavy mitochondrial, a peroxisomal, a
microsomal, and a cytosolic fraction. Among these fractions,
anti-AceCS1 antibody detected an immunoreactive band of 78 kDa
exclusively in the cytosolic fraction (Fig.
6, top panel). In contrast, a
71-kDa protein detected by an anti-AceCS2 antibody was present mainly
in the light and heavy mitochondrial fractions (Fig. 6, middle
panel). Together with the solubility in hypotonic buffer of AceCS2
in transfected COS cells and the presence of a CoA pool in the
mitochondrial matrix, the cell fractionation data indicate that AceCS2
is a mitochondrial matrix enzyme. An approximately 55-kDa band detected with anti-AceCS2 antibody (Fig. 6, middle panel) may be a
degradation product of AceCS2 because it was colocalized with AceCS2,
and proteolytic digestion of purified bovine AceCS2 with trypsin, subtilisin BPN', and chymotrypsin generated a common 56-kDa fragment resistant to these proteases (15).
Incorporation of [14C]Acetate in Transfected
Cells--
To evaluate the role of AceCS2, we incubated
AceCS2-transfected COS cells with [14C]acetate and
analyzed the incorporation of 14C into CO2 and
lipids. As shown in Fig. 7A,
transfection of AceCS2 in COS cells resulted in an approximately 4-fold
induction of AceCS activity. Consistent with the induction of AceCS
activity, incorporation of 14C into CO2 in
AceCS2-transfected cells increased approximately.5-fold over the
relatively low incorporation by parental vector-transfected cells (Fig.
7B). In contrast, induction of 14C incorporation
into lipids by AceCS2 transfection was only 1.6-fold. These data
suggest that the major function of AceCS2 is to produce acetyl-CoA for
oxidation through the tricarboxylic acid cycle to produce ATP
and CO2 in the mitochondrial matrix.
In the current study, we have isolated and characterized two
cDNAs encoding functionally distinct AceCSs. One, designated AceCS1, is a cytosolic enzyme identical to that described by Luong et al. (12); the other, AceCS2, is a mitochondrial matrix
enzyme, having a putative mitochondrial targeting signal at the N
terminus of its primary amino acid sequence. Consistent with the
presence of the putative signal sequence, subcellular fractionation
indicated that AceCS2 is located in the mitochondrial matrix.
Purification of murine AceCS1 and AceCS2 revealed that the two enzymes
preferentially utilized acetate with similar affinity among C2-C5
short and medium chain fatty acids. Although there are numerous studies
showing acetate utilization in the hepatic mitochondria, apparently no AceCS2 transcripts were detected in the liver. Hepatic carnitine acetyltransferase, which catalyzes the reversible transfer of short
chain acyl groups between CoA and carnitine, may play a role in the
transportation of cytosolic acetyl-CoA into the mitochondrial matrix.
Although the two enzymes exhibit similar affinity for acetate, the
regulation of the two enzymes is completely different. Luong et
al. (12) have demonstrated that AceCS1 is a cytosolic enzyme that
provides acetyl-CoA for the synthesis of fatty acids and cholesterol.
Consistent with the function of AceCS1, it is regulated by sterol
regulatory element-binding proteins, key transcriptional factors that
mediate cholesterol and fatty acid synthesis in the liver (23). In
contrast, AceCS2 is abundant in the heart and skeletal muscle and
induced under ketogenic conditions. The [14C]acetate
incorporation study revealed that AceCS2 produces acetyl-CoAs that are
utilized mainly for oxidation. These results suggested that AceCS2
plays a role in the production of energy under ketogenic conditions,
such as starvation and diabetes.
Under ketogenic conditions, relatively large amounts of fatty
acid-derived free acetate are released from the liver (24). Consistent
with the increased production of acetate, plasma levels of acetate are
also increased by starvation and in patients with diabetes (25, 26).
Furthermore, the hepatic activity of cytosolic acetyl-CoA hydrolase is
also induced under ketogenic conditions (27). The acetate released from
the liver under ketogenic conditions requires AceCS to be metabolized
in extrahepatic tissues. Based on the abundant expression of AceCS2 in
the heart and skeletal muscle, its absence from the liver, and its
marked induction under ketogenic conditions, it is strongly suggested
that AceCS2 plays a key role in the metabolism of acetate for energy
production under ketogenic conditions. Consistent with our hypothesis,
AceCS2 is an abundant protein in the heart of ruminant mammals (15, 28), where large amounts of acetate are produced by microorganisms from
plant fibers in the rumen (1).
Although the mechanism by which the expression of AceCS2 is induced
under ketogenic conditions remains unclear, our current data provide
evidence for the presence of two pathways for the metabolism of acetate
in animals. Further studies are necessary to elucidate the precise
function and regulation of the two enzymes and to determine any
disorders caused by their absence.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Purification of AceCS1 and AceCS2
80 °C.
80 °C.
with isopropyl
-D-thiogalactopyranoside and purified with a
glutathione-Sepharose 4B column (Amersham Pharmacia Biotech). The
purified glutathione S-transferase fusion protein was
digested with thrombin and subjected to SDS-polyacrylamide gel
electrophoresis, and the resulting thrombin-cleaved 16-kDa fragment was
used as an antigen.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Amino acid sequences of bovine and murine
AceCS2. Panel A, the amino acid sequences of
bovine (bAceCS2) and murine AceCS2 (mAceCS2) deduced from the cDNAs
are compared with that of murine AceCS1 (mAceCS1). Amino acids are
numbered on the right. Gaps (indicated with dots)
are inserted to allow alignment that gives the highest identity.
Identical amino acids are highlighted by solid black. The
potential mitochondrial targeting sequence found at the N terminus of
bovine and murine AceCS2 is denoted with open boxes. A
conserved sequence motif among the luciferase family is indicated by a
solid underline. Panel B, rooted
phylogenetic tree of the members of the AceCS family. The tree was
generated using the neighbor joining program Clustal X (29).
Confidence levels were determined by the bootstrapping method (1,000 replicas). Bootstrap values greater than 950 indicate a significant
relationship.
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Fig. 2.
SDS-PAGE of purified murine AceCS1 and
AceCS2. The two enzymes were purified as described under
"Experimental Procedures." 5 µg of the purified enzymes was
subjected to 10% SDS-PAGE and stained with Coomassie Brilliant Blue.
The positions of molecular size markers are indicated on the
left.
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Fig. 3.
Substrate specificities and kinetic
properties of purified AceCS1 and AceCS2. Panel
A, the enzyme activity was determined by the
spectrophotometric method as described under "Experimental
Procedures" with 1 µg of the purified enzyme and the standard
reaction mixture, except that the indicated short chain fatty acid
(final concentration = 1 mM) was used. Enzyme activity
is expressed as a percentage of that obtained with acetate as a
substrate. The specific activities of the purified AceCS1 and AceCS2
obtained with acetate were 26.8 ± 0.14 and 11.9 ± 0.11 µmol/min/mg, respectively. The data are means ± S.D. of
triplicate determinations. Panel B, assays were
performed with the indicated concentrations of acetate and propionate,
as described above. Data points represent the mean of three
determinations obtained in one experiment. The values for apparent
Km were calculated from Lineweaver-Burk
plots.
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Fig. 4.
Tissue expression of AceCS2 transcripts.
2 µg of poly(A) RNA from the indicated murine tissues was probed with
32P-labeled murine AceCS1 (top panel) and AceCS2
(middle panel). The filters were exposed to Kodak X-Omat
AR-5 film with an intensifying screen at 80 °C for 48 h. The
same samples were subsequently hybridized with a control probe for
mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH ;
bottom panel) and exposed to Kodak X-Omat AR-5 film with an
intensifying screen at
80 °C for 6 h.
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Fig. 5.
Induction of AceCS2 mRNA under ketogenic
conditions. Panels A and B,
effects of fasting on the levels of AceCS1 and AceCS2 in mouse heart
(panel A) and skeletal muscle (panel
B). Total RNA was isolated from heart and skeletal muscle of
nonfasted control mice (C = control) and mice fasted
for 48 h (F = fasted). 15-µg aliquots of total
RNA were subjected to Northern blot analysis. A typical autoradiogram
(48-h exposure) is shown. Northern blot analysis with a
glyceraldehyde-3-phosphate dehydrogenase probe was also carried out for
normalization (data not shown). The radioactivity in each animal was
quantified using a bioimaging analyzer with various exposure times and
normalized to the glyceraldehyde-3-phosphate dehydrogenase signal. The
values are the means for six animals ± S.D., relative to the
mRNA level in control mice (set at 100). * indicates
p < 0.05; ** indicates p < 0.01 compared with the control. Panel C, expression in
Zucker diabetic fatty rats. 15-µg aliquots of total RNA from skeletal
muscle of 6-month-old male Zucker diabetic fatty rats
(Z = Zucker) and their normal littermate males
(n = normal) were subjected to Northern blot analysis
as described above. A typical autoradiogram (48-h exposure) is shown.
The values are the means for four animals ± S.D., relative to the
mRNA level in control animal (set at 100). * indicates
p < 0.05 compared with the control.
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Fig. 6.
Subcellular localization of AceCS2. A
postnuclear fraction prepared from murine kidneys was processed further
into the indicated fractions as described under "Experimental
Procedures." 10 µg of protein from the indicated fractions was
subjected to SDS-PAGE, blotted onto a nitrocellulose membrane, and
detected by anti-AceCS1 and anti-AceCS2 antibodies. Immunoblottings
with specific antibodies for NADPH P-450 reductase (a microsome
marker), prohibitin (a mitochondria marker), and catalase (a
peroxisomal marker) were carried out as controls.
View larger version (18K):
[in a new window]
Fig. 7.
[14C]Acetate Incorporation by
AceCS2-transfected cells. Panel A, AceCS
activities in transfected cells. 72 h after transfection, AceCS2
and parental vector transfected cells were harvested for measurement of
AceCS activity. Panel B,
[14C]acetate incorporation into CO2 and
lipids. 72 h after transfection, cells were incubated with
[14C]acetate as described under "Experimental
Procedures," and 14C in CO2 and lipids was
determined. Bars represent S.D. of quadruplicate
determinations.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Dr. Ian Gleadall for review of the manuscript and Yumiko Takei for excellent technical assistance.
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FOOTNOTES |
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* This work was supported by Grant RFTF97L00803 from the Japan Society for the Promotion of Science.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB046741 and AB046742.
To whom correspondence should be addressed: Tohoku
University Gene Research Center, 1 1 Tsutsumidori-Amamiya, Aoba,
Sendai 981-8555; Japan. Fax: 81-22-717-8877; Email:
yama@biochem.tohoku.ac.jp.
Published, JBC Papers in Press, January 9, 2001, DOI 10.1074/jbc.M008782200
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ABBREVIATIONS |
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The abbreviations used are: AceCS, acetyl-CoA synthetase; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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