Acetyl-CoA Synthetase 2, a Mitochondrial Matrix Enzyme Involved in the Oxidation of Acetate

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.AB046741AB046742 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.AB046741AB046742 acetyl-CoA synthetase polyacrylamide gel electrophoresis 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. 1Bremer J. Osmundsen H. Numa S. Fatty Acid Metabolism and Its Regulation. Elsevier Science Publisher, Amsterdam1984: 113-154Google Scholar). 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 (2Crabtree B. Souter M.-J. Anderson S.E. Biochem. J. 1989; 257: 673-678Crossref PubMed Scopus (26) Google Scholar). 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 (3Sterri S.H. Fonnum F. J. Neurochem. 1980; 35: 249-254Crossref PubMed Scopus (47) Google Scholar, 4Carroll P.T. Brain Res. 1997; 753: 47-55Crossref PubMed Scopus (20) Google Scholar). AceCS from various microorganisms has revealed that the enzyme belongs to the firefly luciferase superfamily (5Toh H. Protein Sequences Data Anal. 1990; 3: 517-521PubMed Google Scholar, 6Toh H. Protein Sequences Data Anal. 1991; 4: 111-117PubMed Google Scholar), which includes mammalian long chain acyl-CoA synthetases, ACS1–ACS5 (7Fujino T. Yamamoto T. J. Biochem. ( Tokyo ). 1992; 111: 197-203Crossref PubMed Scopus (121) Google Scholar, 8Fujino T. Kang M.J. Suzuki H. Iijima H. Yamamoto T. J. Biol. Chem. 1996; 271: 16748-16752Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 9Kang M.J. Fujino T. Sasano H. Minekura H. Yabuki N. Nagura H. Iijima H. Yamamoto T.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2880-2884Crossref PubMed Scopus (208) Google Scholar, 10Suzuki H. Kawarabayasi Y. Kondo J. Abe T. Nishikawa K. Kimura S. Hashimoto T. Yamamoto T. J. Biol. Chem. 1990; 265: 8681-8685Abstract Full Text PDF PubMed Google Scholar, 11Oikawa E. Iijima H. Suzuki T. Sasano H. Sato H. Kamataki A. Nagura H. Kang M.J. Fujino T. Suzuki H. Yamamoto T.T. J. Biochem. ( Tokyo ). 1998; 124: 679-685Crossref PubMed Scopus (132) Google Scholar), 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 (5Toh H. Protein Sequences Data Anal. 1990; 3: 517-521PubMed Google Scholar,6Toh H. Protein Sequences Data Anal. 1991; 4: 111-117PubMed Google Scholar). 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. (12Luong A. Hannah V.C. Brown M.S. Goldstein J.L. J. Biol. Chem. 2000; 275: 26458-26466Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar) 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 molecular biology and immunochemical techniques were performed essentially as described by Sambrook et al. (13Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) and Harlow and Lane (14Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar), 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 1m 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. Bovine AceCS was purified from heart as described previously (15Ishikawa M. Fujino T. Sakashita H. Morikawa K. Yamamoto T. Tohoku J. Exp. Med. 1995; 175: 55-67Crossref PubMed Scopus (14) Google Scholar). 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). 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)-Latex™ (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 Taq™ (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 (16Okayama H. Berg P. Mol. Cell. Biol. 1983; 2: 161-170Crossref Scopus (871) Google Scholar). 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. 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 (17Takai T. Ohmori H. Biochim. Biophys. Acta. 1990; 1048: 105-109Crossref PubMed Scopus (68) Google Scholar). 3e days after DNA transfection, the cells were harvested for purification and [14C]acetate incorporation (see below). 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 (18Tanaka T. Hosaka K. Numa S. Methods Enzymol. 1981; 71: 334-341Crossref PubMed Scopus (23) Google Scholar). 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 mmMgCl2, 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 mmdithiothreitol, 15 mm MgCl2, 10 mmATP, 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 (19Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). All purification steps were carried out at 0–4 °C, and the results are summarized in TableI.Table IPurification of AceCS1 and AceCS2Enzyme and stepTotal proteinTotal activitySpecific activityRecoverymgμmol/minμmol/min/mg%AceCS1230,000 ×g1363672.710060% (NH4)2SO472.42663.772.7DEAE-Sepharose FF23.01988.653.9Phenyl-Sepharose FF6.313020.735.4Blue Sepharose FF1.019.620.75.3Sephacryl S-2000.410.426.82.8AceCS2230,000 ×g13687.30.810060% (NH4)2SO430.367.62.377.4Q-Sepharose FF1.06.36.67.2Phenyl-Sepharose FF0.85.78.76.5Blue Sepharose FF0.33.811.24.32nd Q-Sepharose FF0.22.011.92.3Crude sonicated extracts were prepared from COS cells transfected with either AceCS1 or AceCS2. Enzyme activities were measured at 37 °C by the isotopic method as described under "Experimental Procedures." Open table in a new tab Crude sonicated extracts were prepared from COS cells transfected with either AceCS1 or AceCS2. Enzyme activities were measured at 37 °C by the isotopic method as described under "Experimental Procedures." 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 mmphenylmethylsulfonyl 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 0–1m 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 −80 °C. 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 −80 °C. To obtain an anti-murine AceCS1, a glutathioneS-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 inEscherichia coli DH5α with isopropyl β-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. 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 (20Iijima H. Fujino T. Minekura H. Suzuki H. Kang M.J. Yamamoto T. Eur. J. Biochem. 1996; 242: 186-190Crossref PubMed Scopus (64) Google Scholar). IgG fractions were prepared by affinity chromatography on protein A-Sepharose (Amersham Pharmacia Biotech). 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 × gfor 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 (21Miyazawa S. Hashimoto T. Yokota S. J. Biochem. ( Tokyo ). 1985; 98: 723-733Crossref PubMed Scopus (120) Google Scholar). The pellet of each spin was suspended in a volume of buffer B equal to the volume of the supernatant from the same spin. 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. 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). In the previous study, we purified and characterized AceCS from bovine heart (15Ishikawa M. Fujino T. Sakashita H. Morikawa K. Yamamoto T. Tohoku J. Exp. Med. 1995; 175: 55-67Crossref PubMed Scopus (14) Google Scholar). 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. (12Luong A. Hannah V.C. Brown M.S. Goldstein J.L. J. Biol. Chem. 2000; 275: 26458-26466Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). 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 (7Fujino T. Yamamoto T. J. Biochem. ( Tokyo ). 1992; 111: 197-203Crossref PubMed Scopus (121) Google Scholar, 8Fujino T. Kang M.J. Suzuki H. Iijima H. Yamamoto T. J. Biol. Chem. 1996; 271: 16748-16752Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 9Kang M.J. Fujino T. Sasano H. Minekura H. Yabuki N. Nagura H. Iijima H. Yamamoto T.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2880-2884Crossref PubMed Scopus (208) Google Scholar, 10Suzuki H. Kawarabayasi Y. Kondo J. Abe T. Nishikawa K. Kimura S. Hashimoto T. Yamamoto T. J. Biol. Chem. 1990; 265: 8681-8685Abstract Full Text PDF PubMed Google Scholar, 11Oikawa E. Iijima H. Suzuki T. Sasano H. Sato H. Kamataki A. Nagura H. Kang M.J. Fujino T. Suzuki H. Yamamoto T.T. J. Biochem. ( Tokyo ). 1998; 124: 679-685Crossref PubMed Scopus (132) Google Scholar). Fig. 1 A 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.1 B). 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 (22Nakai K. Horton P. Trends Biochem. Sci. 1999; 24: 34-36Abstract Full Text Full Text PDF PubMed Scopus (1838) Google Scholar) predicted that AceCS2 contains a potential mitochondrial targeting signal at the N terminus (Fig. 1 A). 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 × gsupernatant 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 (12Luong A. Hannah V.C. Brown M.S. Goldstein J.L. J. Biol. Chem. 2000; 275: 26458-26466Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). 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). Using the purified preparation, the fatty acid chain length preferences of AceCS1 and AceCS2 were determined. As shown in Fig.3 A, 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. 3 B). The calculated Kmvalues 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). 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 (12Luong A. Hannah V.C. Brown M.S. Goldstein J.L. J. Biol. Chem. 2000; 275: 26458-26466Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar) (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). Luong et al. (12Luong A. Hannah V.C. Brown M.S. Goldstein J.L. J. Biol. Chem. 2000; 275: 26458-26466Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar) 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.5 A, right panel), and skeletal muscle (6.5-fold, p < 0.01, Fig.5 B, right panel). In contrast, the AceCS1 mRNA level in the skeletal muscle decreased by ∼50% (p < 0.05, Fig. 5 B, left panel), whereas no significant changes were seen in the heart (Fig.5 A, left panel). We also analyzed the levels of the two AceCS mRNAs in Zucker diabetic fatty rats. As shown in Fig.5 C, 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. 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 (15Ishikawa M. Fujino T. Sakashita H. Morikawa K. Yamamoto T. Tohoku J. Exp. Med. 1995; 175: 55-67Crossref PubMed Scopus (14) Google Scholar). 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. 7 A, 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.7 B). 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 Luonget al. (12Luong A. Hannah V.C. Brown M.S. Goldstein J.L. J. Biol. Chem. 2000; 275: 26458-26466Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar); 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. (12Luong A. Hannah V.C. Brown M.S. Goldstein J.L. J. Biol. Chem. 2000; 275: 26458-26466Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar) 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 (23Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (3028) Google Scholar). 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 (24Seufert C.D. Graf M. Janson G. Kuhn A. Soling H.D. Biochem. Biophys. Res. Commun. 1974; 57: 901-909Crossref PubMed Scopus (47) Google Scholar). Consistent with the increased production of acetate, plasma levels of acetate are also increased by starvation and in patients with diabetes (25Akanji A.O. Humphreys S. Thursfield V. Hockaday T.D. Clin. Chim. Acta. 1989; 185: 25-34Crossref PubMed Scopus (25) Google Scholar, 26Buckley B.M. Wiliamson D.H. Biochem. J. 1977; 166: 539-545Crossref PubMed Scopus (95) Google Scholar). Furthermore, the hepatic activity of cytosolic acetyl-CoA hydrolase is also induced under ketogenic conditions (27Matsunaga T. Isohashi F. Nakanishi Y. Sakamoto Y. Eur. J. Biochem. 1985; 152: 331-336Crossref PubMed Scopus (24) Google Scholar). 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 (15Ishikawa M. Fujino T. Sakashita H. Morikawa K. Yamamoto T. Tohoku J. Exp. Med. 1995; 175: 55-67Crossref PubMed Scopus (14) Google Scholar,28Webster Jr., L.T. J. Biol. Chem. 1963; 238: 4010-4015Abstract Full Text PDF PubMed Google Scholar), where large amounts of acetate are produced by microorganisms from plant fibers in the rumen (1Bremer J. Osmundsen H. Numa S. Fatty Acid Metabolism and Its Regulation. Elsevier Science Publisher, Amsterdam1984: 113-154Google Scholar). 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. We thank Dr. Ian Gleadall for review of the manuscript and Yumiko Takei for excellent technical assistance.