Functional Characterization of Δ3,Δ2-Enoyl-CoA Isomerases from Rat Liver
2002; Elsevier BV; Volume: 277; Issue: 11 Linguagem: Inglês
10.1074/jbc.m112228200
ISSN1083-351X
AutoresDongyan Zhang, Wenfeng Yu, Brian V. Geisbrecht, Stephen J. Gould, Howard Sprecher, Horst Schulz,
Tópico(s)Metabolism, Diabetes, and Cancer
ResumoThe degradation of unsaturated fatty acids by β-oxidation involves Δ3,Δ2-enoyl-CoA isomerases (enoyl-CoA isomerases) that catalyze 3-cis → 2-trans and 3-trans → 2-trans isomerizations of enoyl-CoAs and the 2,5 → 3,5 isomerization of dienoyl-CoAs. An analysis of rat liver enoyl-CoA isomerases revealed the presence of a monofunctional enoyl-CoA isomerase (ECI) in addition to mitochondrial enoyl-CoA isomerase (MECI) in mitochondria, whereas peroxisomes contain ECI and multifunctional enzyme 1 (MFE1). Thus ECI, which previously had been described as peroxisomal enoyl-CoA isomerase, was found to be present in both peroxisomes and mitochondria. This enzyme seems to be identical with mitochondrial long-chain enoyl-CoA isomerase (Kilponen, J.M., Palosaari, P.M., and Hiltunen, J.K. 1990. Biochem. J. 269, 223–226). All three hepatic enoyl-CoA isomerases have broad chain length specificities but are distinguishable by their preferences for one of the three isomerization reactions. MECI is most active in catalyzing the 3-cis → 2-trans isomerization; ECI has a preference for the 3-trans → 2-trans isomerization, and MFE1 is the optimal isomerase for the 2,5 → 3,5 isomerization. A functional characterization based on substrate specificities and total enoyl-CoA isomerase activities in rat liver leads to the conclusion that the 3-cis → 2-trans and 2,5 → 3,5 isomerizations in mitochondria are catalyzed overwhelmingly by MECI, whereas ECI contributes significantly to the 3-trans → 2-trans isomerization. In peroxisomes, ECI is predicted to be the dominant enzyme for the 3-cis → 2-trans and 3-trans → 2-transisomerizations of long-chain intermediates, whereas MFE1 is the key enzyme in the 2,5 → 3,5 isomerization. The degradation of unsaturated fatty acids by β-oxidation involves Δ3,Δ2-enoyl-CoA isomerases (enoyl-CoA isomerases) that catalyze 3-cis → 2-trans and 3-trans → 2-trans isomerizations of enoyl-CoAs and the 2,5 → 3,5 isomerization of dienoyl-CoAs. An analysis of rat liver enoyl-CoA isomerases revealed the presence of a monofunctional enoyl-CoA isomerase (ECI) in addition to mitochondrial enoyl-CoA isomerase (MECI) in mitochondria, whereas peroxisomes contain ECI and multifunctional enzyme 1 (MFE1). Thus ECI, which previously had been described as peroxisomal enoyl-CoA isomerase, was found to be present in both peroxisomes and mitochondria. This enzyme seems to be identical with mitochondrial long-chain enoyl-CoA isomerase (Kilponen, J.M., Palosaari, P.M., and Hiltunen, J.K. 1990. Biochem. J. 269, 223–226). All three hepatic enoyl-CoA isomerases have broad chain length specificities but are distinguishable by their preferences for one of the three isomerization reactions. MECI is most active in catalyzing the 3-cis → 2-trans isomerization; ECI has a preference for the 3-trans → 2-trans isomerization, and MFE1 is the optimal isomerase for the 2,5 → 3,5 isomerization. A functional characterization based on substrate specificities and total enoyl-CoA isomerase activities in rat liver leads to the conclusion that the 3-cis → 2-trans and 2,5 → 3,5 isomerizations in mitochondria are catalyzed overwhelmingly by MECI, whereas ECI contributes significantly to the 3-trans → 2-trans isomerization. In peroxisomes, ECI is predicted to be the dominant enzyme for the 3-cis → 2-trans and 3-trans → 2-transisomerizations of long-chain intermediates, whereas MFE1 is the key enzyme in the 2,5 → 3,5 isomerization. Δ3,Δ2-enoyl-CoA isomerase bovine serum albumin Δ3,5,Δ2,4-dienoyl-CoA isomerase dithiothreitol high-performance liquid chromatography mitochondrial Δ3,Δ2-enoyl-CoA isomerase multifunctional enzyme 1 polyacrylamide gel electrophoresis peroxisomal Δ3,Δ2-enoyl-CoA isomerase 4-morpholinepropanesulfonic acid Both saturated and unsaturated fatty acids are degraded by β-oxidation. However, the degradation of unsaturated fatty acids, in contrast to the breakdown of saturated fatty acids, requires the involvement of several auxiliary enzymes that catalyze the isomerization and reduction of double bonds (reviewed in Ref. 1Kunau W.-H. Dommes V. Schulz H. Prog. Lipid Res. 1995; 34: 267-342Crossref PubMed Scopus (405) Google Scholar). During the β-oxidation of unsaturated fatty acid with even-numbered double bonds, e.g. the 12-cis double bond of linoleic acid, a 3-trans → 2-trans double bond shift takes place, whereas three isomerizations, 3-cis → 2-trans, 3-trans → 2-trans, and 2,5 → 3,5 (see Scheme FS1) occur during the β-oxidation of fatty acids with odd-numbered double bonds,e.g. the 9-cis double bond of oleic acid and linoleic acid. All of these positional and steric isomerizations of double bonds are catalyzed by Δ3-Δ2-enoyl-CoA isomerase (EC 5.3.3.8) (ECI or enoyl-CoA isomerase).1Five mammalian enzymes with enoyl-CoA isomerase activities have been described. They are mitochondrial enoyl-CoA isomerase (MECI) (2Stoffel W. Ecker W. Methods Enzymol. 1969; 14: 99-105Crossref Scopus (30) Google Scholar, 3Palossari P.M. Kilponen J.M. Sormunen R.T. Hassinen I.E. Hiltunen J.K. J. Biol. Chem. 1990; 265: 3347-3353Abstract Full Text PDF PubMed Google Scholar, 4Müller-Newen G. Stoffel W. Biol. Chem. Hoppe-Seyler. 1991; 372: 613-624Crossref PubMed Scopus (32) Google Scholar), mitochondrial long-chain enoyl-CoA isomerase (5Kilponen J.M. Palosaari P.M. Hiltunen J.K. Biochem. J. 1990; 269: 223-226Crossref PubMed Scopus (20) Google Scholar), mitochondrial enoyl-CoA hydratase or crotonase (6Kiema T.-R. Engel C.K. Schmitz W. Filppula S.A. Wierenga R.K. Hiltunen J.K. Biochemistry. 1999; 38: 2991-2999Crossref PubMed Scopus (49) Google Scholar), peroxisomal enoyl-CoA isomerase (PECI) (7Geisbrecht B.V. Zhang D. Schulz H. Gould S.J. J. Biol. Chem. 1999; 274: 21797-21803Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), and multifunctional enzyme 1 (MFE1) (8Palossari P.M. Hiltunen J.K. J. Biol. Chem. 1990; 265: 2446-2449Abstract Full Text PDF PubMed Google Scholar). According to the available information, the first three enoyl-CoA isomerase activities are present in mitochondria, and the last two enzymes are associated with peroxisomes. Structural information about this group of enzymes has been growing rapidly as reflected by the recent publications of crystal structures for rat crotonase (9Engel C.K. Mathieeu M. Zeelen J.Ph. Hiltunen J.K. Wierenga R.K. EMBO J. 1996; 15: 5135-5145Crossref PubMed Scopus (171) Google Scholar) and yeast PECI (10Mursula A.M. van Aalten D.M.F. Hiltunen J.K. Wierenga R.K. J. Mol. Biol. 2001; 309: 845-853Crossref PubMed Scopus (45) Google Scholar). These two enzymes are hexameric proteins with similar patterns of folding even though they exhibit low sequence homology and catalyze different reactions. The specific metabolic functions of enoyl-CoA isomerases are poorly defined, especially because it was demonstrated that more than one isomerase is present in either mitochondria or peroxisomes and that collectively these enzymes catalyze three distinct reactions in β-oxidation. Unanswered questions about the identity of enoyl-CoA isomerases, their subcellular localizations, and their substrate specificities have prompted the present investigation. Nycodenz, CoASH, NAD+, NADH, benzamidine hydrochloride, CM-cellulose, di(ethylhexyl)phthalate, acyl-CoA oxidase from Arthrobacter sp., and most standard biochemicals were purchased from Sigma. Fatty acid-free bovine serum albumin (BSA) was from Life Science Resources, Milwaukee, WI. Diketene, crotonic anhydride, hexanal, dodecanal, 3-trans-hexenoic acid, 3-hexyn-1-ol, and other standard chemicals were purchased from Aldrich. 3-Octyn-1-ol was purchased from Lancaster Synthesis Inc., Windham, NH. Matrix Gel Red A was purchased from Amicon, Danvers, MA. Dithiothreitol was purchased from Fisher. 5-cis-Octenoic acid, 3-cis-tetradecenoic acid, and 5-cis-tetradecenoic acid were kindly provided by Dr. Howard Sprecher, Ohio State University. Hydroxylapatite, the dye reagent for protein assays, polyacrylamide ready gels, and the materials for immunoblotting, including the goat anti-rabbit IgG conjugated with alkaline phosphatase, were bought from Bio-Rad. Sep-Pak C18cartridges were purchased from Waters. Iodixanol (Optiprep) was from Nycomed Pharma AS, Oslo, Norway. The antiserum to rat peroxisomal multifunctional enzyme 1 was a kind gift of Dr. Ronald Wanders, University of Amsterdam, Netherlands. Rabbit antisera against mitochondrial and peroxisomal enoyl-CoA isomerases were raised by Pocono Rabbit Farms and Laboratory, Canadensis, PA. Male Sprague-Dawley rats were purchased from Taconic Farms, Germantown, NY. Bovine liver enoyl-CoA hydratase (crotonase) (11Steinman H. Hill R.L. Methods Enzymol. 1965; 35: 136-151Crossref Scopus (120) Google Scholar), pig heart 3-ketoacyl-CoA thiolase (12Schulz H. Staak H. Methods Enzymol. 1981; 71: 398-403Crossref PubMed Scopus (25) Google Scholar), recombinant pig liver 3-hydroxyacyl-CoA dehydrogenase (13He X.-Y. Yang S.-Y. Biochim. Biophys. Acta. 1998; 1392: 119-126Crossref PubMed Scopus (14) Google Scholar), and recombinant rat mitochondrial Δ3,5,Δ2,4-dienoyl-CoA isomerase (dienoyl-CoA isomerase) (14Zhang D. Liang X. He X.Y. Alipui O.D. Yang S.Y. Schulz H. J. Biol. Chem. 2001; 276: 13622-13627Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar) were purified as described. 3-trans-Octenoic acid and 3-trans-tetradecenoic acid were synthesized from malonic acid and hexanal and dodecanal, respectively, according to Boxer and Linstead (15Boxer S.E. Linstead R.P. J. Chem. Soc. 1931; : 740-751Crossref Scopus (39) Google Scholar). 3-cis-Hexenoic acid and 3-cis-octenoic acid were synthesized from 3-hexyn-1-ol and 3-octyn-1-ol, respectively, according to Stoffel and Ecker (2Stoffel W. Ecker W. Methods Enzymol. 1969; 14: 99-105Crossref Scopus (30) Google Scholar). Acetoacetyl-CoA was synthesized from diketene and CoASH according to White and Jencks (16White H. Jencks W.P. J. Biol. Chem. 1976; 251: 1688-1699Abstract Full Text PDF PubMed Google Scholar). Crotonyl-CoA was synthesized from crotonic anhydride and CoASH according to Weeks and Wakil (17Weeks G. Wakil S.J. J. Biol. Chem. 1968; 243: 1180-1189Abstract Full Text PDF PubMed Google Scholar). 2-trans,5-cis-Octadienoyl-CoA and 2-trans,5-cis-tetradecadienoyl-CoA were synthesized from 5-cis-octenoic acid and 5-cis-tetradecenoic acid according to Shoukry and Schulz (18Shoukry K. Schulz H. J. Biol. Chem. 1998; 273: 6892-6899Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Fatty acyl-CoA derivatives of all other fatty acids were prepared by the mixed anhydride method as described by Fong and Schulz (19Fong J.C. Schulz H. Methods Enzymol. 1981; 71: 390-398Crossref PubMed Scopus (90) Google Scholar). All substrates for enoyl-CoA isomerase assays were purified by HPLC. A Waters μBondapak C18 column (30 × 3.9 mm) attached to a Waters gradient HPLC system was used for this purpose. The absorbance of the effluent was monitored at 254 nm. Separation was achieved by linearly increasing the acetonitrile/H2O (9:1, v/v) content of the 50 mm ammonium phosphate buffer (pH 5.5) from 10 to 40% (for C6 substrates), from 10 to 50% (for C8 substrates), or from 10 to 70% (for C14 substrates) at a flow rate of 2 ml/min. Desired fractions were collected, and the organic solvent was evaporated under vacuum on a rotatory evaporator. Sep-Pak C18 cartridges were used to concentrate the substrates after HPLC purification. After the substrates were absorbed onto the Sep-Pak columns, they were eluted with 3 ml of methanol. Methanol was evaporated under vacuum before the substrates were redissolved in H2O. Concentrations of acyl-CoAs were determined spectrophotometrically by quantifying CoASH with Ellman's reagent (20Ellman G.L. Arch. Biochem. Biophys. 1959; 82: 70-77Crossref PubMed Scopus (21624) Google Scholar) after cleaving the thioester bonds with NH2OH at pH 7.0 (19Fong J.C. Schulz H. Methods Enzymol. 1981; 71: 390-398Crossref PubMed Scopus (90) Google Scholar). Enoyl-CoA isomerases were assayed spectrophotometrically by measuring the absorbance increase at either 263 or 280 nm on a Gilford recording spectrophotometer at 25 °C. A typical assay mixture contained 0.2 m KPi (pH 8.0), 35 μm substrate, BSA (0.2 mg/ml), and enzyme. Extinction coefficients of 6,700 m−1cm−1 at 263 nm or 4,400 m−1cm−1 at 280 nm were used to calculate rates. Enoyl-CoA isomerase activities associated with multifunctional enzyme 1 or fractions obtained by separating mitochondrial, peroxisomal, or tissue extracts were assayed by a coupled assay (21Binstock J.F. Schulz H. Methods Enzymol. 1981; 71: 403-411Crossref PubMed Scopus (100) Google Scholar), in which the isomerization of 35 μm 3-enoyl-CoA to 2-enoyl-CoA was coupled to the hydration of the latter compound by crotonase, the NAD+-dependent dehydrogenation of the 3-hydroxyacyl-CoA intermediate by 3-hydroxyacyl-CoA dehydrogenase, and the thiolytic cleavage of the resultant 3-ketoacyl-CoA by 3-ketoacyl-CoA thiolase. Formation of NADH (ε = 6,220m−1 cm−1) was the basis for calculating rates of isomerization. When 2,5-octadienoyl-CoA or 2,5-tetradecadienoyl-CoA was used as substrate in the enoyl-CoA isomerase assay, dienoyl-CoA isomerase was added as a coupling enzyme. A typical assay mixture contained 0.2 m KPi (pH 8.0), 35 μm substrate, dienoyl-CoA isomerase (0.25 units/ml), BSA (0.2 mg/ml), and an aliquot of enzyme. An extinction coefficient of 27,800 m−1 cm−1(22Yang S.-Y. Cuebas D. Schulz H. J. Biol. Chem. 1986; 261: 15390-15395Abstract Full Text PDF PubMed Google Scholar) was used to calculate rates. Enoyl-CoA hydratase (19Fong J.C. Schulz H. Methods Enzymol. 1981; 71: 390-398Crossref PubMed Scopus (90) Google Scholar), 3-hydroxyacyl-CoA dehydrogenase (21Binstock J.F. Schulz H. Methods Enzymol. 1981; 71: 403-411Crossref PubMed Scopus (100) Google Scholar), catalase (23Baudhuin P. Beaufay H. Rahman-Li Y. Sellinger O.Z. Wattiaux R. Jacques P. de Duve C. Biochem. J. 1964; 92: 179-184Crossref PubMed Scopus (485) Google Scholar), and malate dehydrogenase (24Ochoa S. Methods Enzymol. 1955; 1: 735-739Crossref Scopus (469) Google Scholar) activities were determined by established procedures. Enzymes were diluted with 50 mm KPi(pH 7.0) containing BSA (1 mg/ml). One unit of enzyme activity is defined as the amount of enzyme that catalyzes the conversion of 1 μmol of substrate to product in 1 min. Protein concentrations were determined as described by Bradford (25Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) with BSA as standard. For the kinetic characterizations of ECI and MECI with 3-enoyl-CoAs as substrates, absorbance changes at 280 nm were recorded because the basal absorbance at 263 nm was too high at elevated substrate concentrations. Rates were measured at five or six substrate concentrations, and averages of three assays were used for each point. Preanalyses were performed to determine appropriate substrate and enzyme concentration ranges. Kinetic parameters (K mand V max) were obtained by nonlinear curve fitting using the SigmaPlot 2000 program. Values ofk cat, the catalytic center activity, were calculated using the reported subunit molecular masses of 40.4 (7Geisbrecht B.V. Zhang D. Schulz H. Gould S.J. J. Biol. Chem. 1999; 274: 21797-21803Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), 78.5 (26Osumi T. Ishii N. Hijikata M. Kamijo K. Ozasa H. Furuta S. Miyazawa S. Londo K. Inoue K. Kagamiyama H. Hashimoto T. J. Biol. Chem. 1985; 260: 8905-8910Abstract Full Text PDF PubMed Google Scholar), and 29 kDa (4Müller-Newen G. Stoffel W. Biol. Chem. Hoppe-Seyler. 1991; 372: 613-624Crossref PubMed Scopus (32) Google Scholar) for ECI, MFE1, and MECI, respectively. Mitochondria and a light mitochondrial fraction were prepared from rat livers as described by Nedergaard and Cannon (27Nedergaard J. Cannon B. Methods Enzymol. 1979; 69: 390-398Google Scholar) and de Duve et al. (28de Duve C. Pressman B.C. Gianetto R. Wattiaux R. Appelmans F. Biochem. J. 1955; 60: 604-617Crossref PubMed Scopus (2573) Google Scholar), respectively. Adult male Sprague-Dawley rats (240–260 g) were used. They were kept on a standard chow and then fasted for 24 h before their livers were removed. Mitochondria and peroxisomes were separated by Nycodenz density gradient centrifugation of a light mitochondrial fraction as described (29He X.-Y. Shoukry K. Chu C. Yang J. Sprecher H. Schulz H. Biochem. Biophys. Res. Commun. 1995; 215: 15-22Crossref PubMed Scopus (36) Google Scholar). For this purpose, a 30% (w/v) solution of Nycodenz containing 1 mm EDTA, 5 mm Hepes (pH 7.3), and 0.1% ethanol was prepared, and 21 ml of this solution were placed in a 30-ml ultracentrifuge tube on top of 1.5 ml of a 60% sucrose cushion. A density gradient was generated by centrifugation at 60,000 × g in a T865 small angle rotor on a DuPont RC70 ultracentrifuge at 4 °C for 24 h. A light mitochondrial fraction (∼15 mg of protein in 1.5 ml) was layered on top of the gradient followed by 1.5 ml of a cover solution of a 3-fold diluted isolation buffer containing 0.25 msucrose, 1 mm EDTA, 0.1% ethanol, and 10 mmTris (pH 7.4). This was followed by centrifugation at 76,000 ×g for 1 h at 4 °C. Fractions of 1.6 ml each were collected from the bottom of the tube, diluted 2-fold with isolation buffer, and centrifuged at 17,500 × g for 20 min. Pellets from each fraction were redissolved in 100 μl of isolation buffer for further analysis. Peroxisomes were purified by centrifugation of a light mitochondrial fraction on a preformed continuous gradient of iodixanol (Optiprep) as described by van Veldhoven et al. (30Van Veldhoven P.P. Baumgart E. Mannaerts G.P. Anal. Chem. 1996; 237: 17-23Google Scholar, 31Osumi T. Hashimoto T. Biochem. Biophys. Res. Commun. 1979; 89: 580-584Crossref PubMed Scopus (151) Google Scholar). The gradient was prepared in 30-ml centrifuge tubes from equal volumes of iodixanol (20%, w/v) containing 0.41 m sucrose, 1.2 mm EDTA, 0.12% ethanol, and 6 mm Mops (pH 7.2) and iodixanol (40%, w/v) containing 0.14 m sucrose, 0.8 mm EDTA, 0.08% ethanol, and 4 mm Mops (pH 7.2), by use of a gradient mixer and a peristaltic pump. After 20 ml of the gradient mixture had been placed in each tube, 2 ml of iodixanol (50%, w/v) containing 25 mm sucrose, 0.5 mm EDTA, 0.05% ethanol, and 2.5 mm Mops (pH 7.2) were delivered to the bottom of the tube by use of a long syringe needle. Three ml of light mitochondria (12 mg of protein/ml) were carefully layered on top of the gradient. The tubes were then centrifuged at 105,000 × g for 1 h in a T865 fixed angle rotor at 4 °C using the slow acceleration and deceleration mode. Fractions were collected from the bottom after slowly inserting a thin glass tube through the bottom of the tube. Marker enzyme activities for mitochondria and peroxisomes were assayed. Peroxisomal fractions were combined and diluted 2-fold with isolation buffer before they were harvested by centrifugation at 17,500 × g for 20 min. Portions (4 g) of rat liver were homogenized in 16 ml of 10 mm KPi (pH 7.4) containing 0.2 mKCl, 0.5 mm EDTA, 1 mm benzamidine, and 0.5 mm DTT (buffer A). The homogenates were sonicated and centrifuged at 100,000 × g for 1 h. The supernatants were dialyzed against 20 mm KPi(pH 7.0) containing 0.5 mm benzamidine and 0.5 mm DTT (buffer B). After dialysis, the samples were applied to hydroxylapatite columns (1.5 × 15 cm) equilibrated with buffer B at a flow rate of 10 ml/h. The proteins bound to the column were eluted with a linear gradient made up of 200 ml each of 20 mm and 500 mm KPi (pH 7.0) containing 0.5 mm benzamidine and 0.5 mm DTT. Also enoyl-CoA isomerases present in peroxisomes (15 mg of protein) and mitochondria (130 mg of protein) were separated by this procedure after sonicating and centrifuging the organelle suspensions before the resultant soluble extracts were loaded onto hydroxylapatite columns and eluted by a KPi gradient from 20 to 500 mm. Adult Sprague-Dawley rats were fed rodent chow containing 2% (w/w) di(ethylhexyl)phthalate for 2 weeks before being sacrificed. For the purification of MECI (3Palossari P.M. Kilponen J.M. Sormunen R.T. Hassinen I.E. Hiltunen J.K. J. Biol. Chem. 1990; 265: 3347-3353Abstract Full Text PDF PubMed Google Scholar), mitochondria isolated from rat liver (28de Duve C. Pressman B.C. Gianetto R. Wattiaux R. Appelmans F. Biochem. J. 1955; 60: 604-617Crossref PubMed Scopus (2573) Google Scholar) were sonicated in 20 mm KPi (pH 7.0) containing 0.5 mmDTT, 1 mm EDTA, 0.5 mm benzamidine, and 0.5 mm phenylmethylsulfonyl fluoride (buffer A) and centrifuged at 100,000 × g for 1 h. The supernatant was applied to a Matrix Gel Red A column (2.5 × 12 cm) previously equilibrated with buffer A. The column was washed with buffer A and then was developed with a gradient made up of 100 ml of buffer A and 100 ml of buffer A containing 1.2 m KCl. Fractions containing enoyl-CoA isomerase activity were combined. After dialysis overnight against 50 mm KPi (pH 6.0) containing 0.5 mm DTT, 0.5 mm benzamidine, 10% glycerol (buffer B), the sample was applied to a CM-cellulose column (1.5 × 6 cm) previously equilibrated with buffer B. Mitochondrial enoyl-CoA isomerase was eluted with a linear gradient made up of 25 ml of buffer B and 25 ml of buffer B containing 0.4 m KCl. For the purification of MFE1 (33Bjerrum O.J. Schafer-Nielson Dunn M.J. Electrophoresis 1986. VCH Publishers Inc., Deerfield Beach, FL1986: 315-327Google Scholar), a liver from a rat treated with di(ethylhexyl)phthalate was homogenized in 1:5 (w/v) of 10 mm K3PO4 containing 1 mm EDTA, 1 mm EGTA, 1 mmbenzamidine, 0.5 mm DTT, and 0.5 mmphenylmethylsulfonyl fluoride with a Polytron tissue homogenizer. The suspension was sonicated 10 times for 20 s each at 4 °C before being centrifuged at 100,000 × g for 1 h. The supernatant was adjusted to pH 7.0 before being applied to a phosphocellulose column (2.5 × 20 cm) previously equilibrated with 50 mm KPi (pH 7.0) 0.5 mmbenzamidine, 0.5 mm DTT (buffer C). The column was eluted with a linear gradient made up of 200 ml of 50 mmKPi in buffer C and 200 ml of 500 mmKPi in buffer C. Active fractions were combined and fractionated with (NH4)2SO4. The precipitate formed between 200 and 400 g/liter of (NH4)2SO4 was dialyzed overnight against buffer B before being applied to a CM-cellulose column (1.5 × 8 cm) previously equilibrated with buffer B. The enzyme was eluted with a linear gradient of 100 ml of 50 mmKPi in buffer B and 100 ml of 200 mmKPi in buffer B. Active fractions were combined and concentrated. Samples were treated with equal volumes of SDS sample buffer and subjected to SDS-PAGE on either gradient (4–20%) or 10% ready gels (32Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). Proteins were transferred to a nitrocellulose membrane by semi-dry blotting (33Bjerrum O.J. Schafer-Nielson Dunn M.J. Electrophoresis 1986. VCH Publishers Inc., Deerfield Beach, FL1986: 315-327Google Scholar) using the semi-dry transfer cell from Bio-Rad. Proteins remaining on the gel were visualized by staining with Coomassie Blue. The membrane was blocked with 5% dry milk for 1 h before being incubated with a 500-fold diluted rabbit antiserum for 1 h. After incubating the membrane with goat anti-rabbit IgG conjugated with alkaline phosphatase for 1.5 h, it was developed with a staining mixture containing the alkaline phosphatase substrate until the antigen bands were visible (34Blake M.S. Johnston K.H. Russell-Jones G.J. Gotschlich E.C. Anal. Biochem. 1984; 136: 175-179Crossref PubMed Scopus (1631) Google Scholar). The expression and purification of recombinant peroxisomal enoyl-CoA isomerase was done as described in principle by Geisbrecht et al. (7Geisbrecht B.V. Zhang D. Schulz H. Gould S.J. J. Biol. Chem. 1999; 274: 21797-21803Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). For the expression of His6-PECI, freshly transformed BL21 (DE3) cells harboring the pT7His-PECI plasmid were grown at 30 °C with vigorous shaking (225 rpm) for 12–14 h in 50 ml of LB media supplemented with kanamycin sulfate (25 μg/ml) and sterile 1% dextrose. After this incubation period, 7.5 ml of the preculture cell suspension were diluted into 500 ml of 2YT media containing kanamycin sulfate (25 μg/ml) and sterile 0.2% dextrose. This culture was grown with vigorous shaking at 18 °C until the A 600 reached 0.5, at which time the induction of protein expression was initiated by the addition of 1 mm isopropyl-β-d-thiogalactoside. Following growth of the induced culture for 18 h, cells were harvested and sonicated in 50 ml of buffer A (20 mmKPi (pH 7.8) containing 500 mm NaCl, and 5 mm 2-mercaptoethanol) and centrifuged at 100,000 ×g for 1 h. The supernatant was diluted to a final volume of 200 ml with buffer A and was applied at a rate of 2 ml/min to a 5-ml bed of ProBond-agarose at 4 °C. The column was washed with 5 volumes of buffer A and then with 20 volumes of buffer B (20 mm KPi (pH 6.0) containing 500 mmNaCl, and 5 mm 2-mercaptoethanol). Following these washing steps, the His6-PECI was eluted from the bed using an imidazole step gradient in buffer B. One column volume each of buffers containing 50, 250, 350, and 500 mm imidazole was applied sequentially to the resin bed. The eluants were collected, and each fraction was assayed for enoyl-CoA isomerase. Fractions containing high activities were pooled, and the His6-PECI present was precipitated slowly by the addition of solid ammonium sulfate to 0.4 g/ml. The aim of this study was the characterization of all enoyl-CoA isomerases that are present in rat liver and the determination of their contributions to the total enoyl-CoA isomerase activities in both mitochondria and peroxisomes. Mitochondrial long-chain enoyl-CoA isomerase had been detected by Kilponen et al. (5Kilponen J.M. Palosaari P.M. Hiltunen J.K. Biochem. J. 1990; 269: 223-226Crossref PubMed Scopus (20) Google Scholar) who separated it from MECI and peroxisomal MFE1 by chromatography on hydroxylapatite. However, they did not purify it any further. Because this enoyl-CoA isomerase was more active with 3-trans-dodecenoyl-CoA than with 3-trans-hexenoyl-CoA as substrate, they named it long-chain enoyl-CoA isomerase. They also concluded that it had a mitochondrial localization. We repeated the separation of a soluble extract from rat liver on hydroxylapatite, but we assayed each fraction with 3-trans-octenoyl-CoA and 3-cis- octenoyl-CoA because the ratio of activities obtained with these two substrates aids in the identification of different enoyl-CoA isomerases. Shown in Fig.1 is the result of this experiment. The activity pattern with 3-trans-octenoyl-CoA as substrate is similar to that observed by Kilponen et al. (5Kilponen J.M. Palosaari P.M. Hiltunen J.K. Biochem. J. 1990; 269: 223-226Crossref PubMed Scopus (20) Google Scholar) who concluded that the enoyl-CoA isomerase eluted first from the column was a novel isomerase, which they named long-chain enoyl-CoA isomerase. The activity pattern obtained with 3-cis-octenoyl-CoA as substrate was quite different. The isomerase activity present in fractions 13–18 is easily missed, whereas the existence of two isomerase activities, presumably corresponding to MFE1 and MECI, in fractions 20–40 is more clearly revealed than with the 3-trans substrate. A trans/cis activity ratio of close to 2 determined for fractions 13–16 is similar to that of PECI, which has a trans/cis activity ratio of 2 in contrast to MECI and MFE1 with trans/cis activity ratios below 1. The presence of PECI in fractions 13–18 was confirmed by immunoblotting (data not shown). Thus, it seems that mitochondrial long-chain enoyl-CoA isomerase is identical with PECI. If mitochondrial long-chain enoyl-CoA isomerase and PECI are the same enzyme, PECI must be present in both mitochondria and peroxisomes. To confirm this prediction, a light mitochondrial fraction was prepared from a rat liver homogenate and subjected to centrifugation on a Nycodenz density gradient. Fractions were assayed for catalase and malate dehydrogenase to localize peroxisomes and mitochondria, respectively. Fractions were also analyzed by immunoblotting with antibodies to MECI and PECI. The results shown in Fig.2 demonstrate that MECI was present only in mitochondria (fractions 7–11), whereas PECI was detected in both peroxisomes (fractions 1–5) and mitochondria (fractions 7–11). Because the dual localization of PECI contradicts the reported unique association of this enzyme with peroxisomes, further confirmation was sought. For this purpose, an extract from isolated rat liver mitochondria was subjected to chromatography on hydroxylapatite. The results shown in Fig. 3 demonstrate the presence of at least two enoyl-CoA isomerases in mitochondria. The isomerase that was eluted first had a trans/cis activity ratio of ∼2 and was recognized by an antibody to PECI. Hence, this enoyl-CoA isomerase was PECI. The enoyl-CoA isomerase corresponding to the second peak was identified as MECI because it had atrans/cis activity ratio below 1 and was detected with antibodies raised against MECI. A similar experiment was also carried out with a soluble extract from rat liver peroxisomes, which had been purified by centrifugation on an iodixanol density gradient. The results are shown in Fig. 4. Again, two peaks of enoyl-CoA isomerase activity were detected. The isomerase that was eluted first from the column was PECI as indicated by itstrans/cis activity ratio of ∼2 and because of its recognition by antibodies to PECI. The second isomerase peak was due to MFE1 as demonstrated by immunoblotting and co-elution of enoyl-CoA isomerase, enoyl-CoA hydratase, and l-3-hydroxyacyl-CoA dehydrogenase activities. The possibility that MFE1 may bind either MECI or PECI and thereby acquire enoyl-CoA isomerase activity prompted an experiment in whi
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