THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 992 by The Amerian Soiety for Biohemistry and Moleular Biology, In. Vol. 267, No. 2, Issue of January 5, pp. 027-033,992 Printed in U.S.A. Novel Fatty Aid &Oxidation Enzymes in Rat Liver Mitohondria I. PURIFICATION AND PROPERTIES OF VERY-LONG-CHAIN ACYL-COENZYME A DEHYDROGENASE* (Reeived for publiation, June 27, 99) Kaoru IzaiS, Yasushi UhidaS, Tadao OriiS, Shigehiro YamamotoQ, and Takashi Hashimotoll From the $Department of Pediatris, Gifu University Shool of Mediine, Gifu, Gifu 500, the $Department of Pediatris, Shimoshizu National Hospital and Sanatorium, Yotsukaido, Chiba 284, and the VDepartment of Biohemistry, Shinshu University Shool of Mediine, Matsumoto, Nagano 390, Japan Freeze-thawed rat liver mitohondria were extensively washed with potassium phosphate, ph 7.5, and the residue was extrated with 0 mm potassium phosphate, ph 7.5,% (w/v) sodium holate, 0.6 M KCl. The four &oxidation enzyme ativities of the washes and the last extrat were assayed with substrates of various arbon hain lengths. Our data suggest that the last extrat ontains a novel ayl-coa dehydrogenase and long-hain 3-hydroxyayl-CoA dehydrogenase. A novel ayl-coa dehydrogenase was purified. The moleular masses of the native enzyme and the subunit were estimated to be 60 and 7 kda, respetively. One mole of enzyme ontained 2 mole of FAD. These properties and immunohemial properties of the enzyme differed from those of three other ayl-coa dehydrogenases: short-, medium-, and long-hain ayl- CoA dehydrogenases. Carbon hain length speifiity of the enzyme differed from that of other ayl-coa dehydrogenases. The enzyme was ative toward CoA esters of long- and very-long-hain fatty aids, but not toward those of medium- and short-hain fatty aids. The speifi enzyme ativity was >0 times that of long-hain ayl- CoA dehydrogenase when palmitoyl-coa was used as substrate. We propose the name very-long-hain ayl- CoA dehydrogenase for this enzyme. Mitohondrial fatty aid B-oxidation is one of the main energy-yielding metaboli proesses, and enzymes involved in this system have been well haraterized. Enzymes suh as long-hain ayl-coa synthetase and arnitine palmitoyltransferase are loated in the mitohondrial membrane, whereas those involved in the @-oxidation yle are loated in the matrix. Most of these matrix enzymes have been extrated by soniation or freeze/thawing in the absene of detergent. We usually extrated these enzymes with phosphate buffer in the absene of detergent after disruption of the mitohondrial struture by freeze/thawing (-4). In this study, frozen rat liver mitohondria were thawed and washed with phosphate buffers, and then the residue was extrated with buffer ontaining a high onentration of KC * This work was supported in part by a grant-in-aid for sientifi researh from the Ministry of Eduation, Siene, and Culture of Japan, Grant 2-A from the National Center of Neurology and Psyhiatry of the Ministry of Health and Welfare of Japan, a researh grant for intratable diseases from the Ministry of Health and Welfare of Japan, and a grant from the Uehara Memorial Foundation. The osts of publiation of this artile were defrayed in part by the payment of page harges, This artile must therefore be hereby marked advertisement in aordane with 8 U.S.C. Setion 734 solely to indiate this fat. 027 and detergent. The four @-oxidation enzyme ativities of the extrat were assayed using substrates of various arbon hain lengths. A high palmitoyl-coa dehydrogenase ativity was found in the last extrat. The purified enzyme differed from the short-, medium-, and long-hain ayl-coa dehydrogenases with respet to moleular and atalyti properties. EXPERIMENTAL PROCEDURES Materials Crotonyl-CoA and aetoaetyl-coa were prepared with the use of anhydrides (5, 6). Other CoA esters of saturated, monounsaturated, and 3-hydroxy fatty aids were prepared by the mixed anhydride method (7). 3-Ketoayl-CoAs were prepared enzymatially (8). Ar- ahidoyl-coa, behenoyl-coa, and lignoeroyl-coa were obtained from Sigma. The purified @-oxidation enzymes and their antibodies were prepared as desribed previously: ayl-coa oxidase (9), enoyl-coa hydratase (2) and enoyl-coa hydratase/3-hydroxyayl-coa dehydrogenase bifuntional protein (2, lo), short-hain 3-hydroxyayl-CoA dehydrogenase (3), and mitohondrial and peroxisomal 3-ketoayl- CoA thiolases (4). Proteins used as moleular mass markers in moleular sieve hromatography and sodium dodeyl sulfate-polyarylamide gel eletrophoresis (SDS-PAGE) were from Bio-Rad and Boehringer Mannheim. Ultrogel AA34 was purhased from LKB Produkter, and Sephadex G-50 was from Pharmaia LKB Biotehnology In. DEAE-Toyopearl was obtained from Toyo Soda Manufaturing Co. (Tokyo). Hydroxylapatite and phosphoellulose were supplied by Naalai Tesque, In. (Kyoto, Japan). All other reagents were of the purest analytial grade available. Enzyme Assays Ayl-CoA dehydrogenase ativity was assayed in terms of the redution of 2,6-dihloroindophenol as an eletron aeptor andphenazine methosulfate as an intermediate eletron arrier at 30 C (). The reation mixture ontained, in a volume of ml, 50 mm potassium phosphate, ph 7.4,30 PM palmitoyl-coa, 35 pm 2,6-dihloroindophenol, mm N-ethylmaleimide, and enzyme. The reation was started by addition of.6 DM phenazine methosulfate. The molar absorption oeffiient at 600 nm was 2,000. Other ativities of fatty aid oxidation enzymes were determined aording to the ited soures: enoyl-coa hydratase (2), 3-hydroxyayl-CoA dehydrogenase (3), and 3-ketoayl-CoA thiolase (4). One unit of enzyme is defined as the amount that onverts @mol of substrate/min at 30 C. Assay and Analysis of Protein Protein onentration was determined by modifiation () of the method of Lowry et al. (2). SDS-PAGE was arried out using a 0% The abbreviations used are: SDS-PAGE, sodium dodeyl sulfatepolyarylamide gel eletrophoresis; ETF, eletron transfer flavoprotein; DEHP, di-(2-ethylhexyl) phthalate; HEPES, 4-(2-hydroxyethyl)--piperazineethanseulfoni aid.
028 Novel Very-long-hain Ayl-CoA Dehydrogenase gel (3). Immunoblot analysis was performed aording to Towbin et ai. (4). Purifiation of Short-, Medium-, and Long-hain Ayl-CoA Dehydrogenases and Eletron Transfer Flavoprotein Short-, medium-, and long-hain ayl-coa dehydrogenases were purified as desribed previously (). The long-hain ayl-coa dehydrogenase preparation was further purified by repeating hydroxylapatite olumn hromatography as desribed by Ikeda et al. (5) to remove a small amount of ontaminating medium-hain ayl-coa dehydrogenase. Eletron transfer flavoprotein (ETF) was purified as desribed (l), and the final preparation was stored in 50 mm HEPES, ph 7.8, 0% (v/v) ethylene glyol (6), onditions under whih ETF was stable and full ativity was maintained for more than several months. Purifiation of New Ayl-CoA Dehydrogenase Male Wistar rats (220-250 g) were fed a diet ontaining 2% (w/w) di-(2-ethylhexyl) phthalate (DEHP) for 2-4 weeks beause the ativities of ayl-coa dehydrogenases inreased with suh treatment (see "Results and Disussion"). All proedures (exept the heat step) were arried out at 4 "C. Step : Preparation of Rat Liver Mitohondria-Subellular frationation of liver was done as desribed by de Duve et al. (7), exept that the heavy and light mitohondrial frations were not separated. The mitohondrial fration was resuspended in 0.25 M surose ontaining mm EDTA and entrifuged. The supernatant and the mirosome-rih fluffy layer were removed. The mitohondrial pellets obtained from 00 g of liver were suspended in 50 mm potassium phosphate, ph 7.5, in a final volume of 50 ml and kept at -20 "C overnight. This frozen fration ould be stored for > month without a signifiant loss of enzyme ativity. Step 2: Extration-The frozen mitohondrial fration (50 ml) was thawed by mixing with 250 ml of 50 mm potassium phosphate, ph 7.5, ontaining mm phenylmethylsulfonyl fluoride. After standing for 30 min on ie, the mixture was entrifuged at 00,000 X g for 30 min at 4 "C. The supernatant was disarded, and the pellet was suessively washed by homogenization in a glass homogenizer with a Teflon pestle and entrifugation with 300 ml of 0 mm potassium phosphate, ph 7.5, and 0 mm potassium phosphate, ph 7.5, ontaining 0.% sodium holate. The washed pellet was homogenized with 50 ml of 0 mm potassium phosphate, ph 7.5, % sodium holate, 0.5 M KC, 0.% (w/v) hexamethylphosphori triamide, 2 mm meraptoethanol, mm EDTA, 0 p~ FAD. The suspension was kept for 30 min on ie and entrifuged at 00,000 X g for 30 min. All buffer solutions used in the following proedures ontained 0.% hexamethylphosphori triamide, 2 mm meraptoethanol, mm EDTA. Step 3: Heat and Ammonium Sulfate Frationation-The extrat was divided into five or six portions and heated to 5-52 "C with stirring in hot water (-75-80 "C), kept for 30 s at this temperature, and then rapidly ooled in ie water. The denatured proteins were removed by entrifugation. Ammonium sulfate was added to the supernatant (50 g/liter). The mixture was entrifuged. The pellet was disarded, and the ammonium sulfate was added to the supernatant (0 g of the original extrat/liter). After entrifugation, the preipitate was dissolved in 5 ml of 0 mm potassium phosphate, ph 7.5,0.2% (w/v) Tween 20,lO p~ FAD and dialyzed in a thin dialysis tube (ellulose tubing, '/32 inh; Viskase Sales Corp.) against the same buffer for 2 h with a hange of buffer. Step 4: First Phosphoellulose Column Chromatography-The ph of the dialyzed enzyme solution was adjusted to ph 7.5 and then diluted with a solution of 0.2% Tween 20, 0 PM FAD to give the same ondutivity as that of 0 mm potassium phosphate, ph 7.5. The diluted enzyme solution showing a slight turbidity was applied to a phosphoellulose olumn (2.5 X 6 m) that had been equilibrated with dialysis buffer. The enzyme was eluted with 80 ml of a 0-200 mm linear gradient of potassium phosphate, ph 7.5, ontaining 0.2% Tween 20, 0 p~ FAD. When ative frations were olleted, the latter parts were not saved beause a ontaminating 68-kDa polypep- tide was removed at this step and at Step 6. Step 5: DEAE-Toyopearl Column Chromatography-Ative frations were pooled, diluted %fold with 0.2% Tween 20, and applied to a DEAE-Toyopearl olumn (.5 X m) that had been washed with 30 mm potassium phosphate, ph 7.5, 0.2% Tween 20. A 30-300 mm linear gradient of potassium phosphate, ph 7.5, in 60 ml of 0.2% Tween 20 was used for elution of the enzyme. Step 6: Seond Phosphoellulose Column Chromatography-Ative frations were pooled, diluted 5-fold with 0.2% Tween 20, and then applied to a phosphoellulose olumn (.0 X 3 m). The enzyme was eluted with 0 mlof a 20-200 mm linear gradient of potassium phosphate, ph 7.5, ontaining 0.2% Tween 20. Ative frations were subjeted to SDS-PAGE. Frations not ontaining the 68-kDa polypeptide were pooled. Preparation of Antibody against New Enzyme The purified enzyme (0.2 mg) was emulsified with an equal volume of Freund's omplete adjuvant, and the emulsion was injeted into the axillary regions of a rabbit. Four weeks later, a booster ontaining 0.2mgof enzyme in the emulsion was injeted into the axillary regions, subutaneous sites on the dorsum, and hip musle. Blood was taken from a arotid artery 2 weeks after the booster injetion. The antibody was partially purified by preipitation with ammonium sulfate and dialyzed against 0.5 M NaCl, 0 mm potassium phosphate, ph 7.5. RESULTS AND DISCUSSION Extration of,&oxidation Enzymes-Male Wistar rats (250-300 g) were fed laboratory how for 2 weeks with or without DEHP. The hepati mitohondria were suspended in 50 mm potassium phosphate, ph 7.5, and frozen at -20 "C overnight. The frozen mitohondria were thawed; and 50 mm potassium phosphate, ph 7.5, was added to give a final onentration of 0 mgof protein/ml. EDTA, meraptoethanol, and hexamethylphosphori triamide were added to give final onentration of mm, 2 mm, and 0.%, respetively. Thereafter, we added these reagents to all buffers used in the extration experiment. The suspension was entrifuged at 00,000 x g for h. The pellet was sequentially extrated with various buffers at a volume equal to that of the starting suspension. Table I summarizes typial results. The last extrats of the ontrol and DEHP groups exhibited muh higher ayl-coa dehydrogenase ativities with palmitoyl-coa than with otanoyl-coa or butyryl-coa when ompared to the other extrats. The arbon hain length speifiity of the ayl-coa dehydrogenase of the last extrat differed from those of short-, medium-, and long-hain ayl-coa dehydrogenases (). These observations suggested the presene of a novel ayl-coa dehydrogenase. Proliferation of hepati mitohondria and an inrease in some of the mitohondrial &oxidation enzymes were observed after the administration of a peroxisome proliferator suh as lofibrate and DEHP (8-20). The ativities of short-, medium-, and long-hain ayl-coa dehydrogenases of the first extrat were inreased -2-fold/g of liver after the administration of DEHP, as reported elsewhere (20). The palmitoyl-coa dehydrogenase ativity of the last extrat was also higher in the DEHP group than in the ontrol. The enoyl-coa hydratase ativity of the DEHP group was partly due to the peroxisomal bifuntional protein. Upon treatment with DHEP, the bifuntional protein was markedly inreased; and part of this enzyme leaked out during homogenization and preipitated to mitohondria and mirosomes under onditions of low ioni strength, suh as surose solution (2). The presene of another enoyl-coa hydratase was not suspeted. It was lear that long-hain 3-hydroxyayl-CoA dehydrogenase, as estimated by the ratio of ativities with 3-ketopalmitoyl-CoA and aetoaetyl-coa, was solubilized in the last extrat and that this enzyme was markedly inreased by DEHP treatment. The purifiation and properties of this enzyme are desribed in the aompanying paper (2). Purifiation of New Ayl-CoA Dehydrogenase-A summary of the purifiation of a novel ayl-coa dehydrogenase is shown in Table. The results of SDS-PAGE of the purified
Novel Very-long-hain Ayl-CoADehydrogenase 029 TABLE I Sequential extration of fatty aid @-oxidation enzymesfrom rat liver mitohondria Frozen mitohondria from ontrol and DEHP-fed rat livers kept at -20 "C overnight were thawed and then sequentially extrated with 50 mm potassium phosphate, ph 7.5; 0 mm potassium phosphate, ph 7.5; 0 mm potassium phosphate, ph 7.5,0.% sodium holate; and 0 mm potassium phosphate, ph 7.5, %sodium holate, 0.5 M KCl. Cl-C4 are sequential extrats from the ontrol rat liver mitohondria, and Dl-D4 are sequential extrats from the DEHP-fed rat liver mitohondria. The ativities of four 8-oxidation enzymes of the extrats were assayed using substrates with various lengths of arbon hain (C4,CS, and C6). Control mitohondria extrats DEHP-treated Enzyme D3 l 2 mitohondria extrats 4 D2 D3 D4 unitslrnl units/rnl Ayl-CoA dehydrogenase 0.238 0.3 0.095 4 C8 6 0.048 0.005 0.022 0.042 0.004 0.034 0.032 0.003 0.095 0.365 0.85 0.43 0.07 0.00 0.048 0.053 0.008 0.032 0.038 0.005 0.43 Enoyl-CoA hydratase 4 C8 C6 3-Hydroxyayl-CoAdehydrogenase 4 C8 0.25 6 0.09 0. 3-Ketoayl-CoA thiolase 4 CS 65 53.3 2.5 7.8 6.9.3 0.3.7.6.0 0.5 3.3 6.2 0.8 0.22 Extrat Heat Ammonium sulfate st phosphoellulose DEAE-Toyopearl 2nd phosphoellulose ativity protein units mg 0.7 0. 23.9 7.8 2.2 0.87.00 0.32 0.49 20.0 44.4 4.4 5. 2..0 8 54.8.5 3.9 6.5.0.20 0.23 0.8 0.78 0.3 0.66 0.4.80 2.3 0.69.2 9.0 34.7 mg' 63.0 95 0.069 34.5 54.8 32 0.07 27.0 67 42.9 0.62 22.5 4.5 35.7.55 7.58.93.96 0.88 7. 2.3 0.8 % 00 7 -L 5042-27.8 0.50 0.47 2 koa ativity unit 9.4 3.2 0.7 0.88 TABLE I Summary of the purifiation The mitohondria (total protein was 3600 mg) obtained from 00 g of liver from rats fed adiet ontainingdehp were used. k D a Total Total Speifi Yield step 2.2 4.0. w - - - 97.4 - ra.5 74.7 -- 662-53.0 =4":; -3.0 preparation are shown in Fig.. The enzyme ativity of the extrat was dereased by half or less when stored overnight either at 4 "C or in a frozen state. When the ammonium sulfate fration wasdialyzed overnight, the enzyme ativity was also dereased by about half. The enzyme after the first phosphoellulose olumn hromatography step was relatively stable. Therefore, we usually washed the frozen mitohondria with 50 mm potassium phosphate, ph 7.5, and 0 mm potassium phosphate, ph 7.5, on the first day and purified the enzyme to Step 4 or 5 on the seond day. The addition of 0.% hexamethylphosphoritriamide 2 mm meraptoethanol, mm EDTA was essential to obtain a high reovery of the enzyme ativity; and FAD exhibited a protetive effet inthe earlier steps of purifiation. Two ontaminating polypeptides were present in the final enzyme preparation. Onewas a 68-kDa polypeptide that eluted in the latter part of the enzyme ativity peak on phosphoellulose olumnhromatography. Therefore, the ative frations at Step 6 were olleted after examination of the separation of the enzyme from this polypeptide by SDSPAGE. This ontaminating protein was removed by repeating phosphoellulose olumn hromatography.the other ontaminant was a 50-kDa polypeptide. This polypeptide remained in the final preparation (Fig. ). Removal of this 50-kDa polypeptide was diffiult even with various olumn hromatographi separations. However, we found that this polypeptide -2.5 FIG.. SDS-PAGE of purified preparations. The enzymes (2.5 pg eah) were analyzed on 0% gel. Lane, new enzyme; lane 2, long-hain ayl-coa dehydrogenase. Proteins used as moleular mass standards were rabbit musle phosphorylase b (97.4 kda), bovine serum albumin (66.2 kda), hen egg white ovalbumin (42.7 kda), bovine arboni anhydrase (3.0 kda), soybean trypsin inhibitor (2.5 kda), rat liver ayl-coa oxidase (omponent A, 74.7 kda; omponent B, 53.0 kda (22)), rat liver enoyl-coa hydratase/3-hydroxyayl-coa dehydrogenase (78.5 kda (23)), and rat liver peroxisomal 3-ketoaylCoA thiolase (4.0 kda (24)). The estimated moleular masses are indiated 7 kda for the subunit of the new enzyme, 50 kda for the ontaminant as desribed in the text, and 42 kda for the subunit of long-hain ayl-coa dehydrogenase. ould be partially separated from the enzyme on a hydroxylapatite olumn or a alium phosphate gel/ellulose olumn in the presene of %(w/v) n-heptyl /3-D-thiogluoside.The enzyme preparation at Step 6 was diluted 0-fold with %nheptyl /3-D-thiogluoside and applied to a hydroxylapatite olumn. The olumn was eluted with a linear gradient of 20300 mm potassium phosphate, ph 7.5, ontaining %detergent. This proedure had to be arried out rapidly, and the eluates from the olumn were diluted -5-fold with buffer ontaining %Tween 20. The enzyme exhibited a lower ativity in buffer ontaining n-heptyl @-D-thiogluoside,and
030 Novel Very-long-hain Ayl-CoA Dehydrogenase full ativity was restored when the enzyme solution was immediately added to 5 or 0 volumes of buffer ontaining %Tween 20. The distribution of the enzyme ativity oinided with that of the 7-kDa polypeptide (but not the 50kDa polypeptide). Thus, the 7-kDa polypeptide was the subunit of the enzyme, and the 50-kDa polypeptide was not related to theenzyme ativity. We did not use this proedure for purifiation of the enzyme beause the enzyme was labile, and separation was not satisfatory. These 68- and 50-kDa polypeptides were thought to derive from the 7-kDa polypeptide beause quantities of the 68and 50-kDa polypeptides in the final preparation were larger when the enzyme waspurified without phenylmethylsulfonyl fluoride at Step 2. The antibody was purified using the 7kDa polypeptide to examine the relationship between the 7kDa polypeptide and these68- and 50-kDa polypeptides. The purified enzyme preparation was subjeted to SDS-PAGE and blotted onto anitroellulose filter. The filter was stained with Amido Blak. The part ontaining the 7-kDa polypeptide was ut out. The antibody was immunopurified with this filter strip and used for immunoblot analysis. The ratio of the intensities of the immunoblot signals for the 7-kDa and 68and 50-kDa polypeptides was similar to that of the protein staining of the gel after SDS-PAGE. When freshly prepared homogenate or mitohondria was used at theamount giving a strong signal for the 7-kDa polypeptide, no signal or only faint signals for the 68- and 50-kDa polypeptides appeared. The yield of the enzyme was 7% of the original amount at Step 6, and the speifi enzyme ativity with palmitoyl-coa was -2 units/mg (Table ). The degree of purity of the preparation at Step 6 is shown in Fig.. The amount of ontaminating 50-kDa polypeptide of this preparation, determined by measuring Coomassie Blue dye eluted from the gel, was -5% of the total proteins. Nearly the full ativity of the purified preparation was maintained for month, and the ativity dereased to half after being stored at -20 "C for 3 months. Moleular Properties-The native moleular mass of the enzyme was estimated by gel hromatography using Ultrogel AA 34 and Sephadex G-50. The buffer used in this experiment ontained 0.5 M KC and 0.2% Tween 20. Therefore, proteins suh as atalase and ferritin ould not be used as moleular mass markers beause of a shift in the elution positions of these proteins in thepresene of both the detergent and 0.5 M KCI. The enzyme ativity peak orresponded to a moleular mass of 50 kda (Fig. 2). The apparent moleular mass of the subunit estimated by SDS-PAGE and Coomassie Blue staining was 7 kda (Fig. ).Thus, this enzyme is probably a homodimer. Immunohemial Properties-Fig. 3 summarizes the results of titration with the antibodies of the new enzyme and longhain ayl-coa dehydrogenase.the new enzyme was titrated ompletely with the antibody against this enzyme, but longhain ayl-coa dehydrogenase was nottitrated with this amount of the antibody. Inversely, the antibody against longhain ayl-coa dehydrogenase titrated only this enzyme, not the new enzyme. Monospeifiity of the antibody against the new enzyme was examined by immunoblot analysis using rat liver homogenates. The main immunoblot signal band orresponding to the 7-kDa polypeptide and a very faint band orresponding to the 68-kDa polypeptide appeared, and no other band was evident. Fig. 4 indiates the lak of ross-reativity between the new enzyme and long-hain ayl-coa dehydrogenase. Determination and Identifiation of FAD-The enzyme preparation at Step 6was used for FAD determination andto 50 42 60 Elution volume. ml FIG. 2. Estimation of moleular weight by Ultrogel AA 34 olumn hromatography. A olumn (.5 X 45 m) was equilibrated and eluted with 0 mm potassium phosphate, ph 7.5, 0.2% Tween 20, 0.5 M KCI, 0.% hexamethylphosphori triamide, 2 mm meraptoethanol, mm EDTA. The following standards (0)were used for alibration of the olumn:, rabbit musle pyruvate kinase (M,= 237,000); 2, rabbit musle latate dehydrogenase (M, = 40,000); 3, pig heart malate dehydrogenase (M, = 70,000); and 4, rat liver 3hydroxyayl-CoA dehydrogenase (M.= 65,000). The position of the peak of ativity of the new enzyme is shown (0). B A 2r- 0 0.2 0.4 0.6 0.8 Antibody, mg FIG.3. Immunotitration of newenzyme and long-hain ayl-coa dehydrogenase. The reation mixture used for titration of the new enzyme ontained 50 mm potassium phosphate, ph 7.5, 0.2% Tween20,0.% hexamethylphosphori triamide, 2 mm meraptoethanol, mm EDTA; and that for long-hain ayl-coa dehydrogenase ontained 50 mm potassium phosphate, ph 7.5. The fixed amount of the enzyme was mixed with various amounts of the antibody and kept at room temperature for 30 min. After entrifugation, an aliquot of the supernatant was used for enzyme assay. A, titration with the antibody against the new enzyme: 0, new enzyme (5 pg); 0,long-hain ayl-coa dehydrogenase (5 pg). B, titration with the antibody against long-hain ayl-coa dehydrogenase: 0, new enzyme (0 pg); 0, long-hain ayl-coa dehydrogenase (0 pg). A 2 2 B FIG.4. Immunoblot analysis. A, antibody against the new enzyme; B, antibody against long-hain ayl-coa dehydrogenase. Lane, new enzyme (20 ng); lane 2, long-hain ayl-coa dehydrogenase (20 ng).
Very-long-hain Dehydrogenase Ayl-CoA Novel 03 observe absorbane harateristis. Purity of the preparation used for this experiment was 92% as determined by the amounts of Coomassie Blue dye eluted from the stained gel strips after SDS-PAGE. The protein was deproteinized with trihloroaeti aid, and the supernatant was neutralized with KZHP04. The absorbane of this solution at 450 nm was measured. Based on the molar extintion oeffiient of FAD at this wavelength (,300 M m ) (25), the ontent of FAD was alulated to be 0.94 mol/mol of the subunit of the enzyme using a value of 7 kda for the moleular mass of the subunit and the purity of this preparation. FAD was extrated with phenol and subjeted to thin-layer hromatography (9). A single fluoresent spot orresponding to FAD was deteted. The spetral maxima and their ratio for the new enzyme were 277,377, and 453 nm and 3.6:0.82:.0, respetively. These values differed from those of short-, medium-, and long-hain ayl-coa dehydrogenases (). Effet of ETF-Ayl-CoA dehydrogenase atalyzes the reation with the formation of a trans double bond between C- 2 and C-3 of the thioester substrates. After redution by the substrate, the dehydrogenase transfers reduing equivalents to another flavoprotein, ETF, whih ommuniates with the eletron transport hain at the ETF-oenzyme Q oxidoredutase reation. The experimental onditions were examined for assay of the new ayl-coa dehydrogenase ativity with ETF in plae of phenazine methosulfate. The reation was started by addition of phenazine methosulfate in the standard assay as desribed under Experimental Proedures. The effet of N- ethylmaleimide on the new ayl-coa dehydrogenase during preinubation was examined using a higher onentration of N-ethylmaleimide than that used for the standard assay. The ativity of the new enzyme was dereased to half after a 5- min preinubation at 5 mm N-ethylmaleimide in the absene of ayl-coa, but the ativity was ompletely retained in the presene of 30 PM palmitoyl-coa. Dihloroindophenol on- entration used in the standard assay reahed near saturation when the ativity was measured with ETF. Addition of FAD at 0 MM during the assay did not affet the ativity of the new enzyme. ETF was not inativated by short-time exposure to N-ethylmaleimide (). Therefore, the effet of ETF was indued under standard assay onditions, exept that the reation was started by adding ETF in plae of phenazine methosulfate. Fig. 5 summarizes the ativities of the new ayl- 0 0.5.o.5 ETF, p M FIG. 5. Effet of ETF. The ativities of the new enzyme (4 pg) and long-hain ayl-coa dehydrogenase (0 pg) were assayed with 30 pltm palmitoyl-coa in the presene of various onentrations of ETF as an eletron mediator to dihloroindophenol. 0, new enzyme; 0, long-hain ayl-coa dehydrogenase. CoA dehydrogenase and long-hain ayl-coa dehydrogenase with ETF as an eletron mediator to 2,6-dihlorolindophenol. Apparent K,,, values of the new enzyme and long-hain ayl- CoA dehydrogenase for ETF were 0.83 and.5 PM, respetively. The new enzyme ativity at the saturated level of ETF was about one-fourth of the value measured by the standard assay method using phenazine methosulfate, whereas the long-hain ayl-coa dehydrogenase ativity with ETF was.6-fold that measured using phenazine methosulfate. Apparent K,,, values of the new enzyme and long-hain ayl-coa dehydrogenase for phenazine methosulfate were 0.9 and.5 mm, respetively. Substrate Speifiity-Three straight-hain ayl-coa dehydrogenases are present in mammalian mitohondria with overlapping speifiities: long-, medium-, and short-hain substrates. Fatty aid hain length speifiity of the new enzyme differed from that of these three ayl-coa dehydrogenases, as summarized in Fig. 6. The new enzyme exhibited its highest ativity with palmitoyl-coa as substrate. No ativity was observed when ayl-coas with shorter than 8- arbon hain lengths were used. A very low ativity was found with substrates having 0- and 2-arbon hain lengths. This enzyme did exhibit signifiant ativity with substrates having 20-, 22-, and 24-arbon hain lengths. Therefore, we propose the name very-long-hain ayl-coa dehydrogenase to distinguish it from other ayl-coa dehydrogenases. A group of severe hereditary peroxisomal diseases in man has been deteted (26-30). One of the biohemial harateristis of these diseases is impairment in peroxisomal @-oxidation of very-long-hain fatty aids (>22 arbons), whih are presumed to be predominantly degraded in peroxisomes. The presene of two ativating enzymes is known. Long-hain ayl-coa synthetase is present in mirosomes, mitohondria, and peroxisomes; and very-long-hain ayl-coa synthetase is loated in mirosomes and peroxisomes, but not in mitohondria. However, even a highly purified long-hain ayl-coa synthetase preparation exhibited ativity with very-longhain fatty aids, albeit the ativity was low when ompared to the ativity with long-hain fatty aids. The findings of the presene of the new ayl-coa dehydrogenase and a new trifuntional protein desribed in the aompanying paper (2) suggest that studies need to be done to determine whether mitohondria have a subsidiary role in the oxidation of verylong-hain fatty aids. Effets of Detergents-The effets of various detergents were examined (Fig. 7). The new enzyme ativity was inreased in the presene of low onentrations of detergents in the reation mixture, but dereased in the presene of higher onentrations. The effets of the detergents may be partly related to the interation of the enzyme and the detergent beause the effets differed from those on long-hain ayl- CoA dehydrogenase. Effet of ph-as shown in Fig. 8, the enzyme ativity was higher at a higher ph for both enzymes. The effet of ph was muh more marked for the new enzyme. The onentrations of the buffers were fixed at 50 mm in this experiment. No marked hanges in the ph-ativity relationship were observed when the onentrations of the buffers were varied from 20 to 200 mm. Loalization of Enzyme-The mitohondrial frations of ontrol and DEHP-fed rat livers were subjeted to surose gradient entrifugation; and the palmitoyl-coa dehydrogenase ativities of the purified mirosomal, mitohondrial, and peroxisomal frations were determined after removal of longhain ayl-coa dehydrogenase with the antibody. This remaining ativity was onfirmed to be due to the new enzyme
032 Novel Very-long-hain Ayl-CoA Dehydrogenase A B FIG. 6. Veloityofayl-CoA dehydrogenase as funtion of substrate onentration. A, assays were arried out using 0.5 pg of new enzyme and various onentrations of the ayl- CoA substrates: lauroyl-coa (0), myristoyl-coa (O), palmitoyl-coa (0), stearoyl-coa (B), arahidoyl-coa (A), behenoyl-coa (A), and lignoeroyl-coa (0). B, omparison of the arbon hain length speifiities of the new enzyme (0) and long-hain ayl-coa dehydrogenase (0) at a fixed substrate onentration of 0 UM. 0 20 30 Ayl-CoA,PM 6 8 0 2 4 6 8 20 22 24 Carbon hain length A 9 200 00 Conentration, %(v/v) FIG. 7. Effet of detergents onayl-coadehydrogenase. Assays were arried out using 30 pm palmitoyl-coa as substrate in the presene of various onentrations of the detergent. A, long-hain ayl-coa dehydrogenase (5 pg); B, new enzyme (0.5 pg). 0, Tween 20; 0, Triton X-00; A, sodium holate; 0, heptyl thiogluoside; B, otyl thiogluoside. by titration with the antibody against the new enzyme. Most of the new enzyme ativity was reovered in the mitohondrial fration. A low level of the new enzyme ativity was found in the mirosomal and peroxisomal frations. These ativities were thought to be a ontamination of the mitohondria beause ratios of the dehydrogenase ativity to the glutamate dehydrogenase ativity were muh the same as those determined for the mitohondria. Nearly the same intensities of immunoblot signals for the new enzyme were observed for these three organelles when the amounts of the samples used were fixed to give the same glutamate dehydrogenase ativity. Subfrationation of the mitohondria was performed aording to the method of Lipsky and Pedersen (9). Most of the new enzyme ativity was reovered in the inner membrane fration. The new enzyme was onsidered to be loated in the inner membrane as dedued from a omparison to the distribution of marker enzymes: monoamine oxidase, adenylate kinase, ATPase, and glutamate dehydrogenase (data not shown). ^t I I I I I o 6.5 7.0 7.5 8.0 8.5 PH FIG. 8. Effet of ph on ativities of new enzyme and longhain ayl-coa dehydrogenase. Assays were arried out with 0.4 pg of new enzyme (0, A) and 4.5 pg of long-hain ayl-coa dehydrogenase (0, A) using 30 p~ palmitoyl-coa as substrate. Potassium phosphate (50 mm) (0,O) and Tris-C (50 mm) (A, A) were used. The presene of the new enzyme in various tissues was studied. As a marker enzyme for the mitohondria, we determined the mitohondrial enoyl-coa hydratase ativity for the following reasons. ) This enzyme is a member of the mitohondrial fatty aid @-oxidation yle. 2) The enzyme ativity is high and is readily differentiated from the peroxisomal bifuntional protein beause of its heat stability. 3) The ontent of this enzyme in the liver remained unhanged after the administration of DEHP. The ratios of the new dehydrogenase ativity to thenoyl-coa hydratase ativity was nearly the same for liver, kidney, heart, and leg musles. The ratio was inreased -2-fold only for liver after the administration of DEHP. The results of immunoblot analysis oinided with those of the enzyme ativity determination (data not shown). The new ayl-coa dehydrogenase is thought to be loated in the inner membranes of mitohondria of various tissues. This assoiation of the enzyme and the membrane may not be so lose sine -20% of the enzyme was found in the extrat of the frozen and thawed mitohondria in 50 mm potassium
Novel Very-long-hain Dehydrogenase Ayl-CoA 033 phosphate, ph 7.5. The mehanism of this assoiation may (95) J. Biol. Chem. 93, 265-275 be diret binding of the enzyme or indiret binding via some 3. Laemmli$ u. K. (lg70) Nature 2279 680-685 protein linked to the membrane. 4. Towbin, H., Staehelin, T., and Gordon, J. (979) Pro. Nutl. Aad. Si. U. S. A. 76, 4350-4354 Of New Enzyme"The new enzyme showed si@ifiant 5. Ikeda, Y., Dabrowsky, C., and Tanaka, K. (983) J. Biol. Chem. ativity toward long- and very-long-hain ayl-coas. Al- 258, 066-076 though its preise role in fatty oxidation is presently unlear, 6. Husain, M., and Steenkarnp, D. J. (983) Biohem. J. 209, 54- the possibility is raised that this enzyme is involved in the 545 mitohon&ial oxidation of very-long-hain fatty aids. If SO, 7. de Duve,., Pressman, B.., Gianetto, R., Wattiaux, R., and Applemans, F. (955) Biohern. J. 60,604-67 the view that very-long-hain fatty aids are 8. Gear, A. R. L., Albert, A. D., and Bednarek, J. M. (974) J. Biol. preferentially oxidized by peroxisomes may have to be re- Chem. 249,6495-6504 examined. 9. Lipsky, N., G., and Pedersen, P. L. (982) J. Biol. Chern. 257, 473-48 Aknowledgment-We thank M. Ohara for helpful omments. 20. Ozasa, H., Fumta, S., Miyazawa, S., Osumi, T., Mori, M., Miura, S., and Tatibana, M. (984) Eur. J. Biohem. 44,453-4586 REFERENCES 2. Uhida, Y., Izai, K., Orii, T., and Hashimoto, T. (992) J. Biol. Chem. 267, 034-04. FuWta, s., Miyazawa, s., and Hashimoto, T. (98) J. Biohem. 22. Miyazawa, S.., Hayashi, H., Hijikata, M., Ishii, N., Furuta, S., (Tokyo) 90, 739-750 Kagamiyama, H., Osumi, T., and Hashimoto, T. (987) J. Bwl. 2. Furuta, S., Miyazawa, S., Osumi, T., Hashimoto, T., and Ui, N. Chem. 262,83-837 (980) J. Biohem. (Tokyo) 88, 059-070 23. Ishii, N., Hijikata, M., Osumi, T., and Hashimoto, T. (987) J. 3. Osumi, T., and Hashimoto, T. (980) Arh. Biohem. Biophys. Biol. Chem. 262,844-850 203,372-383 24. Hijikata, M., Ishii, N., Kagarniyama, H., Osumi, T., and Hashi- 4. Miyazawa, S., Osumi, T., and Hashimoto, T. (980) Eur. J. moto, T. (987) J. Bwl. Chem. 262,85-858 Biohem. 03,589-596 25. Massey, V. (959) Biohem. Biophys. Ata. 34, 255-256 5. Steinman, H. M., and Hill, R. L. (975) Methods Enzyrnol. 35, 26. Shutgens, R. B. H., Heymans, H. S. A., Wanders, R. J. A., van 36-5 der Bosh, H., and Tager, J. M. (986) Eur. J. Pediatr. 44, 6. Lynen, F., Henning, U., Bublitz, C., Sorbo, B., and Kroplin- 430-440 Rueff, L. (953) Biohem. 2. 330,269-295 27. Moser, H. W. (987) Dev. Neurosi. 9, -8 7. Wieland, T., and Rueff, L. (953) Angew. Chem. Int. Ed. End. 28. Wanders, R. J. A., Heymans, H. S. A,, Shutgens, R. B. H., Barth, 65,86-87 P. G., van den Bosh, H., and Tager, J. M. (988) J. Neurol. 8. Seubert, W., Lamberts, I., Kramer, R., and Ohley, B. (968) Si. 88, -39 Biohim. Biophys. Ata 64, 498-57 29. Lazarow, P. B., and Moser, H. W. (989) in The Metaboli Basis 9. Osumi, T., Hashimoto, T., and Ui, N. (980) J. Biohem. (Tokyo) of Inherited Disease (Sriver, C. R., Beaudet, A. L., Sly, W. S., 87, 735-746 and D., Valle, eds) 6th Ed., pp. 479-509, MGraw-Hill Book 0. Osumi, T., and Hashimoto, T. (979) Biohem. Biophys. Res. Co., New York Commun. 89,580-584 30. Moser, H. W., and Moser, A. B. (989) in The Metaboli Basis of. Markwell, M. A. K., Haas, S. M., Bieber, L. L., and Tolbert, N. Inherited Disease (Sriver, C. R., Beaudet, A. L., Sly, W. S., E. (978) Anal. Biohem. 87, 206-20 and Valle, D., eds.) 6th Ed., pp. 5-532, MGraw-Hill Book 2. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Co., New York