The peroxisomal β-oxidation enzyme system of rat heart. Basal level and effect of the peroxisome proliferator clofibrate

The peroxisomal β-oxidation enzyme system of rat heart. Basal level and effect of the peroxisome proliferator clofibrate

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  See discussions, stats, and author profiles for this publication at: The peroxisomal β -oxidation enzyme systemof rat heart. Basal level and effect of theperoxisome proliferator clofibrate  Article   in  Biochimica et Biophysica Acta · December 1994 DOI: 10.1016/0304-4165(94)90042-6 · Source: PubMed CITATIONS 17 READS 11 3 authors , including:Torgeir FlatmarkUniversity of Bergen 275   PUBLICATIONS   7,753   CITATIONS   SEE PROFILE All content following this page was uploaded by Torgeir Flatmark on 25 November 2014. The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the srcinal documentand are linked to publications on ResearchGate, letting you access and read them immediately.  ELSEVIER Biochimica et Biophysica Acta 1201 (1994) 203-216 BB, Biochi~ic~a et Biophysica A~ta The peroxisomal fl-oxidation enzyme system of rat heart. Basal level and effect of the peroxisome proliferator clofibrate Jon Kvannes, Thor S. Eikhom, Torgeir Flatmark Department of Biochemistry, University of Bergen, ,4rstadveien 19, N-5009 Bergen, Norway Received 16 November 1993; revised 28 March 1994 Abstract Peroxisomes, isolated from homogenates of rat hearts (myocard), contain a fl-oxidation enzyme system which is indistinguishable from that found in liver, but the total capacity of/3-oxidation is only 0.8% of the liver value (expressed per g of tissue). Fatty acyl-CoA oxidase was assayed by an H 202 based fluorescent assay avoiding important interfering side reactions. The presence of polypeptides with electrophoretic and immunological properties similar to the /3-oxidation enzymes of liver peroxisomes, was demonstrated by immuno- blotting using polyclonal antibodies. The level of 72 and 52 kDa subunits of fatty acyl-CoA oxidase (FAO), quantitated by an anti-FAOl_16 peptide antibody, was only 1% of the level in liver (expressed per g of tissue). Immunoblots of one-dimensional (l-D) SDS-PAGE of rat heart and liver peroxisomal fractions revealed a 60 kDa subunit of the fatty acyl-CoA oxidase in addition to the known 72 and 52 kDa subunits. Immunoblots of two-dimensional (2-D) IEF/SDS-PAGE revealed that all subunits are strongly basic polypeptides, with a microheterogeneity, which probably represents deamidations of the polypeptides. The 2-D immunoblot also revealed another group of polypeptides with M r 72 kDa of less basic isoelectric point, possibly representing an isoform of fatty acyl-CoA oxidase. Substrate specificity studies revealed the highest Vma values with Clo-C12. For the very long-chain fatty acids C2o-C24, the monoenes revealed much higher Vm~ values than the saturated fatty acids. Administration of the classical peroxisome proliferator clofibrate resulted in a similar increase in the fatty acyl-CoA oxidase activity and the 72 and 52 kDa subunits of FAO in the heart. The response (activity) was found to change from 2.2-fold increase in young (34 days) to ll.l-fold increase in adult (76 days) rats. In contrast to liver, where the ratio of the increase in FAO mRNA to the increase in FAO activity was about 4, this ratio in heart was about 0.5. Keywords: Peroxisome; Beta-oxidation; Enzyme induction; Enzyme stabilization; Clofibrate; Immunoblotting; (Heart); (Liver); (Rat) 1. Introduction It is now well established that peroxisomes of rat liver have the capacity of shortening fatty acids by a /3-oxida- tion mechanism which is different from that of mito- chondria [1]. This shortening system seems to be particu- larly important in the metabolism of very long-chain fatty Abbreviations: Anti-Px, polyclonal rabbit antiserum against highly purified rat liver peroxisomes; BSA, bovine serum albumin; Brij 58, polyoxyethylene 20 cetylether; ECL, enhanced chemi-luminescence; DMSO, dimethylsulfoxide; FAO, fatty acyl-CoA oxidase (EC; IEF, isoelectric focusing; IHD, A3, A2-enoyl-CoA somerase, 2-enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase; HRP, horseradish peroxi- dase; NEM, N-ethylmaleimide; 4-OH-PAA, p-hydroxyphenylacetic acid; PBS, phosphate-buffered saline; PBS-T, phosphate-buffered saline con- taining Tween 20; PHFO, partially hydrogenated fish oil; SDS-PAGE, sodium dodecylsulfate polyacrylamide gelelectrophoresis * Corresponding author. Fax: + 47 55 206400. 0304-4165/94/$07.00 © 1994 Elsevier Science B.V. All fights reserved SSDI 0304-4165(94)00072-6 acids, such as 22:1, which are poorly oxidized by mito- chondria (for review, see [2]). The liver peroxisomes are also responsible for the shortening of the side-chain of cholesterol in bile acid synthesis (for review, see [3]), and it has been shown that long-chain dicarboxylic acids are also shortened by the same peroxisomal system [4]. In a number of other tissues peroxisomes have been reported [5-12] to possess a fl-oxidation enzyme system similar to that in liver, including the 'microperoxisomes' of the brown adipose tissue of rat and guinea pig [7-10] and the heart muscle of guinea pig [5] and rat [11,12]. In the heart muscle the conclusions were based on measurement of low specific activities of palmitoyl-CoA oxidase [5] or of cyanide insensitive palmitoyl-CoA dependent dehydro- genase [12]. Some inherent problems of these spectropho- tometric assays have to be considered, however, particu- larly in tissues of low specific activity and significant contribution of interfering reactions, e.g., a time-dependent  204 J. Kvannes et aL /Biochimica et Biophysica Acta 1201 1994) 203-216 decrease in light scattering induced by addition of the substrate and reoxidation of NADH [10] and the presence of interfering reducing agents [13]. Therefore, an assay of the individual enzymes of the pathway is required in order to get a more clear idea of the actual levels and the potential capacity of fl-oxidation in the peroxisomes of the heart muscle. Administration of the hypolipidemic drug clofibrate has been reported to result in a small (2.7-fold) increase in the capacity for cyanide insensitive palmitoyl-CoA dependent dehydrogenase activity in rat heart peroxisomes [12] and to result in an increased chain shortening of 22:1 fatty acids in the perfused rat heart [14]. It has also been reported to induce a proliferation of the 'microperoxisomes' in the heart [15]. In the present study a comparison has been made of peroxisomal fractions isolated from the heart and liver of male rats. Based on measurements of enzyme activities, SDS-PAGE electrophoresis and immunoblots, it is con- cluded that the peroxisomes of rat heart muscle contain a /3-oxidation enzyme system which is indistinguishable from that found in liver, but the specific activity and total capacity of /3-oxidation are much lower. Treating the rats with clofibrate for 10 days resulted in a significant in- crease in fatty acyl-CoA oxidase (FAO) activity in both heart and liver. An age variation study revealed that in the heart the basal FAO activity decreased with age whereas the response to clofibrate administration increased, includ- ing the level of the FAO p72 and p52 subunits. A similar age-dependent response was observed in the liver. 2. Materials and methods 2.1. Chemicals Poly(vinylpyrrolidone)-coated colloidal silica particles (Percoll) and Sepharose 4B were supplied by Pharmacia Fine Chemicals AB (Uppsala, Sweden). 125I-labelled pro- tein A (affinity purified) was from Amersham (UK). HRP-conjugated goat anti-rabbit IgG antibody (GAR-HRP) was from Bio-Rad (Richmond, CA). p-Hydroxyphenyl- acetic acid, trypsin (treated with L-l-tosylamide-2-phenyl- ethylchloromethyl ketone (TPCK) to inhibit chymotrypsin) and most other fine chemicals were supplied by Sigma (St. Louis, MO). Horseradish peroxidase (isoenzyme C) was obtained from Boehringer-Mannheim (Germany), bovine serum albumin (fatty acid free) from ICN ImmunoBiologi- cals (Costa Mesa, CA), sodium azide, perhydrol and DMSO from Merck (Darmstadt, Germany), N-ethylmaleimide from Koch-Light (Colnbrook, UK), and sucrose from BDH Limited (Poole, UK). Clofibrate (ethyl-p-chloropheno- xyisobutyrate) was a gift from Weiders Farmascytiske A/S (Oslo, Norway). Ponceau S solution was from Serva (Heidelberg, Germany). 2.2. Animals Male Wistar rats were obtained from M~llegaard (Den- mark). They were given daily subcutaneous injections of 65 mg undiluted clofibrate per 100 g body weight for 10 days, and were fed a standard pellet diet (R3 from Ewas A/B, Stidert[ilje, Sweden) ad libitum. At the time of killing, the body weight of the control group was 150 + 2.3 g (mean + S.E.), corresponding to an age of 41 days. The body weight of the clofibrate group was 132 + 3.6 g. For the studies on age variation three different age groups were used. Their age at the time of killing was 34, 55 and 73 days, respectively. These were treated according to the same regimen; but clofibrate was diluted in soybean oil before injection. The youngest group of rats were still weanling until killing. 2.3. Preparation of a peroxisome enriched fraction from rat heart Rat heart peroxisomal fractions were prepared essen- tially as described in [11] using a homogenization medium containing 0.25 M sucrose, 0.5 mM EGTA and 5 mM Hepes (pH 7.4). An RC-5B refrigerated superspeed cen- trifuge and a HB-4 rotor rmi n = 6.2 cm and rma x = 14.4 cm) from Sorvall was used for subcellular fractionations. A centrifugal effect of 8.55 • 108 rad 2 s -1 (11500 × g, 13 min) was used to sediment a combined fraction containing debris, nuclei and mitochondria; and 6.12.109 rad 2 s -1 (11500 × g, 93 min) to sediment a peroxisomal fraction. The peroxisome enriched fraction from each heart was resuspended in 1 ml homogenization medium per g my- ocard, and samples were immediately stored in liquid nitrogen. 2.4. Isopycnic gradient centrifugation The heart peroxisomal fraction, obtained by differential centrifugation, was subjected to equilibrium density cen- trifugation in a gradient self-generated from a homoge- neous solution of 35% (v/v) polyvinylpyrrolidone-coated colloidal silica particles (Percoll) in 0.25 M sucrose, pH adjusted to 7.2 by sulfuric acid [11]. A Beckman L8 centrifuge with a 50 Ti rotor was used rmi n = 3.8 and rma x = 8.2 cm). The sample (in a total volume of 0.3 ml) was loaded on top of the Percoll-0.25 M sucrose solution, and after centrifugation (26 800 × g, 75 min at 4°C) the gradient was fractionated as described [11]. The density of each fraction (0.26 or 0.52 ml) collected from the gradient, was determined gravimetrically at 20°C. Samples were stored as small aliquots at -25°C for assay of enzyme activities (see below). The thiolase activity attributed to peroxisomes and contaminating mitochondria was esti- mated by a linear combination of marker enzymes specific for the two organelles [16].  J. Kvannes et al. / Biochimica et Biophysica Acta 1201 1994) 203-216 205 2.5. Preparation of a peroxisome enriched fraction from rat liver 1 g of liver tissue was fine-minced in ice-cold sucrose medium and homogenized in a Potter-Elvehjem glass-teflon homogenizer [17]. The HB-4 rotor and RC-5B refrigerated superspeed centrifuge were used for centrifugation. For control rats a subcellular fraction, enriched in peroxisomes, was obtained by differential centrifugation using the cen- trifugal effect of 4.54.107 rad 2 s- 1 (719 × g, 10 min) and 2.85.109 rad 2 s -1 (11500 × g, 30 min) for the sedimen- tation of debris and nuclear material and a large granule fraction containing the total peroxisomal population, re- spectively [18,19]. Samples were stored at -80°C. Liver peroxisomes of clofibrate treated rats show a high degree of polydispersity [18] and are therefore centrifuged at a higher centrifugal effect. The debris and nuclear fraction was sedimented as above (HB-4 rotor, 719 X g for 10 min). The large granular fraction was then sedimented using a time integral of 1.065 • 101° rad 2 s- 1 (26 200 x g, 56 min) in a Beckman 50.2 Ti rotor (rmi n = 5.44 cm and rma x = 10.79 cm) [18] and a Beckman L8-70 preparative ultracentrifuge. For immunization of rabbits a highly purified fraction of rat liver peroxisomes was obtained by a combination of differential centrifugation and gradient centrifugation in a Metrizamide gradient [20] using the Beckmann VTi 50 vertical rotor. 2.6. Antibodies A polyclonal rabbit antiserum directed against highly purified rat liver peroxisomes was obtained by injection (in several intracutaneous sites and the inguinal lymph nodes) of 2 mg of total peroxisomal proteins mixed with Freund's complete adjuvant, followed by later intramuscular injec- tions of 1 mg of peroxisomal proteins mixed with Freund's incomplete adjuvant. The rabbits were bled 10-12 days after each injection, erythrocytes were sedimented by cen- trifugation (10000 × g, 20 min) and the serum was then ultracentrifuged (100000 X g, 1 h) and frozen in aliquots at -80°C. For most immunoblotting experiments the anti- serum (referred to as anti-Px) was used without further purification. Immunoblots of control rat liver peroxisome enriched fractions detected by this antiserum clearly re- vealed 9 immunoreactive bands in the M r region 43-85 kDa (Fig. 1, lane 1). An affinity purified antibody directed against an oligopeptide, K N P D L R K E R A S A T F N P, corresponding to the 16 amino acids of the N-terminal sequence of FAO [21] (the N-terminal Met was replaced by Lys), was obtained from the anti-Px anti-serum, i.e., the IgG fraction obtained by ammonium sulfate fractionation [22]. The peptide was coupled to CNBr-activated phenyl- sepharose beads [23], and the monospecific antibody was adsorbed by a standard protocol and eluted with 0.1 M 1 2 3 4 5 6 Fig. 1. Identification of several protein bands which appear in autoradio- grams of 125I-Protein A labelled immunoblots of rat heart peroxisome enriched fractions detected with a polyclonal rabbit antiserum raised against highly purified rat liver peroxisomes (anti-Px). After staining with Ponceau S individual lanes were cut out and cleaved vertically. Then one half was immunodetected with anti-Px and the other half with specific antiserum against a defined peroxisomal protein. When blot processing was complete, the two halves were mounted together for autoradiography. Lane 1 shows a lane with a liver peroxisome enriched fraction for comparison; this was detected in one piece by anti-Px. Lanes 2-6 are heart peroxisomal fractions. Lane 2 shows two lane halves detected separately with anti-Px just to demonstrate the precision of the method. Individual polyclonal rabbit antisera were obtained against 52 kDa FAO subunit (lane 3), the trifunctional enzyme IHD (lane 4), catalase (lane 5) and a 69 kDa membrane protein (lane 6), all isolated from rat liver. glycine-HC1 (pH 2.8). This IgG fraction is referred to as anti-FAO1_16 peptide antibody. The IgG fraction of a polyclonal rabbit antiserum against rat liver catalase was obtained from Prof. Dr. Dr: A. V61kl, Heidelberg, Germany. Polyclonal rabbit antisera against 52 kDa FAO subunit, the trifunctional enzyme IHD and a 69 kDa membrane protein from rat liver peroxisomes were obtained from Dr. W.W. Just, Heidelberg, Germany (the respective proteins eluted from SDS-PAGE gels were used for immunization of rabbits). The antibodies are referred to as anti-52 kDa, anti-IHD and anti-p69, respectively. 2.7. Electrophoresis and immunoblotting SDS-PAGE electrophoresis of peroxisome enriched fractions (Fig. 1) from rat liver and heart was performed at 180 V (5-6 h) in a 10% (w/v) polyacrylamide gel [24], 1 mm thick, using a gel electrophoresis apparatus (GE 2/4) from Pharmacia (Sweden) with water cooling. Electroblot- ting was performed using a Genie blotter with plate elec- trodes (from IDEA Scientific, Minneapolis, MN). The protein was transferred to a nitrocellulose membrane of 0.2 /zm pore diameter (BA 83 from Schleicher and Schuell, Dassel, Germany) in a buffer containing 48 mM Trizma base, 39 mM glycine (pH 9.2) and 20% (v/v) methanol [25]. Transfer of proteins in the relevant M r range was complete after 5.5 h at 300 mA. The nitrocellulose mem- brane was stored overnight in Tris-buffered saline (20 mM Tris base (pH 7.6 with HC1)/137 mM NaC1) with 0.02% (w/v) NaN 3. The protein bands were reversibly stained with Ponceau S solution, the relevant M r range cut out and  206 J. Kvannes et al. /Biochimica et Biophysica Acta 1201 1994) 203-216 used for immunodetection. This consisted of 1 h blocking with phosphate-buffered saline (0.1 M sodium phosphate (pH 7.4)/0.1 M NaC1-PBS) containing 0.1% (v/v) Tween-20 (PBS-T) and 2.5% (w/v) low fat dry milk powder at room temperature, followed by 1 h incubation with anti-Px (dilution 1:150) in a similar solution; the other antisera were diluted 1:200 or 1:300. Thereafter the membrane was washed by several changes of PBS-T and incubated for 1 h in a25I-labelled protein A (0.1 /xCi/ml final concentration in PBS-T). It was then washed in several changes of PBS-T and finally overnight at 4°C in PBS to obtain a low background. The blot was dried and exposed to /3-max film (Amersham) for 1-10 days at room temperature. Films were developed manually and immunoreactivity was quantitated by densitometric scan- ning of the autoradiograms using an Ultroscan XL laser densitometer from LKB Produkter AB (Bromma, Sweden). 2-D IEF/SDS-PAGE was performed as described in [26]. The second dimension SDS-PAGE gel was 2 mm thick, and electroblotting was carried out for 7.5 h at 300 mA. The enhanced chemiluminescence (ECL) system from Amersham was used for the immunodetection using an affinity purified HRP-conjugated goat anti-rabbit IgG anti- body (GAR-HRP) diluted 1:2000 in PBS-T with 2.5% (w/v) dry milk as the second antibody, followed by washing in PBS-T and detection with the ECL reagents according to the standard protocol. The M, of the different protein bands were determined by comparison with a protein standard kit from Pharmacia. by adding palmitoyl-CoA (60 /xM) from a stock solution (2 mM in 35% (v/v) DMSO). The very long-chain fatty acids were dissolved in pure DMSO for solubility reasons. Calculation of Vma x values was performed by non-linear regression analysis [27]. Catalase (EC activity was determined spec- trophotometricaUy at 25°C using the wavelength 265.5 nm [28]. Thiolase activity was assayed at 25°C by measuring the specific absorption band at 233 nm due to the thioester bond [1]. The specific peroxisomal activity of thiolase was corrected for the small mitochondrial contribution as esti- mated by the malate dehydrogenase activity using highly purified mitochondria as a reference. Malate dehydrogenase (EC activity was mea- sured spectrophotometrically according to Bergmeyer [29]. NADPH:cytochrome c reductase was assayed according to Sottocasa et al. [30]. Protein was determined by the Bradford procedure [31] with Bio-Rad dye reagent using bovine serum albumin as a standard. 2.9. cDNA probes The recombinant plasmids PMJ 115 and PMJ 26 con- tain respectively the cDNA sequences of peroxisomal acyl- CoA oxidase and the trifunctional enzyme IHD in the PstI site of PBR 322 [21,32,33]. The recombinants were lin- earized with PstI [17]. 2.8. Assay of marker enzymes and other analytical meth- ods Cyanide insensitive palmitoyl-CoA dependent reduction of NAD ÷ was determined spectrophotometrically as de- scribed by Lazarow [1] except that the incubation medium contained 0.01% (w/v) Brij 58 and 50 /xM FAD, and the concentration of palmitoyl-CoA was 40 /xM [18,19]. The reaction temperature was 25°C (pH 8.0), and no bovine serum albumin was present in the incubation medium [19]. Fatty acyl-CoA oxidase (FAO, EC activity was assayed by a coupled peroxidatic reaction, using horseradish peroxidase (HRP) and p-hydroxyphenylacetic acid (4-OH-PAA) as electron donor [13]. The amount of fluorescent oxidation product was assayed by a LS 50 Luminescence Spectrometer from Perkin Elmer (Beacon- sfield, UK). Settings were Aex 318 nm and Aem 405 nm, excitation ban@ass 2.5 nm and emission ban@ass 10 nm and 5 nm for high and low sensitivity, respectively. The standard incubation mixture contained in a total volume of 2.06 ml: 50 mM Tris-HC1 (pH 8.3), 0.01% (w/v) Brij 58, 1 mM 4-OH-PAA, 10 mM sodium azide, 0.5 mg/ml BSA, 100/xM NEM, 25/zM FAD and 2 /zg/ml (2 U/ml) HRP plus indicated amounts of the peroxisomal fraction. The NEM concentration was 50 /xM for liver and 100 /xM for heart peroxisomal fractions [13]. The reaction was started 2.10. Extraction of total RNA and assay of specific mRNA Rat liver (0.5 g from each of four rats) or rat heart (approx. 1.5 g from four animals) was homogenized and RNA was extracted and purified as previously described [34]. rRNA was intact as judged by agarose gel elec- trophoresis. Labelling of cDNA, hybridization, autoradiog- raphy and quantitation were essentially as previously de- scribed [17]. The membranes were washed according to the Amersham protocol with a final wash in 0.1 X SSPE, 0.1% SDS at room temperature (5 X SSPE is 0.9 M NaC1, 0.05 M sodium phosphate (pH 7.7), 5 mM Na2EDTA). 2.11. Presentation of results Data are presented as mean + S.E. if not otherwise indicated, and analyzed statistically by Student's unpaired t-test. 3. Results 3.1. Isolation of heart peroxisomes The s values and conditions for isolation of rat heart peroxisomes were first described by Norseth et al. [11].
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