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Activation of 3-methyl-branched fatty acids in rat liver
Pergamon
0020-711X(94)E0024-W
ACTIVATION
Inr. J. Biochem. Vol. 26. No. 9, pp. 1095-1101, 1994 Copyright cm 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0020-7 I I X/94 $7.00 + 0.00
OF 3-METHYL-BRANCHED IN RAT LIVER
FATTY
ACIDS
J. C. T. VANHOOREN,’ S. ASSELBERGHS,’ H. J. EYSSEN,’ G. P. MANNAERTS’ and
P. P. VAN VELDHOVEN’* ‘Katolieke Universiteit Leuven, Campus Gasthuisberg-Afdeling Leuven [721. 32-16-345802; Fox 32-16-3456991 and lRega Instituut, Belgium (Received IO December
Farmakologie, Herestraat, B-3000 Minderbroederstraat, B-3000 Leuven,
1993)
Abstract--l. Subcellular fractionation of rat liver revealed that 3-methylmargaric acid, a monobranched phytanic acid analogue, can be activated by mitochondria, endoplasmic reticulum and peroxisomes. 2. Indirect data (effects of pyrophosphate and Triton X-100) suggested that the peroxisomal activation of 3-methylmargaric, 2-methylpalmitic and palmitic acid is catalyzed by different enzymes. 3. Despite many attempts, column chromatography of solubilized peroxisomal membrane proteins so far did not provide more conclusive data. On various matrices, lignoceroyl-CoA synthetase clearly eluted differently from the synthetases acting on 3-methylmargaric, 2methylpalmitic and palmitic acid. The latter three however, tended to coelute together, although often not in an identical manner.
INTRODUCTION
Fatty acids are metabolized by two main pathways: degradation via j-oxidation and esterification with glycerol-3-phosphate or dihydroxyacetone-phosphate resulting in the formation of glycerolipids. Before a fatty acid can enter these pathways, it needs first to be activated to its CoA-ester, a reaction that is catalyzed by acyl-CoA synthetases. Long chain acyl-CoA synthetases, acting on long straight chain fatty acids, are found in the mitochondrial outer membrane, the peroxisomal membrane and the membranes of the endoplasmic reticulum (Krisans et al., 1980; Mannaerts er al., 1982; Shindo and Hashimoto, 1978). The enzymes in the three organelles appear to be identical with respect to their catalytic, molecular and immuno-chemical properties (Hashimoto, 1982; Miyazawa et lzl., 1985; Tanaka et al., 1979) and they seem to have identical amino acid *To whom correspondence should be addressed. Abbreviations: DTT, dithiothreitol; 3-MMA, 3-methylmargaric acid (3-methylheptadecanoic acid); 2-MPA, 2-methylpalmittc acid; Mops, 4-morpholinopropanesulfonic acid.
sequences (Suzuki et al., 1990). There is indirect evidence that these synthetases are also responsible for the activation of long 2-methylbranched fatty acids such as the synthetic 2-MPA (Vanhove et al., 1991) and the naturally occurring pristanic (2,6,10,1Ctetramethylpentadecanoic) acid (Wanders et al., 1992). Very long chain fatty acids such as lignoceric (tetracosanoic) acid are activated by a separate enzyme present in peroxisomal and endoplasmic reticulum membranes (Singh and Poulos, 1988). Evidence for the existence of a separate very long chain acyl-CoA synthetase is based mainly on indirect arguments: absence from mitochondria (Singh and Poulos, 1988) and differential effects of detergents and inhibitors on the activation of long and very long chain fatty acids (Lageweg et al., 1991; Singh and Poulos, 1988; Singh et af., 1992a). 3-Methyl-branched fatty acids such as the synthetic 3-MMA and the naturally occurring phytanic (3,7,11,15tetramethylhexadecanoic) acid cannot be degraded directly via /?-oxidation. They are first oxidatively decarboxylated in a poorly characterized reaction called a-oxidation, that yields CO, and a 1095
J. C. T. VANHOOREN et al
1096
2-methylbranched fatty acid (ZMPA in the case of 3-MMA and pristanic acid in the case of phytanic acid) (Avigan et al., 1966; Tsai et al., 1969) which is then B-oxidized predominantly in peroxisomes (Vanhove et al., 1991; Van Veldhoven et al., 1993). The subcellular localization of cr-oxidation and whether a-oxidation requires the prior conversion of the 3-methyl branched fatty acid to its CoA derivative remain controversial. Studies on a-oxidation in homogenates and subcellular fractions from rat liver and human fibroblasts have shown different effects of CoA ranging from strong inhibition over no effect to marked stimulation (Huang et al., 1992; Muralidharan and Muralidharan, 1987; Muralidharan and Kishimoto, 1984; Skjeldal and Stokke, 1987; Singh et al., 1992b). Activation of phytanic acid-whether involved in a-oxidation or not-has been shown to occur, however (Muralidharan and Muralidharan, 1986). Moreover, in Refsum’s disease, in which cr-oxidation is deficient, accumulated phytanic acid is present in esterified form in triglycerides and phospholipids, implying a prior activation of the branched fatty acid (Molzer et al., 1979; Yao and Dyck, 1987). In a crude cell fractionation study carried out by Muralidharan and Muraldiharan (1986) phytanoyl-CoA synthetase activity was detected in mitochondrial and microsomal fractions from rat liver, but its presence in other cell organelles such as peroxisomes were not examined. We now report that 3-methyl-branched fatty acids are activated not only by mitochondria and endoplasmic reticulum but also by peroxisomes. By means of column chromatography of solubilized peroxisomal membrane proteins, we further tried to reveal the identity of the acyl-CoA synthetases acting on 3-methylbranched, 2-methyl-branched, long straight chain and very long straight chain fatty acids, the four activities known now to reside in peroxisomes. MATERIALS
AND
METHODS
Materials 2-MPA, [I -‘4C]-2-MPA (35 mCi/mmol sp. radioact.), 3-MMA and [ 1-‘4C]-3-MMA (54 mCi/mmol sp. radioact.) were synthesized as described before (Vanhove et al., 1991; Huang et al., 1992). [l-‘4C]-lignoceric acid (49 mCi/mmol sp. radioact.) was prepared according to Singh and Poulos (1988). [1-‘4C]palmitic acid (57 mCi/mmol) was purchased
from New England Nuclear, Bad Homburg, Germany. Coenzyme A, Percoll, Phenyl Sepharose (fast flow) and Blue Sepharose were purchased from Pharmacia Belga, Brussels, Belgium; ATP, ol-cyclodextrin and Thesit (research grade) were obtained from Boehringer Mannheim, Heidelberg, Germany. Nycodenz was from Nycomed, Oslo, Norway. Prepacked hydroxylapatite columns (Econo-Pat HTP cartridge, 5 ml) were from BioRad, Richmond, California, U.S.A. Cell fractionation Homogenates were prepared from livers of male Wistar rats in 0.25 M sucrose-l mM DTT-1 mM EDTA pH 7.2-0.1% (v/v) ethanol and fractionated as described before (Declercq et al., 1984). The light mitochondrial fraction, enriched in peroxisomes and lysosomes, was subfractionated by means of centrifugation in iso-osmotic self-generating Percoll gradients or discontinuous Nycodenz gradients (Vanhove et al., 1991). Highly purified peroxisomes, used for the preparation of membranes, were obtained exactly as described before (Verheyden et al., 1992). In order to increase the yield of peroxisomes, livers from rats kept for two weeks on a standard diet containing 0.3% (w/v) clofibrate were used. Marker enzymes and protein were measured as published before (Declercq et af., 1984; Verheyden et al., 1992). Preparation membranes
and solubilization of peroxisomal
Highly purified peroxisomes obtained as described above were diluted to a protein concentration of approx. 3 mg/ml in 10 mM pyrophosphate buffer pH 9.0- 1 mM EDTA- 1 mM DTT and sonicated 4 times for 15 set with intervals of 15 set at 4°C. This treatment disrupts the peroxisomal membranes and releases the matrix proteins and most of the peripheral membrane proteins (Van Veldhoven et al., 1987). After centrifugation for 1 hr at 100,OOOg at 4’C the membrane pellet was resuspended (final protein concentration approx. 1 mg/ml) in 50mM Tris-HCl buffer pH 7.2-0.5% (w/v) Thesit-0.5 M NaCl, and allowed to stand at 4’C for 30min. In order to prevent the acylCoA synthetases from inactivation the solubilization buffer also contained 5 mM ATP-3 mM MgCl,- 1 mM EDTA- 1 mM DTT. After centrifugation (100,OOOg for 1 hr at 4°C) the solubilized integral membrane proteins were recovered in the supernatant.
Activation
Activation
of fatty
of 3-methyl-branched
acids
The activation of palmitic, 2-MPA and 3MMA by homogenates and subcellular fractions was measured by adding an aliquot (50 p I) of homogenates or subcellular fractions, diluted in homogenization medium, to 200 ~1 of reaction mixture. Final concentrations were 50 mM Mops-NaOH buffer pH 7.4, 4mM ATP, 2.4 mM MgCl,, 2 mM DTT, 0.4 mM CoA, 50 PM [l-‘4C]-fatty acid (2,220-l 1,100 dpm/nmol sp. radioact.) Reactions were incubated for 3 min at 37°C and terminated by the addition of 2 ml of isopropanol/O.I N HCl (l/l-v/v) and phase-separated by adding 4 ml of petroleumether (b.p. 40-60°C). Part of the aqueous phase was counted for radioactivity. Lignoceroyl-CoA synthetase activity in the solubilized peroxisomal fractions and in the column fractions was measured as described by Singh and Poulos (1988). An aliquot (50 ~1) of the fractions, diluted in homogenization or elution buffer, was added to a 150 ~1 reaction mixture containing 50 mM Tris-HCl buffer pH 8.0-5 mM ATP-2 mM MgCI,-150 PM CoA- 300 p M DTT- 5 p M [ 1 - “C]-lignoceric acid: 49 ~Ci/~rnmol (sp. radioact.)-2.5 mg r-cyclodextrin/ml and incubated for 6 min at 37°C. Activation of palmitic, 2-MPA and 3-MMA in the solubilized peroxisomal membrane fractions or the column fractions was measured under the same conditions except that the substrate concentration was increased to
fatty acids
1097
cl-cyclodextrin was 50pM and omitted. Reactions were terminated by the addition of 20 ~1 of 10 M HCl and 1.5 ml of iso(40/ 1O/ l-v/v/v) propanol/heptane/ 10 M HCl and phases were separated by adding 1.5 ml of heptane and 1 ml of water. The aqueous phase was washed twice with 2 ml of heptane and counted for radioactivity.
RESULTS
AND DISCUSSION
In whole liver homogenates the activation of 3-MMA was linear with time for 3 min and proceeded linearly with protein contents up to 75 pg per assay. Kinetic analysis showed an almost first order reaction at substrate concentrations up to 50 PM but above 100 PM severe substrate inhibition was observed. At 50 p M of substrate, the activity in liver homogenates was 2.75 + 0.34 pmol/min.g of liver (n = 4). Analysis of the subcellular fractions, obtained by differential centrifugation, demonstrated that the 3-methylmargaroyl-CoA synthetase activity displayed a multisubcellular localization, similar to that previously described for palmitoyl-CoA synthetase (Krisans et al., 1980; Mannaerts et al., 1982; Shindo and Hashimoto, 1978) 2-methylpalmitoyl-CoA synthetase (Vanhove et al., 1991) and pristanoyl-CoA synthetase (Wanders et al., 1992). The major part of the synthetase activity was recovered in the heavy mitochondrial and microsomal fractions (see _ D
E t
I
IIII
N I 0
I
I
MLP
S
I
I,
50
% OF TOTAL
PROTEIN
100
Fig. 1. Subcellular distribution of 3-methylmargaric acid activation in rat liver. Rat liver was fractionated into a nuclear (N), heavy mitochondrial (M), light mitochondrial (L), microsomal (P) and cytosolic fraction (S) and the fractions were analyzed for protein, marker enzymes and 3-MMA activation. Recoveries of markers were between 85 and 113%. A: glutamate dehydrogenase (mitchondria); B: acid phosphatase (lysosomes); C: catalase (peroxisomes); D: glucose-6-phosphatase (endoplasmic reticulum); E: 3-methylmargaroyl-CoA synthetase.
1098
J. C. T. VANHOORENet a/
Fig. 2. Subfractionation of a light mitochondrial fraction on a Nycodenz step gradient and a self-generating Percoll gradient. A light mitochondrial fraction, prepared by differential centrifugation and derived from 8 g of liver, was subfractionated by centrifugation through a discontinuous Nycodenz gradient (left panel) or by iso-pycnic centrifugation in an iso-osmotic self-generating Percoll gradient (right panel). Fractions were collected starting from the bottom and analysed for protein (A, G). acid phosphatase (B, H), glutamate dehydrogenase (C, I), glucose-6-phosphatase (D, J), catalase (E, K) and 3-methylmargaroyl-CoA synthetase (F, L). Results are expressed as % of total gradient activity or content present in each fraction numbered on the abscissa. Recoveries varied between 65 and 107%.
Fig. 1). However, the activity present in the light mitochondrial fraction was higher than could be attributed to contamination by mitochondria and microsomes together. Further separation of the light mitochondrial fraction on a Percoll gradient or a Nycodenz gradient revealed that 3-methylmargaroyl-CoA synthetase is associated also with peroxisomes (see Fig. 2). From the marker enzyme analysis we calculated that the contribution of mitochondria, peroxisomes and endoplasmic reticulum to the total homogenate synthetase activity is approx. 35, 10 and 45% respectively. During the preparation of this paper, Pahan et al. (1993) published similar findings using phytanic acid as substrate.
The observation that 3-MMA acid can be activated by peroxisomes, endoplasmic reticulum and mitochondria suggests that 3-methylbranched fatty acids are activated by the long chain acyl-CoA synthetase, associated with these three organelles (see introduction). In order to verify this contention, the influence of pH, pyrophosphate and Triton X-100 on the synthetase activities towards palmitic acid, 2-MPA and 3-MMA was screened in purified All three enzymatic activities peroxisomes. showed a similar pH dependency with a broad optimum between 7.5 and 8.5, either in cationic or anionic buffers (data not shown). Low concentrations of pyrophosphate, an inhibitor of
Activation
of 3-methyl-branched
different microsomal synthetases (Schepers et al., 1989) blocked the peroxisomal activation of 3-MMA almost completely (20% residual activity at 1 mM). The activation of 2-MPA was somewhat less affected (38% residual activity at 1 mM) but that of palmitic acid was only moderately inhibited (66% residual activity at 1 mM). Differential effects were seen also upon addition of Triton X-100 to the assay mixtures. Whereas only a modest stimulation of palmitoyl-CoA synthetase was observed with this detergent (1.7-fold), the activation of 2-MPA and 3-MMA was increased approx. IO-fold (see Fig. 3A). In order to find out whether the observed differential stimulations could be related to the (chain length of the substrates, the effect of Triton X-100 on the peroxisomal activation of stearic acid, which possesses the same number of carbon atoms as 3-MMA was also determined. The stimulation was 1.5fold resembling that observed with palmitate and certainly much smaller than that measured with 3-MMA. In a further attempt to exclude differential effects due to physicochemical interactions between the different fatty acids and the detergent micelles, synthetase activities were also studied in mitochondria, purified as described
125-
1099
fatty acids
by Declercq et al. (1984). The shape of the detergent activation curves was different for each fatty acid (Fig. 3B), and did not resemble those seen with purified peroxisomes. Indirectly, these data suggest that separate enzymes may be involved in the activation of branched and straight chain fatty acids. Based on different kinetics of heat inactivation Muralidharan and Muraldiharan (1987) concluded that palmitoylCoA and phytanoyl-CoA synthetase activities present in rat liver microsomes reside also within different enzymes, while Singh and associates recently provided evidence, based on the differential effects of antibodies and Triton X-100, that in peroxisomes from human fibroblasts phytanic and palmitic acid are activated by distinct enzymes (Pahan et al., 1993). On the other hand, Wanders et al. (1992) using competition experiments and immunotitration, believe that in rat liver microsomes pristanic acid is recognized by the long chain acyl-CoA synthetase. Clearly, the final (dis)proof of the identity of the synthetases has to rely on more direct approaches. Since we succeeded in solubilizing these enzymes in an active state from highly purified peroxisomal membranes, their behaviour on various columns was screened
.
LOO_/\
*
0
0.1 %
(W/V)
0.2 TRITON
X-100
0.3
0.1
0 %
(W/V)
0.2 TRITON
0.3
X-100
Fig. 3. Effect of Triton X-100 on peroxisomal and mitochondrial acyl-CoA synthetase activities. Synthetase activities were measured in purified peroxisomes (A) and purified mitochondria (B) with palmitic acid (0) 2-MPA (A), 3-MMA (m), and stearic acids (0) in the absence and presence of increasing concentrations of Triton X-100.
J. C. T. VANHOOREN et al.
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FRACTION NUMBER
Fig. 4. Separation of solubihzed peroxisomal acyl-CoA synthetases on hydroxylapatite. Solubilized peroxisomal membrane proteins (700 pl; approx. 0.7 mg of protein) were loaded on a hydroxylapatite cartridge, equilibrated at 4’C with 10 mM K-phosphate buffer (pH 7.2). 1mM DTT, 2 mM MgCI, and 0.1% (w/v) Thesit, and eluted with a linear gradient of 10~400 mM K-phosphate buffer (pH 7.2) at 0.5 ml/min. The elution buffer contained also 1 mM DTT, 2 mM MgCI, and 0. I % (w/v) Thesit. Fractions of 2 ml were collected and analyzed for phosphate (-- -). lignoceroyl-CoA synthetase (a; rec. 20%), palmitoyl-CoA synthetase (m; rec. 47%) 2-methylpalmitoyl-CoA synthetase (0; rec. 39%) and 3-methylmargaroyl-CoA synthetase (0; rec. 5 1%).
using palmitic, 2-MPA, 3-MMA and lignoceric acids as the substrates. Lignoceroyl-CoA synthetase consistently eluted at higher phosphate concentration from the hydroxylapatite column than the three other synthetase activities, which seemed to co-elute, although often not in an identical fashion (Fig. 4). The synthetases, bound to Phenyl Sepharose in the presence of ammonium sulphate, eluted upon lowering the salt concentration in broad and skewed peaks, complicating the interpretation. Nevertheless, the elution profiles of the four enzyme activities could not be superimposed. On Blue Sepharose no separation was achieved (data not shown). Taken together, our results show that 3-methyl-branched fatty acids can be activated by peroxisomes and they provide direct evidence that long chain and very long chain fatty acids are activated by separate peroxisomal enzymes. In addition, we cannot exclude the possible existence of multiple activating enzymes, discriminating the position of the branch in isoprenoid-derived fatty acids. Interestingly, acyl-CoA oxidases, responsible for the following step in peroxisomal fatty acid breakdown, are able to recognize a 2-methyl branch (Van Veldhoven et al., 1992), but do not act on 3-methyl-branched compounds (Van Veldhoven
and Mannaerts, unpublished data). Whether the peroxisomal activation of 3-methyl-branched fatty acids has physiological consequences for a-oxidation remains questionable. In peroxisomes, isolated from lymphoblasts from Refsum patients, we were not able to show a specific defect in the activation of 3-MMA (Vanhooren, Mannaerts and Van Veldhoven, unpublished data). Acknowledgements-This work was supported by grants from the “Geconcerteerde Onderzoekacties van de Vlaamse Gemeenschap”, from the Belgian “Fends voor Geneeskundig Onderzoek” and from the “Onderzoeksfonds van de Katholieke Universiteit Leuven”.
REFERENCES Avigan J., Steinberg D., Gutman A., Mize C. E. and Mime G. W. (1966) Alpha-decarboxylation, an important pathway for degradation of phytanic acid in animals. Biochem. biophys. Res. Commun. 24, 838-845. Declercq P. E., Haagsman H. P., Van Veldhoven P. P., Debeer L. J., van Golde L. M. G. and Mannaerts G. P. (1984) Rat liver dihydroxyacetone-phosphate acyltransferases and their contribution to glycerolipid synthesis, J. biol. Chem. 259, 2064-2075. Hashimoto T. (1982) Comparison of enzymes of lipid P-oxidation in peroxisomes and mitochondria. In Peroxisomes in Biology and Medicine (Edited by Fahini H. D. and Sies H.), pp. 97-104. Springer, Berlin.
Activation
of 3-methyl. -branched
Huang S., Van Veldhoven P. P., Vanhoutte F., Parmentier G., Eyssen H. J. and Mannaerts G. P. (1992) a-Oxidation of 3-methyl-substituted fatty acids in rat liver. Archs Biochem. Biophys. 296, 214-223. Krisans S. K., Mortensen R. M. and Lazarow P. B. (1980) Acyl-CoA synthetases in rat liver peroxisomes. J. biol. Chem. 255, 9599-9607. Lageweg W., Wanders R. J. A. and Tager J. M. (1991) Topography of very long chain fatty acid activity in peroxisomes from rat liver. Eur. J. Biochem. 196, 519-523. Mannaerts G. P., ‘Van Veldhoven P.. Vanbroekhoven A., Vandenbroek G. and Debeer L. J. (1982) Evidence that peroxisomal acyl-CoA synthetase is located at the cytoplasmic of the peroxisomal membrane. side Biochem. J. 204, 17-23. Miyazawa S., Hashiomoto T. and Yokota S. (1985) Identity of long-chain acyl-CoA synthetase of microsomes, mitochondria ancl peroxisomes in rat liver. J. Biochem. 98, 723-733. Molzer B., Bernheimer H.. Barolin G. S., Hofinger E. and Lenz H. (1979) Di-, mono- and nonphytanoyl triglycerides in the serum: a sensitive parameter of the phytanic acid accumulation in Refsum’s disease. C/in. Chim. Acta 91, 133sl40. Muralidharan V. B. and Kishimoto Y. (1984) Phytanic acid r-oxidation in rat liver. /. hiol. Chem. 259, 13021ll3026. Muralidharan F. N. and Muralidharan V. B. (1986) Phytanoyl-CoA ligase activity in rat liver. Biochem. Int. 13, 123- 130. Muralidharan F. N. and Muralidharan V. N. (1987) Phytanic acid alpha oxidation in rat liver: studies on alpha hydroxylation. Int. J. B&hem. 19, 663-670. Pahan K.. Cofer J., Baliga P. and Singh 1. (1993) Identification of phytanoyl-CoA ligase as a distinct acylCoA ligase in peroxisomes from cultured human skin libroblasts. FEBS Left. 322, lOll104. Schepers L., Casteels M., Verheyden K., Parmentier G., Asselberghs S.. Eyssen H. J. and Mannaerts G. P. (1989) Subcellular distribution and characteristics of trihydroxycoprostanoyl-CoA synthetase in rat liver. Biochem. J. 257, 22 I -229. Shindo Y. and Hashimoto T. (1978) Acyl-Coenzyme A synthetase and fatty acid oxidation in rat liver peroxisomes. J. Biochem. 84, 1I777 I 18 1. Smgh H. and Poulos A. (1988) Distinct long chain and very long chain fatty acyl-CoA synthetases in rat liver peroxisomes and microsomes. Archs Biochem. BiophJs. 266, 486-495. Singh I., Lazo O., Dhaunsi G. S. and Contreras M. (1992a) Transport of fatty acids into human and rat liver peroxisomes. f. biol. Chem. 267, 13306613313.
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Singh I., Lazo 0.. Kalipada P. and Singh A. K. (1992b) Phytanic acid a-oxidation in human cultured skin fibroblasts. Biochim. Biophys. Acra 1180,221-224. Skjeldal 0. H. and Stokke 0. (1987) The subcellular localization of phytanic acid oxidase in rat liver. Biochim. Biophys. Acta 921, 38-42. Suzuki H., Kawarabayasi Y., Kondo J., Abe T., Nishikawa K., Kimura S., Hashimoto T. and Yamamoto T. (1990) Structure and regulation of rat long chain acyl-CoA synthetase. J. biol. Chem. 265, 8681-8685. Tanaka T., Hosaka K., Hoshimaru M. and Numa S. (1979) Purification and properties of long-chain acyl-coenzyme A synthetases from rat liver. Eur. J. Biochem. 98, 165-l 72. Tsai S. U., Avigan J. and Steinberg D. (1969) Studies on the alpha-oxidation of phytanic acid by rat liver mitochondria. J. biol. Chem. 244, 2682-2692. Vanhove G., Van Veldhoven P. P., Vanhoutte F., Parmentier G., Eyssen H. J. and Mannaerts G. P. (1991) Mitochondrial and peroxisomal p-oxidation of the branched chain fatty acid Z-methylpalmitate in rat liver. J. biol. Chem. 266, 24670-24675. Van Veldhoven P. P., Just W. W. and Mannaerts G. P. (1987) Permeability of the peroxisomal membrane to cofactors of p-oxidation: evidence for the presence of a pore-forming protein. J. biol. Chem. 262, 4310-4318. Van Veldhoven P. P., Vanhove G., Asselberghs S., Eyssen H. J. and Mannaerts G. P. (1992) Substrate specificities of rat liver peroxisomal acyl-CoA oxidases: palmitoylCoA oxidase (inducible acyl-CoA oxidase), pristanoylCoA oxidase (non-inducible acyl-CoA oxidase) and trihydroxycoprostanoyl-CoA oxidase. J. biol. Chem. 267, 20065-20074. Van Veldhoven P. P., Huang S., Eyssen H. J. and Mannaerts G. P. (1993) The deficient degradation of synthetic 2- and 3-methyl-branched fatty acids in fibroblasts from patients with peroxisomal disorders. J. Inherited Mefab. Dis. 16, 381-391. Verheyden K., Fransen M., Van Veldhoven P. P. and Mannaerts G. P. (1992) Presence of small GTP-binding proteins in the peroxisomal membrane. Biochim. Biophys. Acta 1109, 48-54. Wanders R. J. A., Denis S., van Roermund C. W. T., Jakobs C. and ten Brink H. J. (1992) Characteristics and subcellular localization of pristanoyl-CoA synthetase in rat liver. Biochim. Biophys. Acta 1125, 274-279. Yao J. K. and Dyck P. J. (1987) Tissue distribution of phytanic acid and its analogues in a kinship with Refsum’s disease. Lipids 22, 69-75.
0020-711X(94)E0024-W
ACTIVATION
Inr. J. Biochem. Vol. 26. No. 9, pp. 1095-1101, 1994 Copyright cm 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0020-7 I I X/94 $7.00 + 0.00
OF 3-METHYL-BRANCHED IN RAT LIVER
FATTY
ACIDS
J. C. T. VANHOOREN,’ S. ASSELBERGHS,’ H. J. EYSSEN,’ G. P. MANNAERTS’ and
P. P. VAN VELDHOVEN’* ‘Katolieke Universiteit Leuven, Campus Gasthuisberg-Afdeling Leuven [721. 32-16-345802; Fox 32-16-3456991 and lRega Instituut, Belgium (Received IO December
Farmakologie, Herestraat, B-3000 Minderbroederstraat, B-3000 Leuven,
1993)
Abstract--l. Subcellular fractionation of rat liver revealed that 3-methylmargaric acid, a monobranched phytanic acid analogue, can be activated by mitochondria, endoplasmic reticulum and peroxisomes. 2. Indirect data (effects of pyrophosphate and Triton X-100) suggested that the peroxisomal activation of 3-methylmargaric, 2-methylpalmitic and palmitic acid is catalyzed by different enzymes. 3. Despite many attempts, column chromatography of solubilized peroxisomal membrane proteins so far did not provide more conclusive data. On various matrices, lignoceroyl-CoA synthetase clearly eluted differently from the synthetases acting on 3-methylmargaric, 2methylpalmitic and palmitic acid. The latter three however, tended to coelute together, although often not in an identical manner.
INTRODUCTION
Fatty acids are metabolized by two main pathways: degradation via j-oxidation and esterification with glycerol-3-phosphate or dihydroxyacetone-phosphate resulting in the formation of glycerolipids. Before a fatty acid can enter these pathways, it needs first to be activated to its CoA-ester, a reaction that is catalyzed by acyl-CoA synthetases. Long chain acyl-CoA synthetases, acting on long straight chain fatty acids, are found in the mitochondrial outer membrane, the peroxisomal membrane and the membranes of the endoplasmic reticulum (Krisans et al., 1980; Mannaerts er al., 1982; Shindo and Hashimoto, 1978). The enzymes in the three organelles appear to be identical with respect to their catalytic, molecular and immuno-chemical properties (Hashimoto, 1982; Miyazawa et lzl., 1985; Tanaka et al., 1979) and they seem to have identical amino acid *To whom correspondence should be addressed. Abbreviations: DTT, dithiothreitol; 3-MMA, 3-methylmargaric acid (3-methylheptadecanoic acid); 2-MPA, 2-methylpalmittc acid; Mops, 4-morpholinopropanesulfonic acid.
sequences (Suzuki et al., 1990). There is indirect evidence that these synthetases are also responsible for the activation of long 2-methylbranched fatty acids such as the synthetic 2-MPA (Vanhove et al., 1991) and the naturally occurring pristanic (2,6,10,1Ctetramethylpentadecanoic) acid (Wanders et al., 1992). Very long chain fatty acids such as lignoceric (tetracosanoic) acid are activated by a separate enzyme present in peroxisomal and endoplasmic reticulum membranes (Singh and Poulos, 1988). Evidence for the existence of a separate very long chain acyl-CoA synthetase is based mainly on indirect arguments: absence from mitochondria (Singh and Poulos, 1988) and differential effects of detergents and inhibitors on the activation of long and very long chain fatty acids (Lageweg et al., 1991; Singh and Poulos, 1988; Singh et af., 1992a). 3-Methyl-branched fatty acids such as the synthetic 3-MMA and the naturally occurring phytanic (3,7,11,15tetramethylhexadecanoic) acid cannot be degraded directly via /?-oxidation. They are first oxidatively decarboxylated in a poorly characterized reaction called a-oxidation, that yields CO, and a 1095
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2-methylbranched fatty acid (ZMPA in the case of 3-MMA and pristanic acid in the case of phytanic acid) (Avigan et al., 1966; Tsai et al., 1969) which is then B-oxidized predominantly in peroxisomes (Vanhove et al., 1991; Van Veldhoven et al., 1993). The subcellular localization of cr-oxidation and whether a-oxidation requires the prior conversion of the 3-methyl branched fatty acid to its CoA derivative remain controversial. Studies on a-oxidation in homogenates and subcellular fractions from rat liver and human fibroblasts have shown different effects of CoA ranging from strong inhibition over no effect to marked stimulation (Huang et al., 1992; Muralidharan and Muralidharan, 1987; Muralidharan and Kishimoto, 1984; Skjeldal and Stokke, 1987; Singh et al., 1992b). Activation of phytanic acid-whether involved in a-oxidation or not-has been shown to occur, however (Muralidharan and Muralidharan, 1986). Moreover, in Refsum’s disease, in which cr-oxidation is deficient, accumulated phytanic acid is present in esterified form in triglycerides and phospholipids, implying a prior activation of the branched fatty acid (Molzer et al., 1979; Yao and Dyck, 1987). In a crude cell fractionation study carried out by Muralidharan and Muraldiharan (1986) phytanoyl-CoA synthetase activity was detected in mitochondrial and microsomal fractions from rat liver, but its presence in other cell organelles such as peroxisomes were not examined. We now report that 3-methyl-branched fatty acids are activated not only by mitochondria and endoplasmic reticulum but also by peroxisomes. By means of column chromatography of solubilized peroxisomal membrane proteins, we further tried to reveal the identity of the acyl-CoA synthetases acting on 3-methylbranched, 2-methyl-branched, long straight chain and very long straight chain fatty acids, the four activities known now to reside in peroxisomes. MATERIALS
AND
METHODS
Materials 2-MPA, [I -‘4C]-2-MPA (35 mCi/mmol sp. radioact.), 3-MMA and [ 1-‘4C]-3-MMA (54 mCi/mmol sp. radioact.) were synthesized as described before (Vanhove et al., 1991; Huang et al., 1992). [l-‘4C]-lignoceric acid (49 mCi/mmol sp. radioact.) was prepared according to Singh and Poulos (1988). [1-‘4C]palmitic acid (57 mCi/mmol) was purchased
from New England Nuclear, Bad Homburg, Germany. Coenzyme A, Percoll, Phenyl Sepharose (fast flow) and Blue Sepharose were purchased from Pharmacia Belga, Brussels, Belgium; ATP, ol-cyclodextrin and Thesit (research grade) were obtained from Boehringer Mannheim, Heidelberg, Germany. Nycodenz was from Nycomed, Oslo, Norway. Prepacked hydroxylapatite columns (Econo-Pat HTP cartridge, 5 ml) were from BioRad, Richmond, California, U.S.A. Cell fractionation Homogenates were prepared from livers of male Wistar rats in 0.25 M sucrose-l mM DTT-1 mM EDTA pH 7.2-0.1% (v/v) ethanol and fractionated as described before (Declercq et al., 1984). The light mitochondrial fraction, enriched in peroxisomes and lysosomes, was subfractionated by means of centrifugation in iso-osmotic self-generating Percoll gradients or discontinuous Nycodenz gradients (Vanhove et al., 1991). Highly purified peroxisomes, used for the preparation of membranes, were obtained exactly as described before (Verheyden et al., 1992). In order to increase the yield of peroxisomes, livers from rats kept for two weeks on a standard diet containing 0.3% (w/v) clofibrate were used. Marker enzymes and protein were measured as published before (Declercq et af., 1984; Verheyden et al., 1992). Preparation membranes
and solubilization of peroxisomal
Highly purified peroxisomes obtained as described above were diluted to a protein concentration of approx. 3 mg/ml in 10 mM pyrophosphate buffer pH 9.0- 1 mM EDTA- 1 mM DTT and sonicated 4 times for 15 set with intervals of 15 set at 4°C. This treatment disrupts the peroxisomal membranes and releases the matrix proteins and most of the peripheral membrane proteins (Van Veldhoven et al., 1987). After centrifugation for 1 hr at 100,OOOg at 4’C the membrane pellet was resuspended (final protein concentration approx. 1 mg/ml) in 50mM Tris-HCl buffer pH 7.2-0.5% (w/v) Thesit-0.5 M NaCl, and allowed to stand at 4’C for 30min. In order to prevent the acylCoA synthetases from inactivation the solubilization buffer also contained 5 mM ATP-3 mM MgCl,- 1 mM EDTA- 1 mM DTT. After centrifugation (100,OOOg for 1 hr at 4°C) the solubilized integral membrane proteins were recovered in the supernatant.
Activation
Activation
of fatty
of 3-methyl-branched
acids
The activation of palmitic, 2-MPA and 3MMA by homogenates and subcellular fractions was measured by adding an aliquot (50 p I) of homogenates or subcellular fractions, diluted in homogenization medium, to 200 ~1 of reaction mixture. Final concentrations were 50 mM Mops-NaOH buffer pH 7.4, 4mM ATP, 2.4 mM MgCl,, 2 mM DTT, 0.4 mM CoA, 50 PM [l-‘4C]-fatty acid (2,220-l 1,100 dpm/nmol sp. radioact.) Reactions were incubated for 3 min at 37°C and terminated by the addition of 2 ml of isopropanol/O.I N HCl (l/l-v/v) and phase-separated by adding 4 ml of petroleumether (b.p. 40-60°C). Part of the aqueous phase was counted for radioactivity. Lignoceroyl-CoA synthetase activity in the solubilized peroxisomal fractions and in the column fractions was measured as described by Singh and Poulos (1988). An aliquot (50 ~1) of the fractions, diluted in homogenization or elution buffer, was added to a 150 ~1 reaction mixture containing 50 mM Tris-HCl buffer pH 8.0-5 mM ATP-2 mM MgCI,-150 PM CoA- 300 p M DTT- 5 p M [ 1 - “C]-lignoceric acid: 49 ~Ci/~rnmol (sp. radioact.)-2.5 mg r-cyclodextrin/ml and incubated for 6 min at 37°C. Activation of palmitic, 2-MPA and 3-MMA in the solubilized peroxisomal membrane fractions or the column fractions was measured under the same conditions except that the substrate concentration was increased to
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cl-cyclodextrin was 50pM and omitted. Reactions were terminated by the addition of 20 ~1 of 10 M HCl and 1.5 ml of iso(40/ 1O/ l-v/v/v) propanol/heptane/ 10 M HCl and phases were separated by adding 1.5 ml of heptane and 1 ml of water. The aqueous phase was washed twice with 2 ml of heptane and counted for radioactivity.
RESULTS
AND DISCUSSION
In whole liver homogenates the activation of 3-MMA was linear with time for 3 min and proceeded linearly with protein contents up to 75 pg per assay. Kinetic analysis showed an almost first order reaction at substrate concentrations up to 50 PM but above 100 PM severe substrate inhibition was observed. At 50 p M of substrate, the activity in liver homogenates was 2.75 + 0.34 pmol/min.g of liver (n = 4). Analysis of the subcellular fractions, obtained by differential centrifugation, demonstrated that the 3-methylmargaroyl-CoA synthetase activity displayed a multisubcellular localization, similar to that previously described for palmitoyl-CoA synthetase (Krisans et al., 1980; Mannaerts et al., 1982; Shindo and Hashimoto, 1978) 2-methylpalmitoyl-CoA synthetase (Vanhove et al., 1991) and pristanoyl-CoA synthetase (Wanders et al., 1992). The major part of the synthetase activity was recovered in the heavy mitochondrial and microsomal fractions (see _ D
E t
I
IIII
N I 0
I
I
MLP
S
I
I,
50
% OF TOTAL
PROTEIN
100
Fig. 1. Subcellular distribution of 3-methylmargaric acid activation in rat liver. Rat liver was fractionated into a nuclear (N), heavy mitochondrial (M), light mitochondrial (L), microsomal (P) and cytosolic fraction (S) and the fractions were analyzed for protein, marker enzymes and 3-MMA activation. Recoveries of markers were between 85 and 113%. A: glutamate dehydrogenase (mitchondria); B: acid phosphatase (lysosomes); C: catalase (peroxisomes); D: glucose-6-phosphatase (endoplasmic reticulum); E: 3-methylmargaroyl-CoA synthetase.
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J. C. T. VANHOORENet a/
Fig. 2. Subfractionation of a light mitochondrial fraction on a Nycodenz step gradient and a self-generating Percoll gradient. A light mitochondrial fraction, prepared by differential centrifugation and derived from 8 g of liver, was subfractionated by centrifugation through a discontinuous Nycodenz gradient (left panel) or by iso-pycnic centrifugation in an iso-osmotic self-generating Percoll gradient (right panel). Fractions were collected starting from the bottom and analysed for protein (A, G). acid phosphatase (B, H), glutamate dehydrogenase (C, I), glucose-6-phosphatase (D, J), catalase (E, K) and 3-methylmargaroyl-CoA synthetase (F, L). Results are expressed as % of total gradient activity or content present in each fraction numbered on the abscissa. Recoveries varied between 65 and 107%.
Fig. 1). However, the activity present in the light mitochondrial fraction was higher than could be attributed to contamination by mitochondria and microsomes together. Further separation of the light mitochondrial fraction on a Percoll gradient or a Nycodenz gradient revealed that 3-methylmargaroyl-CoA synthetase is associated also with peroxisomes (see Fig. 2). From the marker enzyme analysis we calculated that the contribution of mitochondria, peroxisomes and endoplasmic reticulum to the total homogenate synthetase activity is approx. 35, 10 and 45% respectively. During the preparation of this paper, Pahan et al. (1993) published similar findings using phytanic acid as substrate.
The observation that 3-MMA acid can be activated by peroxisomes, endoplasmic reticulum and mitochondria suggests that 3-methylbranched fatty acids are activated by the long chain acyl-CoA synthetase, associated with these three organelles (see introduction). In order to verify this contention, the influence of pH, pyrophosphate and Triton X-100 on the synthetase activities towards palmitic acid, 2-MPA and 3-MMA was screened in purified All three enzymatic activities peroxisomes. showed a similar pH dependency with a broad optimum between 7.5 and 8.5, either in cationic or anionic buffers (data not shown). Low concentrations of pyrophosphate, an inhibitor of
Activation
of 3-methyl-branched
different microsomal synthetases (Schepers et al., 1989) blocked the peroxisomal activation of 3-MMA almost completely (20% residual activity at 1 mM). The activation of 2-MPA was somewhat less affected (38% residual activity at 1 mM) but that of palmitic acid was only moderately inhibited (66% residual activity at 1 mM). Differential effects were seen also upon addition of Triton X-100 to the assay mixtures. Whereas only a modest stimulation of palmitoyl-CoA synthetase was observed with this detergent (1.7-fold), the activation of 2-MPA and 3-MMA was increased approx. IO-fold (see Fig. 3A). In order to find out whether the observed differential stimulations could be related to the (chain length of the substrates, the effect of Triton X-100 on the peroxisomal activation of stearic acid, which possesses the same number of carbon atoms as 3-MMA was also determined. The stimulation was 1.5fold resembling that observed with palmitate and certainly much smaller than that measured with 3-MMA. In a further attempt to exclude differential effects due to physicochemical interactions between the different fatty acids and the detergent micelles, synthetase activities were also studied in mitochondria, purified as described
125-
1099
fatty acids
by Declercq et al. (1984). The shape of the detergent activation curves was different for each fatty acid (Fig. 3B), and did not resemble those seen with purified peroxisomes. Indirectly, these data suggest that separate enzymes may be involved in the activation of branched and straight chain fatty acids. Based on different kinetics of heat inactivation Muralidharan and Muraldiharan (1987) concluded that palmitoylCoA and phytanoyl-CoA synthetase activities present in rat liver microsomes reside also within different enzymes, while Singh and associates recently provided evidence, based on the differential effects of antibodies and Triton X-100, that in peroxisomes from human fibroblasts phytanic and palmitic acid are activated by distinct enzymes (Pahan et al., 1993). On the other hand, Wanders et al. (1992) using competition experiments and immunotitration, believe that in rat liver microsomes pristanic acid is recognized by the long chain acyl-CoA synthetase. Clearly, the final (dis)proof of the identity of the synthetases has to rely on more direct approaches. Since we succeeded in solubilizing these enzymes in an active state from highly purified peroxisomal membranes, their behaviour on various columns was screened
.
LOO_/\
*
0
0.1 %
(W/V)
0.2 TRITON
X-100
0.3
0.1
0 %
(W/V)
0.2 TRITON
0.3
X-100
Fig. 3. Effect of Triton X-100 on peroxisomal and mitochondrial acyl-CoA synthetase activities. Synthetase activities were measured in purified peroxisomes (A) and purified mitochondria (B) with palmitic acid (0) 2-MPA (A), 3-MMA (m), and stearic acids (0) in the absence and presence of increasing concentrations of Triton X-100.
J. C. T. VANHOOREN et al.
1100
I
1
I
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I
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I
I
5
I
06
I
I
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20
FRACTION NUMBER
Fig. 4. Separation of solubihzed peroxisomal acyl-CoA synthetases on hydroxylapatite. Solubilized peroxisomal membrane proteins (700 pl; approx. 0.7 mg of protein) were loaded on a hydroxylapatite cartridge, equilibrated at 4’C with 10 mM K-phosphate buffer (pH 7.2). 1mM DTT, 2 mM MgCI, and 0.1% (w/v) Thesit, and eluted with a linear gradient of 10~400 mM K-phosphate buffer (pH 7.2) at 0.5 ml/min. The elution buffer contained also 1 mM DTT, 2 mM MgCI, and 0. I % (w/v) Thesit. Fractions of 2 ml were collected and analyzed for phosphate (-- -). lignoceroyl-CoA synthetase (a; rec. 20%), palmitoyl-CoA synthetase (m; rec. 47%) 2-methylpalmitoyl-CoA synthetase (0; rec. 39%) and 3-methylmargaroyl-CoA synthetase (0; rec. 5 1%).
using palmitic, 2-MPA, 3-MMA and lignoceric acids as the substrates. Lignoceroyl-CoA synthetase consistently eluted at higher phosphate concentration from the hydroxylapatite column than the three other synthetase activities, which seemed to co-elute, although often not in an identical fashion (Fig. 4). The synthetases, bound to Phenyl Sepharose in the presence of ammonium sulphate, eluted upon lowering the salt concentration in broad and skewed peaks, complicating the interpretation. Nevertheless, the elution profiles of the four enzyme activities could not be superimposed. On Blue Sepharose no separation was achieved (data not shown). Taken together, our results show that 3-methyl-branched fatty acids can be activated by peroxisomes and they provide direct evidence that long chain and very long chain fatty acids are activated by separate peroxisomal enzymes. In addition, we cannot exclude the possible existence of multiple activating enzymes, discriminating the position of the branch in isoprenoid-derived fatty acids. Interestingly, acyl-CoA oxidases, responsible for the following step in peroxisomal fatty acid breakdown, are able to recognize a 2-methyl branch (Van Veldhoven et al., 1992), but do not act on 3-methyl-branched compounds (Van Veldhoven
and Mannaerts, unpublished data). Whether the peroxisomal activation of 3-methyl-branched fatty acids has physiological consequences for a-oxidation remains questionable. In peroxisomes, isolated from lymphoblasts from Refsum patients, we were not able to show a specific defect in the activation of 3-MMA (Vanhooren, Mannaerts and Van Veldhoven, unpublished data). Acknowledgements-This work was supported by grants from the “Geconcerteerde Onderzoekacties van de Vlaamse Gemeenschap”, from the Belgian “Fends voor Geneeskundig Onderzoek” and from the “Onderzoeksfonds van de Katholieke Universiteit Leuven”.
REFERENCES Avigan J., Steinberg D., Gutman A., Mize C. E. and Mime G. W. (1966) Alpha-decarboxylation, an important pathway for degradation of phytanic acid in animals. Biochem. biophys. Res. Commun. 24, 838-845. Declercq P. E., Haagsman H. P., Van Veldhoven P. P., Debeer L. J., van Golde L. M. G. and Mannaerts G. P. (1984) Rat liver dihydroxyacetone-phosphate acyltransferases and their contribution to glycerolipid synthesis, J. biol. Chem. 259, 2064-2075. Hashimoto T. (1982) Comparison of enzymes of lipid P-oxidation in peroxisomes and mitochondria. In Peroxisomes in Biology and Medicine (Edited by Fahini H. D. and Sies H.), pp. 97-104. Springer, Berlin.
Activation
of 3-methyl. -branched
Huang S., Van Veldhoven P. P., Vanhoutte F., Parmentier G., Eyssen H. J. and Mannaerts G. P. (1992) a-Oxidation of 3-methyl-substituted fatty acids in rat liver. Archs Biochem. Biophys. 296, 214-223. Krisans S. K., Mortensen R. M. and Lazarow P. B. (1980) Acyl-CoA synthetases in rat liver peroxisomes. J. biol. Chem. 255, 9599-9607. Lageweg W., Wanders R. J. A. and Tager J. M. (1991) Topography of very long chain fatty acid activity in peroxisomes from rat liver. Eur. J. Biochem. 196, 519-523. Mannaerts G. P., ‘Van Veldhoven P.. Vanbroekhoven A., Vandenbroek G. and Debeer L. J. (1982) Evidence that peroxisomal acyl-CoA synthetase is located at the cytoplasmic of the peroxisomal membrane. side Biochem. J. 204, 17-23. Miyazawa S., Hashiomoto T. and Yokota S. (1985) Identity of long-chain acyl-CoA synthetase of microsomes, mitochondria ancl peroxisomes in rat liver. J. Biochem. 98, 723-733. Molzer B., Bernheimer H.. Barolin G. S., Hofinger E. and Lenz H. (1979) Di-, mono- and nonphytanoyl triglycerides in the serum: a sensitive parameter of the phytanic acid accumulation in Refsum’s disease. C/in. Chim. Acta 91, 133sl40. Muralidharan V. B. and Kishimoto Y. (1984) Phytanic acid r-oxidation in rat liver. /. hiol. Chem. 259, 13021ll3026. Muralidharan F. N. and Muralidharan V. B. (1986) Phytanoyl-CoA ligase activity in rat liver. Biochem. Int. 13, 123- 130. Muralidharan F. N. and Muralidharan V. N. (1987) Phytanic acid alpha oxidation in rat liver: studies on alpha hydroxylation. Int. J. B&hem. 19, 663-670. Pahan K.. Cofer J., Baliga P. and Singh 1. (1993) Identification of phytanoyl-CoA ligase as a distinct acylCoA ligase in peroxisomes from cultured human skin libroblasts. FEBS Left. 322, lOll104. Schepers L., Casteels M., Verheyden K., Parmentier G., Asselberghs S.. Eyssen H. J. and Mannaerts G. P. (1989) Subcellular distribution and characteristics of trihydroxycoprostanoyl-CoA synthetase in rat liver. Biochem. J. 257, 22 I -229. Shindo Y. and Hashimoto T. (1978) Acyl-Coenzyme A synthetase and fatty acid oxidation in rat liver peroxisomes. J. Biochem. 84, 1I777 I 18 1. Smgh H. and Poulos A. (1988) Distinct long chain and very long chain fatty acyl-CoA synthetases in rat liver peroxisomes and microsomes. Archs Biochem. BiophJs. 266, 486-495. Singh I., Lazo O., Dhaunsi G. S. and Contreras M. (1992a) Transport of fatty acids into human and rat liver peroxisomes. f. biol. Chem. 267, 13306613313.
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Singh I., Lazo 0.. Kalipada P. and Singh A. K. (1992b) Phytanic acid a-oxidation in human cultured skin fibroblasts. Biochim. Biophys. Acra 1180,221-224. Skjeldal 0. H. and Stokke 0. (1987) The subcellular localization of phytanic acid oxidase in rat liver. Biochim. Biophys. Acta 921, 38-42. Suzuki H., Kawarabayasi Y., Kondo J., Abe T., Nishikawa K., Kimura S., Hashimoto T. and Yamamoto T. (1990) Structure and regulation of rat long chain acyl-CoA synthetase. J. biol. Chem. 265, 8681-8685. Tanaka T., Hosaka K., Hoshimaru M. and Numa S. (1979) Purification and properties of long-chain acyl-coenzyme A synthetases from rat liver. Eur. J. Biochem. 98, 165-l 72. Tsai S. U., Avigan J. and Steinberg D. (1969) Studies on the alpha-oxidation of phytanic acid by rat liver mitochondria. J. biol. Chem. 244, 2682-2692. Vanhove G., Van Veldhoven P. P., Vanhoutte F., Parmentier G., Eyssen H. J. and Mannaerts G. P. (1991) Mitochondrial and peroxisomal p-oxidation of the branched chain fatty acid Z-methylpalmitate in rat liver. J. biol. Chem. 266, 24670-24675. Van Veldhoven P. P., Just W. W. and Mannaerts G. P. (1987) Permeability of the peroxisomal membrane to cofactors of p-oxidation: evidence for the presence of a pore-forming protein. J. biol. Chem. 262, 4310-4318. Van Veldhoven P. P., Vanhove G., Asselberghs S., Eyssen H. J. and Mannaerts G. P. (1992) Substrate specificities of rat liver peroxisomal acyl-CoA oxidases: palmitoylCoA oxidase (inducible acyl-CoA oxidase), pristanoylCoA oxidase (non-inducible acyl-CoA oxidase) and trihydroxycoprostanoyl-CoA oxidase. J. biol. Chem. 267, 20065-20074. Van Veldhoven P. P., Huang S., Eyssen H. J. and Mannaerts G. P. (1993) The deficient degradation of synthetic 2- and 3-methyl-branched fatty acids in fibroblasts from patients with peroxisomal disorders. J. Inherited Mefab. Dis. 16, 381-391. Verheyden K., Fransen M., Van Veldhoven P. P. and Mannaerts G. P. (1992) Presence of small GTP-binding proteins in the peroxisomal membrane. Biochim. Biophys. Acta 1109, 48-54. Wanders R. J. A., Denis S., van Roermund C. W. T., Jakobs C. and ten Brink H. J. (1992) Characteristics and subcellular localization of pristanoyl-CoA synthetase in rat liver. Biochim. Biophys. Acta 1125, 274-279. Yao J. K. and Dyck P. J. (1987) Tissue distribution of phytanic acid and its analogues in a kinship with Refsum’s disease. Lipids 22, 69-75.