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Purification and properties of peroxisomal carnitine palmitoyltransferase in chick embryo liver

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530 Biochimica et Biophysica Acta 924 (1987) 530-542 Elsevier BBA 22745 Purification and properties of peroxisomal carnitine palmitoyltransferase in chick embryo liver Satoshi Ishii a, Hidemi Ishii b, Takafumi Watanabe a and Tctsuya Suga a Department of Clinical Biochemistry, Tokyo College of Pharmacy, Tokyo and b Department of Clinical Biochemistry, Faculty o/Pharmaceutical Sciences, Teikyo Universi(v, Kanagawa (Japan) (Received 20 October 1986) (Revised manuscript received 10 March 1987) Key words: Carnitine palmitoyltransferase; Acyltransferase; (Chick embryo liver peroxisome) Peroxisomai carnitine palmitoyltransferase was purified by solubilization using Tween 20 and KCI from the large granule fraction of the liver of clofibrate-treated chick embryo, DEAE-Sephacel and blue Sepharose CL-6B column chromatography. The peroxisomal carnitine palmitoyltransferase was an M r 64000 poly- peptide; the mitochondrial carnitine palmitoyltransferase had a subunit molecular weight of 69 000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The carnitine acetyltransferase was an M r 64000 polypeptide. Antibody against purified peroxisomal carnitine palmitoyltransferase reacted only with per- oxisomal carnitine palmitoyltransferase, but not with mitochondriai carnitine palmitoyltransferase or carni- tine acetyitransferase. In addition, anti-peroxisomai carnitine palmitoyltransferase reacted only with the protein in peroxisomes purified from chick embryo liver by sucrose density gradient centrifugation. Thus, it was confirmed that purified peroxisomal carnitine palmitoyltransferase was a peroxisomal protein. Com- pared with mitochondriai carnitine palmitoyltransferase, peroxisomal carnitine palmitoyltransferase was extremely resistant to inactivation by trypsin. The pH optimum of peroxisomal carnitine palmitoyl- transferase was 8.5, differing from that of mitochondrial carnitine paimitoyitransferase. The K m value of peroxisomal carnitine palmitoyltransferase for palmitoyl-CoA (32 pM) was similar to that of the mitochondrial one, whereas those values for L-carnitine (140 ftM), palmitoyl-L-carnitine (43 pM) and CoA (9 pM) were lower than those of mitochondrial carnitine palmitoyltransferase. Peroxisomal carnitine palmitoyltransferase exhibited similar substrate specificities in both the forward and reverse reactions, with the highest activity toward lauroyl derivatives. Furthermore, this enzyme showed relatively high affinities for long-chain acyl derivatives (C10-C 16) and similar K m values (30-50 p M) for acyI-CoAs, acylcarnitine and CoA, and a constant K m value (approximately 150 pM) for carnitine. These results indicate that peroxisomal carnitine palmitoyltransferase played a role in the modulation of the intracellular CoA/long- chain acyl-CoA ratio at the hatching stage of chicken when long-chain fatty acids are actively oxidized in peroxisomes. Introduction Carnitine acyltransferases catalyze the reversi- ble reaction: acyl-CoA + carnitine = acylcarnitine Correspondence: T. Suga, Department of Clinical Biochem- istry, Tokyo College of Pharmacy, 1432-1 Horinouchi, Hachioji, Tokyo 192-03, Japan. + CoASH, and are classified into three kinds of enzymes: carnitine palmitoyltransferase, carnitine octanoyltransferase and carnitine acetyltransfer- ase. Carnitine palmitoyltransferase, which has a high affinity for long-chain acyl derivatives, is located on both the inner and outer surfaces of the inner mitochondrial membrane [1]. Recently, this 0304-4165/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
Transcript

530 Biochimica et Biophysica Acta 924 (1987) 530-542 Elsevier

BBA 22745

Pur i f i ca t ion and p rope r t i e s of pe rox i soma l carni t ine pa lmi toy l t r ans fe rase

in chick e m b r y o liver

Sa tosh i Ishii a, H i d e m i Ishii b, T a k a f u m i W a t a n a b e a and T c t s u y a Suga a

Department of Clinical Biochemistry, Tokyo College of Pharmacy, Tokyo and b Department of Clinical Biochemistry, Faculty o/Pharmaceutical Sciences, Teikyo Universi(v, Kanagawa (Japan)

(Received 20 October 1986) (Revised manuscript received 10 March 1987)

Key words: Carnitine palmitoyltransferase; Acyltransferase; (Chick embryo liver peroxisome)

Peroxisomai carnitine palmitoyltransferase was purified by solubilization using Tween 20 and KCI from the large granule fraction of the liver of clofibrate-treated chick embryo, DEAE-Sephacel and blue Sepharose CL-6B column chromatography. The peroxisomal carnitine palmitoyltransferase was an M r 64000 poly- peptide; the mitochondrial carnitine palmitoyltransferase had a subunit molecular weight of 69 000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The carnitine acetyltransferase was an M r 64000 polypeptide. Antibody against purified peroxisomal carnitine palmitoyltransferase reacted only with per- oxisomal carnitine palmitoyltransferase, but not with mitochondriai carnitine palmitoyltransferase or carni- tine acetyitransferase. In addition, anti-peroxisomai carnitine palmitoyltransferase reacted only with the protein in peroxisomes purified from chick embryo liver by sucrose density gradient centrifugation. Thus, it was confirmed that purified peroxisomal carnitine palmitoyltransferase was a peroxisomal protein. Com- pared with mitochondriai carnitine palmitoyltransferase, peroxisomal carnitine palmitoyltransferase was extremely resistant to inactivation by trypsin. The pH optimum of peroxisomal carnitine palmitoyl- transferase was 8.5, differing from that of mitochondrial carnitine paimitoyitransferase. The K m value of peroxisomal carnitine palmitoyltransferase for palmitoyl-CoA (32 pM) was similar to that of the mitochondrial one, whereas those values for L-carnitine (140 ftM), palmitoyl-L-carnitine (43 pM) and CoA (9 pM) were lower than those of mitochondrial carnitine palmitoyltransferase. Peroxisomal carnitine palmitoyltransferase exhibited similar substrate specificities in both the forward and reverse reactions, with the highest activity toward lauroyl derivatives. Furthermore, this enzyme showed relatively high affinities for long-chain acyl derivatives (C10-C 16) and similar K m values (30-50 p M) for acyI-CoAs, acylcarnitine and CoA, and a constant K m value (approximately 150 pM) for carnitine. These results indicate that peroxisomal carnitine palmitoyltransferase played a role in the modulation of the intracellular CoA/long- chain acyl-CoA ratio at the hatching stage of chicken when long-chain fatty acids are actively oxidized in peroxisomes.

Introduction

Carnitine acyltransferases catalyze the reversi- ble reaction: acyl-CoA + carnitine = acylcarnitine

Correspondence: T. Suga, Department of Clinical Biochem- istry, Tokyo College of Pharmacy, 1432-1 Horinouchi, Hachioji, Tokyo 192-03, Japan.

+ CoASH, and are classified into three kinds of enzymes: carnitine palmitoyltransferase, carnitine octanoyltransferase and carnitine acetyltransfer- ase. Carnitine palmitoyltransferase, which has a high affinity for long-chain acyl derivatives, is located on both the inner and outer surfaces of the inner mitochondrial membrane [1]. Recently, this

0304-4165/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

enzyme has been purified as a single protein from bovine heart [2] and rat liver [3]. Fritz et al. [4] were the first to partially purify carnitine acetyltransferase from pig heart and they showed that carnitine acetyltransferase reacted specifically with shorter-chain acyl-CoAs. This enzymes has also been purified as a single protein from other animal tissues [2,5].

Isolated peroxisomes from the liver of rat and mouse contain carnitine acetyltransferase and carnitine octanoyltransferase, but not carnitine palmitoyltransferase [3,6-10]. However, it has been recently reported that, in human liver, per- oxisomes contained about 20% of the total activity of liver carnitine palmitoyltransferase but not carnitine octanoyltransferase [11]. We have found that carnitine palmitoyltransferase was present in peroxisomes from chick embryo liver and that the activity in peroxisomes was high in the embryo, but low in adult chicken [12]. Furthermore, our studies concerning localization of the activities of carnitine acyltransferases in chick embryo liver using subfractionation and sucrose density gradi- ent centrifugation showed that the activities of carnitine palmitoyltransferase and carnitine acetyltransferase were located in both mitochon- dria and peroxisomes. The peroxisomal carnitine palmitoyltransferase in chick embryo liver was increased by treatment with clofibrate but mitochondrial carnitine palmitoyltransferase activ- ity did not change [13].

We describe here the purification of per- oxisomal camitine palmitoyltransferase and some properties, in order to establish the existence of carnitine palmitoyltransferase in peroxisomes of chick embryo liver and to clarify the physiological role of this enzyme. We also describe copurifica- tion of other carnitine acyltransferases from the large granule fraction and discuss how many carnitine acyltransferases may be present in chick embryo liver.

Materials and Methods

Acyl-coenzyme A esters, CoA, acylcarnitine es- ter, trypsin and trypsin inhibitor were purchased from Sigma. L-(-)-Carnitine was kindly donated by Earth Pharmaceutical Co., Akoh, Japan. Blue Sepharose CL-6B, DEAE-Sephacel, CM-Sephadex

531

C-50 and Sephadex media were obtained from Pharmacia. DEAE-Toyopearl 650M was obtained from Toyo Soda Mfg. Co., Tokyo, Japan. Matrex Orange A was purchased from Amicon. All other reagents were of analytical grade.

Isolation of large granule fraction. Fertilized eggs (White Leghorn line) were obtained from a poultry farm and were incubated at 37 o C. Chick embryos were treated with clofibrate as previously de- scribed [13], and killed at day 20 of incubation.The livers removed from chick embryos were im- mediately homogenized in ice-cold 0.25 M sucrose with a Potter-Elvehjem Teflon-glass homogenizer. The homogenate was centrifuged at 500 x g for 10 min and the supernatant at 12 500 x g for 20 rain. The resultant pellet (large granule fraction) was re-suspended in buffer A (50 mM potassium phos- phate/0.25 mM EDTA/0.5 mM dithiothreitol (pH 7.5)) and assayed for enzyme activities and pro- tein.

Isolation of organelles. Peroxisomes, mito- chondria and microsomes were isolated by sucrose density gradient centrifugation of light mito- chondrial, heavy mitochondrial and microsomal fraction, respectively, from the liver of clofibrate- treated chick embryo. Heavy and light mitochondrial and microsomal fractions were fractionated by the method of De Duve et al. [14]. Sucrose density gradient centrifugation was car- ried out as previously described [13].

Enzyme and protein assay. Carnitine acyltrans- ferase activities were determined by two systems. In the case of purification, the 5,5'-dithiobis(2- nitrobenzoic acid) (DTNB) method of Bieber et al. [15] using a double beam spectrophotometer at 412 nm, using 100 #M acyl-CoA and 1.25 mM carnitine as substrate at pH 8.0 was utilized. In principle this method is based on the detection of free CoA derived from acyl-CoA during the reac- tion by DTNB. One unit of the activity was taken as the formation of 1 #tool reaction product/min. In the studies on the properties, the activities were determined by the method of Sere et al. [16], measuring the rate of change in the absorbance at 232 nm using a double beam spectrophotometer. The 500 #l reaction mixture contained 0.2 M Tris-HC1/1 mM di th io thre i to l /0 .5 mM EDTA/0.2% Tween 20 and substrates at pH 7.5. The forward reaction was assayed with 100 /~M

532

acyl-CoA and 2 mM carnitine and the reverse reaction was assayed with 500 /~M acylcarnitine and 100 #M CoA. One unit of the activity was expressed as the reduction or the formation of 1 ~mol of acyl-CoA/min. Catalase [17], cytochrome c oxidase [18] and NADPH-cytochrome c re- ductase [19] were assayed by these cited proce- dures. Units of enzyme activities are expressed as previously described [20]. Protein concentration was determined by the method of Lowry et al. [21] with bovine serum albumin as a standard.

Solubilization of carnitine acyltransferase. Large granule fractions from clofibrate-treated chick em- bryo fiver (100 g) were pooled. Tween 20 and KC1 were added to the suspension, at final concentra- tions of 3% and 0.5 M, respectively. The suspen- sion was incubated for 1 h at 4°C, and then centrifuged at 14 500 × g for 1 h. An apricot-col- ored supernatant was collected from between the floating lipid and the pellet.

Separation of peroxisomal and mitochondrial carnitine palmitoyltransferases and carnitine acetyltransferase. The solubilized protein solution was equilibrated with buffer B (10 mM Tris- HC1/0.25 mM EDTA/0.1% Tween/20 0.5 mM dithiothreitol (pH 8.0)) by gel filtration on a Seph- adex G-10 column and then applied to a column (2.6 x 25 cm) of DEAE-Sephacel equilibrated with buffer B. The column was washed with four bed volumes of buf fe r B, and all carni t ine acyltransferases were eluted with 250 mM KC1 in buffer B. The active fractions were pooled, equi- librated with buffer B by gel filtration on a Seph- adex G-25 column and then applied (flow rate 40 ml /h ) to a column (3.2 x 24 cm) of blue Seph- arose CL-6B equilibrated with buffer B. Four bed volumes of buffer B were used to wash the column before elution with a 400 ml linear gradient of 0-400 mM KC1 in buffer B (10 ml fractions) (Fig. 1).

Purification of peroxisomal carnitine palmitoyl- transferase. The peak fractions showing carnitine palmitoyltransferase activity (fractions 73-81) were used for further purification of the enzyme. The fractions were pooled (90 ml), diluted with 270 ml buffer C (10 mM potassium phosphate / 0.25 mM EDTA/0.1% Tween 20/0.5 mM di- thiothreitol (pH.75)), and applied to a column (2 x 13 cm) of DEAE-Sephacel equilibrated with

buffer C. The column was washed with 200 ml of buffer C followed by elution with a linear 0-400 mM KC1 gradient in a total volume of 200 ml (Fig. 2A). Fractions containing the activity were pooled and concentrated to 3 ml using an Amicon PM-30 filter. This preparation was subjected to gel filtration on a column (2.6 x 70 cm) of Seph- adex G-150 equilibrated with buffer C, and 3-ml fractions were collected (Fig. 2B). Fractions show- ing high specific activity of the enzyme were pooled and applied to a column (1.5 x 5 cm) of DEAE- Toyopearl equilibrated with buffer C (Fig. 2C). The column was washed with 50 ml of buffer C and then eluted with a linear 0-100 mM KCI gradient in a total volume of 100 ml. Fractions showing constant specific activity of carnitine pal- mitoyltransferase were pooled.

Purification of mitochondrial carnitine palmitoyl- transferase. The fractions of carnitine palmitoyl- transferase which were not retained on a blue Sepharose CL-6B column (fractions 11-25) were used for further purification of mitochondrial en- zyme. The fractions were pooled and applied to a column (2.5 × 16 cm) of DEAE-Sephacel equi- librated with buffer D (10 mM Tris-HC1/0.25 mM EDTA/0.1% Tween 20/0.5 mM dithiothrei- tol (pH. 8.5)). The column was washed with 300 ml of buffer D, followed by elution with a linear 0-400 mM KC1 gradient in a total volume of 300 ml (Fig. 3A). Fractions containing the activity were pooled, equilibrated with buffer D by gel filtration on a Sephadex G-25 column and applied to a column (2 × 10 cm) of DEAE-Toyopearl equilibrated with buffer D (Fig. 3B). The column was washed with 100 ml buffer D and then eluted with a linear 0-75 mM KC1 gradient in a total volume of 150 ml. Active fractions were pooled and concentrated to 3 ml using an Amicon PM-30 filter. This preparation was subjected to gel filtra- tion on a column (2.6 × 70 cm) of Sephadex G-150 equilibrated with buffer D containing 0.1 M KC1 (3 ml fractions) (Fig. 3C). Fractions showing con- stant specific activity were pooled.

Purification of carnitine acetyltransferase. Frac- tions eluting from a blue Sepharose CL-6B col- umn containing the activity (fractions 82-88) were pooled, dialyzed against buffer E (50 mM potas- sium phosphate/0.25 mM EDTA/0.1% Tween 20/0.5 mM dithiothreitol (pH 7.0)) and applied to

533

a column (2 x 14 cm) of Sephadex G-50 equi- librated with buffer E. The column was washed with 200 ml of buffer E and then eluted with a linear 0-500 mM KC1 gradient in a total volume of 200 ml. Active fractions were pooled and con- centrated to 3 ml using an Amicon PM-30 filter. This preparation was applied to a Sephadex G-150 column (2.6 x 70 cm) equilibrated with buffer E containing 0.1 M KC1 and 3-ml fractions were collected. Fractions containing carnitine pal- mitoyltransferase were pooled, dialyzed overnight against 20 volumes of buffer F (10 mM potassium phosphate/0.05 mM EDTA/0.1% Tween 20/0.5 mM dithiothreitol (pH 7.0)) and applied to a column (2 x 7 cm) of Matrex orange A equi- librated with buffer F. The column was washed with 100 ml of buffer F followed by elution with a linear 0-300 ml KC1 gradient in a total volume of 150 ml. Fractions showing constant specific activ- ity were pooled.

Preparation of antibody. Antibody against puri- fied peroxisomal carnitine palmitoyltransferase was prepared as follows. Approx. 0.7 mg of puri- fied enzyme was emulsified with an equal volume of Freund's adjuvant and injected subcutaneously into the back and foot pads of New Zealand white rabbits. The procedure was repeated four times at weekly intervals, but approx. 0.3-0.4 mg of the enzyme was used from 1 week after the first injection. The antibody against peroxisomal car- nitine palmitoyltransferase was partially purified from the anti-serum, which was collected 1 week after the last injection, by fractionation with am- monium sulfate.

Immunodiffusion and immunoblotting. Double diffusion plates were prepared with 1% agarose in 0.1 M potassium phosphate/0.02% sodium azide/0.15% NaC1 (pH 7.5), and developed over- night at room temperature. Immunblotting was performed according to Towbin et al. [22]. For immunoblot analysis, proteins were transferred electrophoretically from unstained slab gels to a nitrocellulose sheet. The sheet was treated with 125I-labeled protein A and visualized by autoradi- ography.

Other methods. SDS-polyacrylamide gel electro- phoresis was performed by the method Laemmli [23]. The molecular weight of the native form was estimated according to the method of Andrews

[24] by gel filteration on a column (2 x 90 cm) of Sephadex G-150 equilibrated with 0.1 mM potas- sium phosphate (pH 7.5) containing 0.15 M NaC1/0.1% Tween 20/0.25 mM EDTA/0.1 mM dithiothreitol. Trypsin treatment was performed as follows: each of peroxisomal and mitochondrial carnitine palmitoyltransferases, and carnitine acetyltransferase were mixed with various amounts of trypsin (0-0.8 mg/g of enzyme) in 10 mM potassium phosphate (pH 7.5), and incubated for 15 min at 37 o C. The reaction was stopped by the addition of a 4-fold excess of trypsin inhibitor.

Results

Solubilization of carnitine acyltransferase activities More than 85% of carnitine acetyltransferase

activity was solubilized from the large granule fraction using Tween 20 and KC1 at final con- centrations of 3% and 0.5 M, respectively, but only 65% of carnitine palmitoyltransferase activity was released into the 145 000 x g supernatant fluid (Table I). More than half of the solubilized carni- tine acetyltransferase activity was lost during DEAE-Sephacel column chromatography of the solubilized proteins. This step is necessary for obtaining a good separation of peroxisomal and mitochondrial camitine palmitoyltransferase on blue Sepharose CL-6B column chromatography, and the loss of camitine palmitoyltransferase ac- tivity was less than that of camitine acetyl- transferase activity.

Separation of peroxisomal carnitine palmitoyltrans- ferase, mitochondrial carnitine palmitoyltransferase and carnitine acetyltransferase

Fig. 1 shows the blue Sepahrose CL-6B column chromatography profile of carnitine acyltrans- ferases solubilized from large granule fraction from the liver of clofibrate-treated chick embryo. Two elution peaks of carnitine palmitoyltransferase ac- tivity and one elution peak of carnitine acetyltransferase activity were found. The carni- tine octanoyltransferase activity exhibited the elu- tion pattern as combined peaks of both carnitine palmitoyltransferase and carnitine acetyltrans- ferase activities, and no other peaks than those of carnitine palmitoyltransferase and carnitine acetyltransferase were found in the patterns of

534

TABLE I

PURIFICATION OF CARNITINE ACYLTRANSFERASES IN CHICK EMBRYO LIVER

The starting material was 100 g of clofibrate-treated chick embryo liver. Peroxisomal and mitochondrial carnitine palmitoyl- transferase, carnitine and acetyltransferase were separated by the blue Sepharose CL-6B column chromatography as in Fig. 1. Further purification procedures of peroxisomal and mitochondrial carnitine palmitoyltransferase also correspond to Figs. 2 and 3, respectively.

Protein (mg)

Carnitine palmitoyltransferase Carnitine acetyltransferase

recovery specific activity recovery specific activity

(units) (%) (unit/mg) (-fold) (units) (%) (unit/mg) (-fold)

Large granule fraction 6096 262 145,000 x g supernatant 2656 170 DEAE-Sephacel 599 133

Peroxisomal carnitine palmitoyltransferase Blue Sepharose (Frs. 73-81) 44.5 93.5 DEAE-Sephacel 9.20 67.9 Sephadex G-150 4.36 41.9 DEAE-Toyopearl 2.58 34.0

Mitochondrial carnitine palmitoyltransferase Blue Sepharose (Frs. 11-25) 229 31.8 DEAE-Sephacel 38.8 28.5 DEAE-Toyopearl 7.10 18.0 SephadexG-150 3.46 16.1

Carnitine acetyltransferase Blue Sepharose (Frs. 82-88) 18.1 CM-Sephadex 2.56 SephadexG-150 1.51 Matrex orange A 0.63

100 0.043 1 65 0.064 1.5 51 0.222 5.2

36 2.10 49 26 7.38 172 16 9.61 223 13 13.2 307

12 0.139 3.2 11 0.735 17 7 2.54 59 6 4.65 108

292 100 0.048 1 252 86 0.095 2.0 97.6 33 0.163 3.4

93.9 32 5.19 108 56.8 19 22.2 463 33.8 12 22.4 466 22.9 8 36.3 756

2

2,0

H

~ 1,5 ! 20 40 60 80

FRACTION NUMBER

4

3~

0

Fig. 1. Blue Sepharose CL-6B column chromatography of solubilized carnitine acyltransferase activities from the liver of clofibrate-treated chick embryo. The volume of each fraction was 10 ml. The activities of carnitine palmitoyltransferase (o) and carnitine acetyltransferase (O) were determined. The dotted line represents the protein. The KC1 gradient is indi- cated by the solid thin line.

c a r n i t i n e o c t a n o y l t r a n s f e r a s e ac t iv i ty (da ta no t

shown) .

Purification of peroxisomal carnitine palmitoyltrans- ferase

T h e resul ts o f fu r the r p u r i f i c a t i o n o f pe r -

o x i s o m a l c a r n i t i n e p a l m i t o y l t r a n s f e r a s e are s h o w n

in Fig . 2. T h e e lu t ion p a t t e r n s o f ca rn i t i ne pa l - m i t o y l t r a n s f e r a s e ac t iv i ty on three c h r o m a t o -

g r a p h y c o l u m n s were f o u n d as a s ingle peak .

P e r o x i s o m a l c a r n i t i n e p a l m i t o y l t r a n s f e r a s e e lu ted

f r o m a S e p h a d e x G - 1 5 0 c o l u m n at 1 .8-fold v o l u m e

of v o i d v o l u m e (Fig. 2B). D E A E - T o y o p e a r l ch ro -

m a t o g r a p h y of the p r o t e i n o b t a i n e d by gel f i l t ra- t ion on S e p h a d e x G - 1 5 0 c o l u m n revea led a sym-

me t r i ca l p e a k of c o n s t a n t spec i f ic ac t iv i ty (Fig.

2C).

~4 ~3

v-

~0

"3

==3 v

~-2

<1

0

~2

El t~

10 20 30 40 FRACTION NUMBER

i

(B)

Blue dextran

i i

,'%

.A /\ 50 60 70 80

FRACTION NUMBER

(C)

i0 20 30 40 FRACTION NUMBER

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0.6 g ].00 0,4~

0 , 2 ~

1 3 -

0 0 --

c.o

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Fig. 2. Purification of peroxisomal camitine palmitoyl- transferase (CPT). The peak fractions of camitine palmitoyl- transferase bound to a column of blue Sepharose CL-6B, fractions 73-81 of Fig. 1, were pooled and diluted with buffer C described under Materials and Methods. In A, the protein solutions were applied to a column of DEAE-Sephacel and the column was washed with buffer C. Carnitine palmitoyl- transferase activity (e) was eluted with a linear 0-400 mM KC1 gradient in buffer C. The dotted line represents the protein. The KCI gradient is indicated by the solid thin line. In B, fractions 25-30 of A were pooled, concentrated and gel-filtered on a column of Sephadex G-150 equilibrated with buffer C. 3-ml fractions were collected and void volume was determined using blue Dextran. The symbols are the same as those described in A. In C, fractions 73-80 of B were pooled and applied to a DEAE-Toyopearl column. The column was washed with buffer C and then eluted a linear 0-100 mM KC1 gradient in buffer C. The symbols are the same as those described in A.

Purification of mitochondrial carnitine palmitoyl- transferase

Frac t ions of carn i t ine palmitoyl t ransferase which were not retained on a blue Sepharose

535

v

0

"3

~ 2 v

E1 F -

~ 0

"3

(A) , ' " ' . , / "" 2 v / , , - _ ],oo

i0 20 30 40 FRACTION NUMBER

0,8~

75

0 O--

(B) / /

' l

10 20 30 40 FRACTION NUMBER

' B l u e ' ' ' (C) dextran , ~ -I0.3~ '/i I:! ~ 1 . 0

~- 0.

~0.5 O,

0 ~ " - ~ " ' " ~ : = ~ = ~ ' - - ' - ' I 0 40 50 60 70

FRACTION NUMBER

Fig. 3. Purification of mitochondrial carnitine palmitoyl- transferase (CPT). In A, the fractions of camitine palmitoyl- transferase which was not retained on a blue Sepharose CL-6B column, fractions 11-25 of Fig. 1, were pooled and applied to a column of DEAE-Sephacel equilibrated with buffer D described under Materials and Methods. The column was washed with buffer D, carnitine palmitoyltransferase activity (e) was then eluted with a linear 0-400 mM KC1 gradient in buffer D. The dotted line represents the protein. The KCI gradient is indicated by the solid thin line. Fractions 11-18 of A were pooled and equilibrated with buffer D for the gel filtration on a Sephadex G-25 column. In B, the protein in buffer D was applied to a colunm of DEAE-Toyopearl. The column was washed with buffer D and then eluted with a linear 0-75 mM KC1 gradient in buffer D. The symbols are the same as those described in A. Fractions 38-41 of B were pooled and concentrated. In C, this preparation was subjected to gel filtration on a column of Sephadex G-150 equilibrated with buffer D containing 0.1 M KCl. 3-ml fractions were collected. The symbols are the same as those described in A.

CL-6B column (fractions 11-25) were used for further purification of mitochondrial carnitine palmitoyltransferase and the results are shown in

536

Fig. 3. A further 20-fold purification of mitochon- drial carnitine palmitoyltransferase was attained by ion-exchange chromatography on DEAE-Seph- acel and DEAE-Toyopearl (Fig. 3A,B). The chro- matographic behavior of mitochondrial carnitine palmitoyltransferase on a Sephadex G-150 column was different from that of peroxisomal carnitine palmitoyltransferase, because mitochondrial carnitine palmitoyltransferase passed through the column at ].3-fold void volume (Fig. 3C).

Purification of carnitine acetyltransferase A 30-fold purification of carnitine acetyltrans-

ferase was attained by blue Sepharose CL-6B col- umn chromatography (Table I). The elution pat- terns of the activity on three chromatography col- umns resulted in a single peak (data not shown). The last preparation had a 750-fold specific activ- ity of large granule fraction.

Molecular weight Fig. 4 shows the SDS-polyacrylamide gel elec-

trophoresis of purified preparations of per- oxisomal carnitine palmitoyltransferase, mito-

1 2 3 TOP

92,500

66,200

45,000 ......

31,000

BOTTOM

Fig. 4. SDS-polyacrylamide gel electroplaoresas of the purified peroxisomal carnitine palmitoyltransferase (lane 1), mitochon- drial carnitine palmitoyltransferase (lane 2) and carnitine acetyltransferase (lane 3). The enzymes (about 2 t~g each) were analyzed on a 10% acrylamide slab gel. The molecular weight standards are phosphorylase b (92 500), bovine serum albumin (66200), ovalbumin (45000) and carbonic anhydrase (31 000).

i p i n I 1

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dextean 440~ 232~ ~ l ~ !,7~ 4 !

0 L - ® - - e~ -o -o -o -o~ -~e_e -®-~ J -e.o°'~e_J 40 50 60 70 80

FRACTION NUMBER

Fig. 5. Sephadex G-150 gel filtration of purified peroxisomal carnitine palmitoyltransferase (CPT), mitochondrial carnitine palmitoyltransferase and carnitine acetyltransferase (CAT). Purified peroxisomal CPT, mitochondrial CPT and CAT from chick embryo liver were mixed and applied to a column of Sephadex G-150. 3-ml fractions were collected and the activi- ties of CPT (O) and CAT (O) were assayed. The molecular weight standards are ferritin (440000), catalase (232000), al- dolase (158000), bovine serum albumin (67000) and ovalbu- min (43000). Void volume was determined using blue dextran.

chondrial carnitine palmitoyltransferase and carnitine acetyltransferase were estimated to be M r 64 000, 69 000 and 64 000, respectively. In order to compare native molecular weights of per- oxisomal, mitochondrial carnitine palmitoyl- transferases and carnitine acetyltransferase, three purified enzymes were mixed and chromato-

Fig. 6. Immunoprecipitation of purified enzyme and tissue preparations with antibody raised against purified peroxisomal carnitine palmitoyltransferase (CPT). A, center well, 500 fig of anti-peroxisomal CPT. Wells 1, 2, 3, and 5, 10 ~g of per- oxisomal CPT. Well 4, 10 fig of mitochondrial CPT. Well 6, ]0 t~g of carnitine acetyltransferase. B, center well, 500 t~g of anti-peroxisomal CPT. Wells 1, 3 and 5, 10 #g of peroxisomal CPT. Wells 2 and 4, 500 ~g of solubilized proteins from livers of chick embryo and rat, respectively. Rats were fed a 0.25% clofibrate diet for 2 weeks.

graphed on a Sephadex G-150 column (Fig. 5). The elution pattern of carnitine palmitoyltrans- ferase activity was found as two peaks. The first elution peak can be regarded as mitochondrial carnitine palmitoyltransferase from the results in Fig. 3C, and was estimated to be 260 000. Molecu- lar weights of native enzymes of peroxisomal carnitine palmitoyltransferase and carnitine acetyltransferase were estimated to be 66 000 and 64 000, respectively.

Peroxisomal carnitine palmitoyltransferase im- munology

Fig. 6A represents a immunoprecipitation reac- tion between the antibody raised against purified peroxisomal carnitine palmitoyltransferase and purified enzymes of peroxisomal carnitine pal- mitoyltransferase, mitochondrial carnitine pal- mitoyltransferase and carnitine acetyltransferase. Anti-peroxisomal carnitine palmitoyltransferase reacted only with peroxisomal camitine palmitoyl- transferase, but not with mitochondrial carnitine palmitoyltransferase and carnitine acetyltrans- ferase. Fig. 6B shows the reaction between anti- peroxisomal carnitine palmitoyltransferase and proteins solubilized from rivers of chick embryo and rats which were treated with clofibrate. Anti- peroxisomal carnitine palmitoyltransferase reacted with chick embryo liver, but not with rat liver.

From the activities of marker enzymes in Table

TABLE II

PROPERTIES OF ISOLATED ORGANELLES

Peroxisomes, mitochondria and microsomes were isolated as described. Catalase, cytoehrome c oxidase and NADPH-cyto- chrome c reductase were used as markers for peroxisomes, mitochondria and microsomes, respectively. Units of enzyme activities are expressed as previously described [27]. Results are given as units/fraction.

Catalase Cyto- NADPH- Protein chrome c cytochrome (mg/ oxidase c reductase fraction)

Peroxisomal fraction 0.756 0.16 2.60 0.46

Mitoehondrial fraction 0.012 173 3.96 5.47

Mierosomal fraction n.d. 2.44 43.5 5.07

TOP

(A) 1 2 3 4 5

537

(B) 1 2 3 4 5

BOTTOM-" :

Fig. 7. Western blotting of peroxisomal, mitochondrial and microsomal proteins from SDS-polyacrylamide gel. A, gel stained with Coomassie blue; B, autoradiograph of blot of an identical gel with antibody against purified peroxisomal carni- tine palmitoyltransferase (CPT). Lane 1, purified peroxisomal CPT; lane 2, mitochondrial CPT; lanes 3, and 4 and 5, solubilized proteins from isolated peroxisomes, mitochondria and microsomes, respectively, from the liver of clofibrate- treated chick embryo in Table II.

II, it was found that peroxisomes, mitochondria and microsomes were cleraly isolated. Fig. 7A shows the Coomassie blue-stained SDS-poly- acrylamide gel electrophoretic patterns of the solubilized proteins from isolated organelles in Table II. The identical protein bands with per- oxisomal carnitine palmitoyltransferase and mitochondrial carnitine palmitoyltransferase were observed in peroxisomes and mitochondria, re- spectively. Fig. 7B shows the autoradiograms of western blots of SDS-polyacrylamide gel electro- phoresis of the proteins solubilized from isolated organelles using antibody against purified per- oxisomal camitine palmitoyltransferase. The iden- tical protein band with peroxisomal camitine pal- mitoyltransferase was also immunochemically ob- served only in peroxisomes. And besides, since some protein bands, which were different from peroxisomal camitine palmitoyltransferase, were observed in peroxisomes, the antibody raised against peroxisomal carnitine palmitoyltransferase can also react weakly with proteins other than peroxisomal carnitine palmitoyltransferase, and all cross-reactants with anti-peroxisomal carnitine palmitoyltransferase were present in peroxisomes but not in mitochondria or microsomes.

538

Comparison of K m and Vma x values of peroxisomal carnitine palmitoyltransferase and mitochondrial palmitoyltransferase

Table III shows the K m and Vma x values of peroxisomal and mitochondrial carnitine palmito- yltransferase for palmitoyl-CoA, L-carnitine, pal- mitoyl-L-carnitine and CoA in both forward and reverse reactions. The g m values of peroxisomal carnitine palmitoyltransferase for palmitoyl-CoA were similar to that of mitochondrial carnitine palmitoyltransferase in both the absence and pres- ence of Tween 20. But the K m values of per- oxisomal carnitine palmitoyltransferase for L- carnitine, palmitoyl-L-carnitine and CoA were ap- parently different from those of mitochondrial carnitine palmitoyltransferase in both detergent conditions, and peroxisomal carnitine palmitoyl- transferase had about 10-fold higher affinities for L-carnitine and CoA in comparison with mitochondrial carnitine palmitoyltransferase. The Vma x value of peroxisomal carnitine palmitoyl- transferase in the forward reaction was decreased by the addition of Tween 20, while that of mitochondrial carnitine palmitoyltransferase was increased, and peroxisomal carnitine palmitoyl- transferase had a higher Vma x value in the forward reaction in the absence of Tween 20.

Substrate specificity Fig. 8 shows the substrate specificities of per-

oxisomal carnitine palmitoyltransferase, mito- chondrial carnitine palmitoyltransferase and carnitine actetyltransferase using acyl-CoAs or

TABLE III

COMPARISON OF PEROXISOMAL AND MITOCHON- DRIAL CARNITINE PALMITOYLTRANSFERASE (CPT) ON K m VALUES AND Vm~ x VALUES FOR SUBSTRATES

The values obtained in the absence of Tween 20 are shown in parentheses.

Peroxisomal CPT Mitochondrial CPT

Km Vmax Km Vma x (/t M) (un i t / ( # M) u n i t /

mg) mg)

Forward reaction

palmitoyl- CoA 32 (9.1) L-carnitine 140 (150)

Reverse reaction

palmitoyl- -L-carnitine 43 (33) CoA 9

11.1 (29.2) 32 (9.2) 23.6 (11.4) 7.0 (20.5) 1020 (930) 18.4 (16.5)

4.5 (4.8) 70 (250) 13.3 (24.2) 4.2 110 19.1

acylcarnitines of various chain lengths. Per- oxisomal carnitine palmitoyltransferase has a maximum activity with lauroyl derivatives, and exhibited an identical substrate specificity in both the forward and reverse reactions. In mitochon- drial carnitine palmitoyltransferase, the maximum activity was obtained with decanoyl-CoA and myristoylcarnitine, and the substrate specificity in the forward reaction was different from that in the reverse reaction. The substrate specificity of this enzyme was similar to that of purified carnitine

I / (A) PEROXISOMAL CPT

I 25[- o i oL /,\

:o oi: "} 5

< oL4" ' ' ' t , i i~_ 2 4 6 8 I0 12 1416 18

CHAIN LENGTH

(B) MITOCHONDRIAL CPT

10

8 o\ ,"~'~

/4 l , 0

0-~ i I i I I J

2 4 6 8 1012 14 16 18 CHAIN LENGTH

(C) CAT

16 o

8 'Q',,

4 ',

o . . . .

CHAIN LENGTH

Fig. 8. Substrate specificities of purified carnitine acyltransferases. Forward reaction velocities (O ) and reverse reaction velocities (e) were assayed as described under Materials and Methods. CPT, carnitine palmitoyltransferase; CAT, carnitine acetyhransferase.

palmitoyltransferase from bovine heart [25] and rat liver [3]. Carnitine acetyltransferase exhibited a relatively sharp substrate specificity with the highest activity toward acetyl derivatives in both the forward and reverse reactions (Fig. 3C). The substrate specificity of this enzyme was similar to that of carnitine acetyltransferase in pigeon breast muscle [26], but different from that of carnitine acetyltransferase in mammarian tissues, in which the enzyme has a maximum activity with butyryl- and propionyl-CoA [4-5,25,27].

Kinetic constants Tables IV and V show the apparent K m values

of enzymes for all substrates in the forward and reverse reactions. In the forward reaction, the K m values of peroxisomal carnitine palmitoyltrans- ferase for long-chain acyl-CoAs (C10-C16) were similar to those of mitochondrial carnitine pal- mitoyltransferase. In the reverse reaction, the K m values of peroxisomal carnitine palmitoyl- transferase for acylcarnitine and CoA with long- chain acylcarnitines (C10-C16) were lower than those of mitochondrial carnitine palmitoyl-

TABLE IV

K m VALUES OF PEROXISOMAL C AR NIT INE PAL- MITOYLTRANSFERAS E (CPT), M I T O C H O N D R I A L CPT A N D C A R N I T I N E ACETYLTRANSFERASE (CAT) FOR ACYL-CoAs A N D C A R N I T I N E

Assays were carried out as described under Materials and Methods. K m values for L-carnitine were expressed when 100 # M various chain length acyl-CoAs were used as cosubstrates. C2-C18 represent carbon chain lengths of acyl-CoAs. The values are expressed a s / t M .

K m for acyl-CoA K m for carnitine

peroxi- mito- CAT peroxi- mito- CAT somal chon- somal chon- CPT drial CPT drial

CPT CPT

C2°CoA 52 C4-CoA 150 130 450 Cr-CoA 46 160 66 1160 860 Cs-CoA 36 65 94 340 1160 Clo-CoA 14 17 140 1630 C12-CoA 71 72 190 1780 C14-CoA 34 35 190 1710 C16-CoA 32 32 140 1020 C18-CoA 35 520

160 250

1100 2000

539

TABLE V

K m VALUES OF PEROXISOMAL CARNITINE PAL- MITOYLTRANSFERASE (CPT), M I T O C H O N D R I A L CPT A N D C A R N I T I N E A C E T Y L T R A N S F E R A S E F O R A C Y L C A R N I T I N E A N D CoA

Assays were carried out as described under Materials and Methods. K m values for CoA were expressed when 500 #M acyl-oL-carnitines of various chain lengths were used as cosubstrates. The values are expressed as #M.

K m for acylcarnitine K m for CoA

peroxi- mito- CAT peroxi- mito- somal chon- somal chon- CPT drial CPT drial

CPT CPT

CAT

C2-carnitine 800 C4-carnitine 800 890 240 C6-carnitine 1100 1000 900 250 250 Cs-camitine 200 850 670 230 230 C lo-carnitine 80 830 38 160 C12-carnitine 63 190 57 100 C14-carnitine 61 110 33 160 C 16-carnitine 91 130 9 110 C 18 -carnitine 100 70

86 53 78 66

transferase. However, the K m values of per- oxisomal enzyme for acylcarnitine and CoA with medium-chain acyl derivatives (C6-C8) were rela- tively high. Thus, when the long-chain acyl deriva- tives (C10-C16) were used as substrates, per- oxisomal carnitine palmitoyltransferase has high affinities for all substrates in both the forward and reverse reactions, but it has low affinities for substrates in the reverse reaction using medium- chain acyl derivatives. Because DL isomers of acylcarnitines were used as substrates in Table V, the K m values of peroxisomal carnitine palmitoyl- transferase and mitochondrial carnitine palmitoyl- transferase for palmitoylcarnitine were 91 and 130 /zM, respectively, and just 2-fold higher than the corresponding K m values for palmitoyl-L-carnitine in Table III. From these findings, the K m values for L isomers of acylcarnitines must be half those for DE isomers: therefore peroxisomal carnitine palmitoyltransferase has relatively similar K m val- ues (30-50 #M) for acyl-CoA, acylcarnitine and CoA using long-chain acyl derivatives (C10-C16). In carnitine acetyltransferase, the K m values for substrates in the reverse reaction were constant, but in the forward reaction, the lowest K m value

540

for acyl-CoA was obtained with acetyl-CoA. The K m value for carnitine was increased from 160 /~M using acetyl-CoA as cosubstrate to 2000 ttM using octanoyl-CoA, and this is similar to the results with mouse liver carnitine acetyltransferase [281.

Effect of trypsin Since some studies have shown the inactivation

of carnitine acyltransferase by trypsin [28,29], we investigated its effect on purified enzymes of chick embryo liver (Fig. 9). All carnitine acyltransferases were completely inactivated with 800 ~g trypsin/g carnitine acyltransferases. Mitochondrial carnitine palmitoyltransferase was more sensitive to trypsin than peroxisomal carnitine palmitoyltransferase and carnitine acetyltransferase. 50% inactivation of mitochondrial carnitine palmitoyltransferase activity was observed with 10 /~g of trypsin/g of enzyme, while a higher concentration of trypsin (150-200 ~g of trypsin/g of enzyme) was required to obtain 50% inactivation of peroxisomal carni- tine palmitoyltransferase and carnitine acetyl- transferase activities.

pH optima for peroxisomal carnitine palmitoyl- transferase, mitochondrial carnitine palmitoyl- transferase and carnitine acetyltransferase

The pH optima for peroxisomal carnitine pal- mitoyltransferase and carnitine acetyltransferase

100

, , , , , I I ,

P

75 ~',~°~o

0 100 200 300 400 80"0 pG TRYPSIN/p6 ENZYME

Fig. 9. Effect of trypsin on purified carnitine acyltransferases. Peroxisomal carnitine palmitoyltransferase (©), mitochondrial carnitine palmitoyltransferase (e) and carnitine acetyltrans- ferase (A) were incubated with various amounts of trypsin and the reaction stopped by the addition of trypsin inhibitor as described under Materials and Methods.

were 8.5 and 8.0, respectively, while mitochondrial carnitine palmitoyltransferase showed high activ- ity between pH 6.5 and pH 8.0.

Discussion

Peroxisomal carnitine palmitoyltransferase of chick embryo liver was an M r 64 000 polypeptide according to the results of SDS-polyacrylamide gel electrophoresis and gel filtration, whereas mitochondrial carnitine palmitoyltransferase had a subunit molecular weight of 69000, and the apparent molecular weight of the native form was 260000 in the presence of the detergent to main- tain the enzyme solubilized. The molecular prop- erty of peroxisomal carnitine palmitoyltransferase was similar to that of carnitine octanoyltrans- ferase purified from rat liver [3] and mouse liver [28]. However, the substrate specificity of per- oxisomal carnitine palmitoyltransferase was dif- ferent from that of purified carnitine oc- tanoyltransferase [5,7]. And besides, no reaction was detected between antibody raised against purified peroxisomal carnitine palmitoyltrans- ferase and the liver of rat which had been treated with ciofibrate (Fig. 6). Since rat liver contains carnitine octanoyltransferase which is induced by the treatment with clofibrate [8,9], peroxisomal carnitine palmitoyltransferase in chick embryo liver may be also immunologically different from carnitine octanoyltransferase of rat liver.

Our present findings indicate that carnitine acyltransferase in chick embryo liver consists of three enzyme proteins (i.e., peroxisomal and mitochondrial carnitine palmitoyltransferases, and carnitine acetyltransferase), and that peroxisomal carnitine palmitoyltransferase is a protein distinct from mitochondrial carnitine palmitoyltransferase or carnitine acetyltransferase. Thus, peroxisomal carnitine palmitoyltransferase in chick embryo liver is a new peroxisomal enzyme.

All carnitine acyltransferases in chick embryo liver were inhibited by D-carnitine and Ca 2+ ion (data not shown). Peroxisomal carnitine palmit- oyltransferase and mitochondrial carnitine palmit- oyltransferase have similar affinity for palmitoyl- CoA. However, the former has higher affinities for carnitine in the forward reaction and for palmit- oylcarnitine and CoA in the reverse reaction. Fur-

thermore, in the forward reaction in the absence of Tween 20 peroxisomal carnitine palmitoyl- transferase has a higher Irma x value compared with those of mitochondrial palmitoyltransferase. Thus, peroxisomal carnitine palmitoyltransferase can catalyze palmitoyl derivatives enough. The affini- ties of peroxisomal carnitine palmitoyltransferase for carnitine, acylcarnitine and CoA when using long-chain acyl derivatives (C10-C16) as cosub- strate were higher than those of mitochondrial camitine palmitoyltransferase, and both enzymes had similar affinities for long-chain acyl-CoAs (C10-C16). Furthermore, peroxisomal carnitine palmitoyltransferase exhibited an identical sub- strate specificity in both forward and reverse reac- tions, and the substrate specificity of peroxisomal carnitine palmitoyltransferase was corresponded well with that of peroxisomal fl-oxidation in chick embryo liver [20]. The activities of carnitine pal- mitoyltransferase and fatty acyl-CoA oxidase in chick embryo liver peroxisomes were 82 and 52 nmol/min per mg protein, respectively, and those of carnitine palmitoyltransferase and fatty acyl- CoA oxidase in chick embryo liver peroxisomes were 82 and 52 nmol/min per mg protein, respec- tively, which increased by the same extent with the treatment with clofibrate [13]. These findings indi- cate that peroxisomal carnitine palmitoyl- transferase catalyzes actively both the forward and reverse reactions with long-chain acyl derivatives (C10-C16), and that peroxisomal carnitine pal- mitoyltransferase keeps the relation with per- oxisomal fl-oxidation. Peroxisomal carnitine pal- mitoyltransferase may have a role in the coversion of medium chain acyl-CoAs to medium-chain acylcarnitines like carnitine octanoyltransferase of mouse liver [30].

We have reported previously [20] that the con- tents of triacylglycerol and free fatty acid were markedly increased just before the hatching of the chick embryo, and the activity of peroxisomal fl-oxidation was correspondingly increased. Ac- cordingly, the large amount of free fatty acid may be activated to long-chain acyl-CoA and then oxidized by peroxisomal fl-oxidation. If per- oxisomal carnitine palmitoyltransferase is not pre- sent in chick embryo liver, the rapid increase of long-chain acyl-CoA would cause the decrease of the CoA/long-chain acyl-CoA ratio in per-

541

oxisomes and inhibit peroxisomal fl-oxidation [31,32]. However, peroxisomes in chick embryo liver contain carnitine palmitoyltransferase. We have also found that this enzyme catalyzed ac- tively in both forward and reverse reactions with long-chain acyl derivatives, and was not inhibited by the high concentration of palmitoyl-CoA. Therefore, when the content of long-chain acyl- CoA in peroxisomes is high, long-chain acyl-CoA can be converted to long-chain acylcarnitine by peroxisomal palmitoyltransferase and supply free CoA accordingly. When the content of long-chain acyl-CoA is decreased with the proceeding of fl-oxidation, peroxisomal carnitine palmitoyl- transferase can produce long-chain acyl-CoA from long-chain acylcarnitine and CoA which is sup- plied from medium-chain acyl-CoAs and acetyl- CoA. Thus, it is suggested that the physiological role of peroxisomal carnitine palmitoyltransferase is the modulation of the CoA/long-chain acyl-CoA ratio in peroxisomes in order to keep the active oxidation of fatty acid in peroxisomes of chick embryo liver.

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