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Characterization of two D-β-hydroxybutyrate dehydrogenase populations in heavy and light mitochondria from jerboa (Jaculus orientalis) liver 

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Characterization of two D - β -hydroxybutyrate dehydrogenase populations in heavy and light mitochondria from jerboa (Jaculus orientalis) liver

Driss Mountassif

a,

, Mostafa Kabine

a

, Norbert Latruffe

b

, M'Hammed Saïd El Kebbaj

a

aLaboratoire de Biochimie, Faculté des Sciences, Université Hassan II-Aïn Chock, km 8 route d'El Jadida BP. 5366, Casablanca, Morocco

bLBMC (GDR-CNRS no 2583), Faculté des Sciences, 6 Bd Gabriel, Université de Bourgogne, 21000 Dijon cedex, France Received 7 April 2005; received in revised form 15 November 2005; accepted 17 November 2005

Abstract

Mitochondrial membrane-bound and phospholipid-dependent D-β-hydroxybutyrate dehydrogenase (BDH) (EC 1.1.1.30), a ketone body converting enzyme in mitochondria, has been studied in two populations of mitochondria (heavy and light) of jerboa (Jaculus orientalis) liver. The results reveal significant differences between the BDH of the two mitochondrial populations in terms of protein expression, kinetic parameters and physico-chemical properties. These results suggest that theβ-hydroxybutyrate dehydrogenases from heavy and light mitochondria are isoform variants. These differences in BDH distribution could be the consequence of cell changes in the lipid composition of the inner mitochondrial membrane of heavy and light mitochondria. These changes could modify both BDH insertion and BDH lipid-dependent catalytic properties.

© 2005 Elsevier Inc. All rights reserved.

Keywords:D-β-Hydroxybutyrate dehydrogenase; Jerboa (Jaculus orientalis); Heavy and light mitochondria; Isoforms

1. Introduction

Mitochondria represent the main energy producer of the cell.

They are also the sites of production for ketone bodies. These compounds, resulting from the degradation of fatty acids, play a role in the energy metabolism of extrahepatic tissues. The interconversion of ketone bodiesβ-hydroxybutyrate and acet- oacetate is achieved by D-β-hydroxybutyrate dehydrogenase (BDH) (EC 1.1.1.30) first described in dog liver tissue (Wake- man and Dakin, 1909). In eukaryotic cells, BDH is an inner mitochondrial membrane-bound enzyme, tightly associated with the NAD-linked electron transport chain, where its active site is located on the matrix side of mitochondria (Wise and

Lehninger, 1962; Nielsen et al., 1973; Latruffe and Gaudemer, 1974a; Gaudemer and Latruffe, 1975; McIntyre et al., 1978).

BDH is synthesized in the cytoplasm as a precursor with a larger size and post translationally imported into mitochondria involving the processing of its N-terminal presequence (Kante et al., 1987). BDH has been studied in numerous organisms:

Rhodopseudomonas spheroides(Bergmeyer et al., 1967), beef heart (Nielsen et al., 1973), rat liver (Latruffe and Gaudemer, 1974b), ruminant's heart and liver (Cherkaoui-Malki et al., 1992) and dromedary liver (Nasser et al., 2002). The molecular mass of the subunit size of the purified BDH was 31.5 kDa for bovine heart, rat liver, and rat brain mitochondria (Bock and Fleischer, 1975; Vidal et al., 1977; McIntyre et al., 1988; Zhang and Churchill, 1990) and about 67 kDa for dromedary liver (Nasser et al., 2002). Purified BDH is devoid of lipid and can insert spontaneously and unidirectionally into performed phos- pholipid vesicles or natural membranes (McIntyre et al., 1979).

It has previously been proposed that activation of BDH by phosphatidyl–choline (PC) containing liposomes involves an allosteric mechanism (Sandermann et al., 1986), whereby PC enhances coenzyme binding (Rudy et al., 1989). The primary sequence of BDH was initially determined for the enzyme from rat liver (Churchill et al., 1992) and more recently for the

Abbreviations:BDH,D-β-hydroxybutyrate dehydrogenase;DL-BOH,DL-β- hydroxybutyrate; EDTA, ethylenediamine tetraacetic acid; ELISA, enzyme- linked immunosorbent assay; HEPES, 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid; Mes, 4-N-morpholinoethanesulfonic acid; NEM,N-ethylmalei- mide; NAD(H), nicotinamide adenine dinucleotide oxidized/(reduced) forms;

PGO, phenylglyoxal; SDSPAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis; TMB, tetramethyl benzidine; Tris, trishydroxymethyl aminomethane.

Corresponding author. Tel.: +212 22 23 06 80/84; fax: +212 22 23 06 74.

E-mail address:drissmountassif@yahoo.fr (D. Mountassif).

1096-4959/$ - see front matter © 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.cbpb.2005.11.019

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enzyme from human heart (Marks et al., 1992) by cDNA cloning. The mature form of this enzyme consists of 297 amino acids and Northern blot analysis identifies a 1.3-kb mRNA (Marks et al., 1992).

A comparison of the amino acid sequences of BDH with other reported sequences reveals a homology with the super- family of short-chain alcohol dehydrogenases. The N-terminal two-thirds of the enzyme include the coenzyme-binding domain and putative active site conserved residues. The C-terminal third of BDH and other family members show little sequence homol- ogy and this region likely contains elements responsible for the binding of BDH. The catalytic activity of the enzyme is lecithin- dependent (Sekuzu et al., 1961; Gazzoti et al., 1964). As reported by Williamson et al. (1971), in liver, the enzyme catalyses the transformation of acetoacetate intoD-β-hydroxy- butyrate in the presence of NADH, which is then transported through the blood stream to peripheral tissues, i.e. brain, heart, kidney, etc. In extrahepatic tissues,D-β-hydroxybutyrate is con- verted into acetoacetate in the presence of NAD+. Acetoacetate, in turn, is used, after its conversion to acetyl-CoA, by the respiratory chain as fuel for ATP production, or after formation of acetoacetyl-CoA, for fatty acid synthesis. A catalytic mech- anism of the interconversion ofD-3-hydroxybutyrate and acet- oacetate in both liver and peripheral tissues has been previously proposed by our group (El Kebbaj and Latruffe, 1997).

The jerboa (Jaculus orientalis), a nocturnal herbivorous ro- dent living in the subdesert highland of Morocco was chosen for our study for the following reasons. This rodent is an appropri- ate organism to study metabolic regulation due to its remarkable resistance to heat, arid conditions and especially to cold (Hooper and El Hilali, 1972). Also, this rodent is a true hibernator developing obesity by accumulating fat during its prehiberna- tion period. Its fat is eliminated during hibernation leading to a high production ofD-β-hydroxybutyrate, via BDH, to serve as an energy source in addition to carbohydrates (Kante et al., 1990).

The present work deals with the characterization and the comparison ofD-β-hydroxybutyrate dehydrogenase of the two mitochondrial populations. The population of large mitochon- dria we shall refer to as“heavy”(0.85μm in length and 0.5μm in width) and the population of small mitochondria we refer to as“light”(0.35μm in length and 0.25μm in width).

In previous studies, heavy and light mitochondria were isolated from rat liver and combined to get high mitochondria content in order to purify a significant amount of BDH (Latruffe and Gaudemer, 1974b; Gaudemer and Latruffe, 1975; El Kebbaj and Latruffe, 1997). Here, for the first time, we isolated the two mitochondria populations from jerboa liver separately to study their individual properties including BDH activity, content and properties.

The existence in cells of two populations of mitochondria (heavy and light) is well known in rat liver and beef heart (Fleischer et al., 1979). The two mitochondrial populations are distinct both by their lipid and protein compositions. This is probably related to their different stages in the biogenesis pro- cess, since almost all lipid and protein are imported from the cytosol.

The explanations on the biological significance of different BDH isoforms may relate to the differences on lipid composi- tion of the mitochondrial membrane, which would modify both the BDH structure (and as lecithin requiring enzyme, conse- quently its activity) and the pre-BDH import and insertion in the inner mitochondrial membrane.

2. Materials and methods 2.1. Animals

Adult greater Egyptian jerboas (J. orientalis, Erxleben, 1777; Rodentia, Dipodidae) (120–150 g, 4–6 months old), were captured in the area of Engil Aït Lahcen (in subdesert East Moroccan highland). They were adapted to laboratory conditions during 3 weeks at a temperature of 22 °C with food (salad and rat chow), and water ad libitum before they were killed. The light cycle during the experiment was set to 14-h light and 10-h dark.

2.2. Liver mitochondria and mitoplast isolation

The jerboas were decapitated and the liver was rapidly removed for mitochondrial extraction according to Fleischer et al. (1979). This method allows the preparation of the two mitochondrial populations.

The mitoplast (outer mitochondrial membrane-free mito- chondria) preparation was done according to the method de- scribed byKielley and Bronk (1958). Liver mitochondria were swelled in a 20 mM phosphate buffer at 0.5 mL/mg of protein for 30 min at 0 °C. The medium was then centrifuged at 1200×gfor 30 min. The mitoplasts were collected in the pellet.

2.3. Protein assay

Protein content was measured according to the Bradford (1976) procedure, using bovine serum albumin (BSA) as standard.

2.4. Thiol determination

Thiols were assayed according to the technique of Ellman (1959). Briefly, 200 μL of 5% SDS and 795 μL of 20 mM potassium phosphate were added to 5 μL of sample. After 15 min, 100μL of 2 mM 5, 5′-dithiobis-(2-nitrobenzoic acid) (DTNB) were added. After 30 min at 25 °C, the absorbance was measured at 405 nm against a blank containing all reagents except mitochondria. A standard range from 0 to 125 nmol of thiol was established from a stock solution of 2 mM reduced glutathione.

2.5. Phospholipid extraction and composition

Phospholipids were extracted according to the technique of Rouser and Fleischer (1967). One volume of mitoplasts was added to chloroform/methanol/0.8% KCl (13.3:6.7:4.2 v/v/v).

The mixture was homogenized with an UltraTurrax at

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7500 rpm for 3 min. After sedimentation, the organic phase was recovered and 0.8% KCl/methanol/chloroform (47:48:3 v/v/v) was added. The chloroform phase was then concentrated in a rotary evaporator. The amount of phospholi- pids was determined by measuring the phosphorus concentra- tion according toChen et al. (1956).

10 μL of sample were mineralized with 450 μL of 70%

perchloric acid for one h. After 10 min, 4.5 mL of water, 500μL of 2.5% ammonium molybdate and 500 μL of 10% ascorbic acid were added. The mixture was then incubated for 5 min in boiling water for color development. After 10 min of cooling, the absorbance was measured at 820 nm against a blank con- taining all the reagents. A standard range from 0 to 5 μg of phosphorus was established with KH2PO4 at 2μg/mL.

Thin layer chromatography was carried out on dried silica- gel plates (20 cm × 20 cm). Phospholipid phosphorus (40μg) was loaded, and plates were developed with chloroform/meth- anol/water (65:25:4 v/v/v). Spots were revealed with 2,7- dichlorofluoresceine. The bands were scraped off, mineralized and their phosphorus content was measured.

2.6. Enzyme assays

Subcellular marker enzymes were assayed according to the following methods: succinate dehydrogenase (King, 1967) for mitochondria; palmitoyl-CoA oxidase (Lazarow and De Duve, 1976) for peroxisomes; NADPH–cytochrome c reduc- tase (Beaufay et al., 1974) for microsomes; and glyceralde- hyde-3-phosphate dehydrogenase (Serrano et al., 1991) for cytosol.

BDH activity was measured at 37 °C as described by Lehninger et al. (1960), by monitoring NADH production at 340 nm (ε= 6.22 × 103M−1cm−1) using 100μg of protein in a medium containing: 6 mM potassium phosphate at pH 8, 0.5 mM EDTA, 1.27% (v/v) redistilled ethanol, 0.3 mM dithiothreitol, in the presence of 2 mM NAD+ (Sigma- Aldrich) and 2.5 μg rotenone (final addition). The reaction was started by the addition of DL-β-hydroxybutyrate (Sigma) to 10 mM final concentration.

2.7. BDH kinetic studies

Initial velocities were measured at varying the concentration of BOH (from 2.5 to 10 mM) or NAD+ (from 0.5 to 2 mM).

Michaelis constants (Km), dissociation constants (KD) and max- imal velocity for the oxidation of BOH and the reduction of NAD+ by the BDH were obtained by mathematical analysis followingCleland (1963).

2.8. BDH chemical modification

Previous studies published by our laboratory showed that the BDH active site contains one essential cysteine and one essential arginine for BDH activity (Latruffe et al., 1980; El Kebbaj et al., 1980, 1982, El Kebbaj and Latruffe, 1997).

Therefore, we used two chemical modifiers:N-ethylmaleimide (NEM) (Sigma) and phenylglyoxal (PGO) (Aldrich) known to react specifically with cysteyl (Latruffe et al., 1980) and arginyl residues (El Kebbaj et al., 1980). After thawing, liver mito- chondrial protein (100μg) was preincubated for 5 min at 25 °C in buffer (6 mM potassium phosphate pH 8, 0.5 mM EDTA, 1.27% (v/v) redistilled ethanol). The inhibitor was added at zero time. Aliquots were removed at different times during the incubation for the measurement of enzymatic activity.

2.9. Determination of optimal pH- and temperature-dependent BDH activity

The effect of pH on BDH activity was studied in range from pH 4 to pH 10 using a mixture of different buffers (Tris, Mes, HEPES, potassium phosphate and sodium acetate).

Temperature effects were characterized by activation and denaturation processes:

For activation, the buffered medium containing 6 mM po- tassium phosphate pH 8, 0.5 mM EDTA, 1.27% (v/v) redis- tilled ethanol was incubated for 2 min at temperatures from 5 to 80 °C. Then, 2.5 μg of rotenone, 2 mM of NAD+ and 100 μg of protein were added. After 2 min at the temperature studied, the reaction was started immedi- ately by the addition of 20 mM of BOH.

For denaturation, 100μg of liver mitochondrial protein were incubated at temperatures from 5 to 80 °C for 2 min in medium containing 6 mM potassium phosphate pH 8, 0.5 mM EDTA, 1.27% (v/v) redistilled ethanol. Then, 2.5μg of rotenone and 2 mM of NAD+were added and the enzymatic activities were measured by the later addition of 20 mM of BOH after 2 min of incubation at 37 °C.

Table 1

Subcellular marker enzyme activities in two mitochondrial isolated fractions from jerboa liver compared to liver homogenate Homogenate Heavy mitochondrial

fraction

Light mitochondrial fraction

Mitochondria Succinate dehydrogenase (absorbance/min/mg protein) 1.04 ± 0.12 2.25 ± 0.18 [× 2.16]⁎ 1.83 ± 0.21 [× 1.76]⁎

Peroxisomes Palmitoyl-CoA oxydase (nmol/min/mg protein) 7.52 ± 0.47 0.58 ± 0.04 [× 0.07]⁎ 1.48 ± 0.12 [× 0.2]⁎

Microsomes NADPHcytochrome c reductase (nmol/min/mg protein) 79.3 ± 9.44 15.74 ± 3.25 [× 0.2]⁎ None Cytosol Glyceraldehyde-3-phosphate dehydrogenase (μmol/min/mg

protein)

0.89 ± 0.05 None None

Mitochondrial purity 72% 80%

Values are given in specific activities. Numbers in brackets correspond to the variation compared to homogenate. Values are given as means ± S.D. of four separate experiments. For experimental conditions, see Materials and methods.

Variations statistically significant atpb0.01.

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For the kinetic study of BDH thermal stability, several mitochondrial aliquots (100μg) from the same protein ex- tract were incubated at 37 °C and BDH activity was mea- sured at various times of incubation.

A BDH Arrhenius plot was obtained by measuring the enzymatic activity at temperatures from 5 to 40 °C and ana- lyzed as described byRaison (1973).

2.10. Western blotting

After SDS–PAGE (12%) (Laemmli, 1970) and subsequent transfer to nitrocellulose (Towbin et al., 1991), the mitochon- drial proteins (50 μg) were exposed to a 1:100 dilution of monospecific polyclonal anti-BDH antibody (BDH rat liver) and detected with the secondary antibody of anti-rabbit, IgG peroxidase conjugate (diluted 1:2500) (Promega).

Table 2

Characterization of the liver heavy and light mitochondria and lipid composition analysis by thin layer chromatography of the heavy and light mitoplasts Total protein/liver

weight (mg/g)

Total thiol groups/protein (nmol/mg)

Total phospholipid phosphorus/liver weight (mg/g)

Proteins/phospholipid phosphorus ratio

Phospholipids (%)

PE PI/PS CL PC

Heavy mitochondria 92.88 ± 5.76 100 ± 7 10.8 ± 0.9 8.6 28 ± 6 32 ± 5 17 ± 2 23 ± 3

Light mitochondria 83.2 ± 4.8 56⁎± 8 11 ± 1.2 7.56⁎ 33 ± 5 38 ± 4 15 ± 3 14⁎± 1

PE, phosphatidyethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; PC, phosphatidylcholine; CL, cardiolipin. After deposit of 40μg lipid phosphorus of phospholipids, migration and revelation, the amount of phosphorus was measured in each band as described in Materials and methods. Values are given as means ± S.D.

of three independent experiments and have taken into account the mitochondrial fractions purities (Table 1).

⁎pb0.01 and⁎⁎pb0.05 (Student'st-test).

0%

20%

40%

60%

80%

100%

5 15 25 35 45 55 65 75 5 15 25 35 45 55 65 75

T˚C Relative

activity

Activation Denaturation

A

0%

20%

40%

60%

80%

100%

T˚C Relative

activity

Activation Denaturation

B

KH = 16 min-1 KL = 2.4 min-1

0 0.2 0.4 0.6 0.8 1 1.2

0 10 20 30 40 50

Time (min) Log

of relative activity

Light mitochondrial BDH Heavy mitochondrial BDH

C

Fig. 1. Effect of the temperature on the activity of the liver BDH from heavy (A) and light (B) mitochondria. This was followed by the activation and denaturation processes at different temperatures (from 5 to 80 °C). Values are given as means of three separate experiments. Thermal stability of liver BDH activity from heavy and light mitochondria at 37 °C (C). Liver mitochondrial proteins (100μg) were incubated at 37 °C and the BDH activity was measured at various incubation times.

Values are given as means ± S.D. of three separated experiments.

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2.11. Enzyme-linked immunosorbent assay

The ELISA method was performed according to Kemeny (1991). Mitochondrial proteins (10 μg) were exposed to 1:100 dilution of monospecific polyclonal anti-BDH antibody (BDH rat liver) and detected with the secondary antibody of anti-rabbit, IgG peroxidase conjugate (diluted 1:2500) (Pro- mega) and the absorbance at 410 nm was measured with an ELISA reader after addition of tetramethyl benzidine (TMB) (Sigma).

2.12. Zymogram gel

It was carried out in 5% (w/v) acrylamide gel under non- denaturing conditions. The gel was stained for in situ location of NAD-linked BDH activity bands by incubation for one h in the standard reaction mixture for the oxidation reaction con- taining NAD+ (2 mM) supplemented with 20 μM phenazine methosulphonate and 1 mM p-nitroblue tetrazolium chloride.

The enzyme activity band (50μg) was located by the appear- ance of a deep purple band of the resulting insoluble forma- zan salt.

2.13. Statistical analysis

In each assay, the experimental data represent the mean of three independent assays ± S.E.M. Means were com- pared using Student's t-test. Differences were considered significant at pb0.05 and very significant at the level pb0.01.

3. Results

3.1. Biochemical characterization of mitochondrial populations of jerboa

We isolated two mitochondrial populations, heavy and light fractions, from liver of active jerboa. InTable 1, we report the relative activities of subcellular marker enzymes in the two mitochondrial fractions. As Table 1 show, the two fractions present high mitochondria content with a low contamination of peroxisomes, microsomes and cytosol. From the data, the purity is estimated at 72% and 80% for heavy and light mito- chondria, respectively.

Table 2provides data related to the protein and phospho- rus content of these mitochondria. The protein yield is diffe- rent in the two mitochondrial populations. This is corroborated with variations in the content of thiol group titration gen- erally considered a good method to determine protein con- tent. No difference was observed concerning phosphorus content.

The content of inner membrane phospholipids of mitochon- dria differs between the two mitochondrial fractions with the amount of phosphatidylcholine being significantly greater in heavy mitochondria than in light mitochondria. No differences were observed for phosphatidylinositol, phosphatidylserine and cardiolipin.

3.2. Determination of BDH physicochemical parameters from heavy and light mitochondria

The optimal pH of the BDH activity of the two mitochon- drial populations is identical at pH 8 (not shown). Fig. 1 shows that the optimal temperature for BDH activity is near 35 °C for heavy mitochondria (A) and 40 °C for light mitochondria (B).

The incubation of the heavy and light mitochondria at 37 °C (Fig. 1C) leads to a high stability in light mitochondria as shown by the thermal inactivation kinetic constant ratio KH/ KL of 0.15. This effect has been also observed at other tem- peratures tested, i.e. stored at−20 °C, 4 °C and 28 °C (results not shown). Interestingly, the Arrhenius plots (Fig. 2) show a break near 30 °C only for BDH from heavy mitochondria.

BDH activity was also measured with two other position isomers ofD-β-hydroxybutyrate, i.e.D-α-hydroxybutyrate and

D-γ-hydroxybutyrate at 10 mM final concentration (data not shown). The results reveal that BDH activity is 40% lower for the γ-hydroxybutyrate in heavy mitochondria than the light

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

3 .1 3.2 3.3 3.4 3.5 3.6

1/T (10-3)˚K-1 Log of

specific activity

A

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

3.1 3.2 3.3 3.4 3.5 3.6

1/T (10-3)˚k-1 Log of

specific activity

B

Fig. 2. Arrhenius plots of the liver BDH from heavy (A) and light (B) mito- chondria were obtained by measuring the enzymatic activity using 100μg of liver mitochondrial protein at various temperatures (from 5 to 40 °C). Values are given as means ± S.D. of three separated experiments.

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ones, but for theα-hydroxybutyrate, no significant differences were observed.

Fig. 3reports comparative BDH sensitivity of the heavy and light mitochondria towards amino acid reagents NEM and PGO.

BDH is strongly and rapidly inactivated by both reagents and the inactivation constants (n= stoichiometry, K2= inactivation kinetic constant of second order) depend on the time of incuba- tion and on the reagent concentrations. They are not signifi- cantly different for the two BDH of mitochondrial origin: BDH from the heavy mitochondria is inactivated with NEM,n= 1.09, K2= 40 min1 mM1 and n= 1.38, K2= 25 min1 mM1 for inactivation by PGO. For the BDH from the light mitochondria, n= 1.13, K2= 43.5 min1 mM1 and n= 1.13, K2= 28 min1 mM−1for NEM and PGO, respectively.

BDH kinetic parameters from the heavy and light mitochon- dria (Vmax,KmBOH,KmNAD+ and KDNAD+) are reported in Table 3. Interestingly, the specific activity of the BDH from the heavy mitochondria is 2.77-fold higher than that from the light mitochondria. Moreover, the kinetic constants with respect to the NAD+and BOH are higher in the heavy mitochondria than in the light ones (× 1.8 for theKmNAD+, × 1.4 for theKDNAD+,

× 2.1 for KmBOH). To explain these differences, SDS–PAGE was carried out (Fig. 4A) and the amount of BDH polypeptide of the two mitochondrial populations was estimated both by Western blotting (Fig. 4B) and by ELISA (Fig. 4C). This amount is always higher in the heavy mitochondria than in the light mitochondria. The above results are in accordance with those observed when BDH activity was measured in non-denaturing conditions electrophoresis (Zymogram gel) (Fig. 4D).

4. Discussion

To purify mitochondrial BDH from eukaryotic cells with a high yield, we previously used a mixed mitochondrial popula- tion (heavy and light) (Latruffe and Gaudemer, 1974a; Gaude- mer and Latruffe, 1975; Latruffe et al., 1980; El Kebbaj et al., 1980, 1985; El Kebbaj and Latruffe, 1997). To our knowledge, no study of BDH has been separately performed on each of these distinct populations until now.

1%

10%

100%

0 10 20 30 40

Incubation time (min) BDH

activity (%)

2 µM 1,5 µM 1 µM 0,5 µM [NEM]

A

1%

10%

100%

0 10 20 30 40

Incubation time (min) BDH

activity (%)

[NEM]

B

10%

100%

0 10 20 30 40 0 10 20 30 40

Incubation time (min) BDH

activity (%)

1 µM 1.33 µM 1.66 µM 2 µM [P GO]

C D

10%

100%

Incubation time (min) BDH

activity

(%) [PGO]

2 µM 1,5 µM 1 µM 0,5 µM

1 µM 1.33 µM 1.66 µM 2 µM

Fig. 3. Time course of the liver BDH inactivation from heavy and light mitochondria by various concentrations ofN-ethylmaleimide [NEM] (A,B) or phenyglyoxal [PGO] (C,D) (semilog plot). Liver mitochondrial proteins (100μg) were preincubated for 5 min at 25 °C in a phosphate buffer pH 8. PGO or NEM was added at zero time of incubation. Aliquots were removed at different times for the measurement of enzymatic activity. Aliquots of the control assay were removed at the same time in order to calculate the percentage of residual BDH activity. Values are given as means of three separate experiments.

Table 3

Determination of the kinetic parameters of the liver BDH from heavy and light mitochondria

BDH parameters from

KmNAD+ (mM)

KmBOH (mM)

KDNAD+ (mM)

Vmax(nmol NADH/min/mg of protein) Heavy mitochondria 0.21 ± 0.01 1.60 ± 0.22 1.16 ± 0.50 0.61 ± 0.05 Light mitochondria 0.39⁎± 0.03 3.36⁎± 0.33 1.66 ± 0.33 0.22⁎± 0.01 Experiments were varying NAD concentration (0.5; 1; 1.5 and 2 mM) or BOH concentration (2.5; 5; 7.5 and 10 mM). Values are given as means ± S.D. of three independent experiments.Vmaxvalues have been calculated according to the mitochondrial fraction purities (Table 1).⁎pb0.01 (Student'st-test).

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In this work, we report a comparative study of BDH from heavy and light mitochondria, in terms of physicochemical properties, kinetic parameters and content. The interest of working on jerboa (J. orientalis) is that this animal is a true hibernator (El Hilali and Veillat, 1979) developing obesity by accumulating fat during its prehibernation period. Its fat is eliminated during hibernation leading to a high production of

D-β-hydroxybutyrate to serve as an energy source in addition to carbohydrates (Kante et al., 1990). This study on BDH focuses on jerboa in its active state.

The data reveal interesting differences between BDH from heavy and light liver mitochondria. We found that BDH activity is higher in heavy mitochondria than in light ones (Table 3).

This might be caused either by the amount of BDH, which is different between the mitochondrial populations, or by the fact that the organelle phospholipid composition change induces variation in BDH activity which is a lipid-dependent enzyme.

Indeed, the amount of BDH in heavy mitochondria was also found to be higher than in light ones. This was confirmed by Western blotting, ELISA and Zymogram gel (Fig. 4B–D).

Secondly, the phospholipid composition of heavy and light mitoplasts (Table 2) is significantly different especially concerning phosphatidylcholine, an activator of BDH (El Keb- baj et al., 1985; Loeb-Hennard and McIntyre, 2000).

Thus, the differences of BDH activity between the two populations of mitochondria appear to be due to both the amounts of BDH present and to differences in phospholipid composition.

Previous studies on the interactions of BDH with phospho- lipids (Gazzoti et al., 1964; McIntyre et al., 1978; Berrez et al., 1984; El Kebbaj et al., 1985; Kante et al., 1990; Loeb-Hennard and McIntyre, 2000) have demonstrated that the composition of membrane phospholipids induced structural modifications of BDH. Taking this into account, we have estimated the physi- cochemical and kinetic characteristics of BDH from heavy and light liver mitochondria. BDH of these mitochondria shows an identical optimal pH of 8 (data not shown). Thermal parameters reveal interesting differences: 1) the optimal temperature is about 35 °C for the BDH from the heavy mitochondria while it is 40 °C for the BDH from the light ones (Fig. 1); 2) the BDH from the heavy mitochondria is less thermally stable than that from the light ones; 3) Arrhenius plots (Fig. 2) show a break near 30 °C only for heavy mitochondria BDH. These observa- tions combine to suggest that the two types of mitochondrial BDH are different in terms of conformation, possibly resulting from different insertion. This conformational change is also confirmed by the study of BDH stereospecificity differences towards 3 isomers of D-hydroxybutyrate, i.e. α-, β- and γ- hydroxybutyrate (data not shown).

Comparable effects of two chemical modifying reagents (NEM and PGO) suggest that the two BDH have a similar catalytic site (Fig. 3).

The determination of kinetic parameters indicated that the heavy and the light mitochondrial BDH behave differently (Table 3). Indeed, the affinity of the heavy mitochondria with respect to the NAD+and BOH is stronger than that of the light 29 kDa

20.1 kDa 45 kDa

3 2 1 36 kDa 66 kDa

24 kDa 14.2 kDa

A

BDH (31.5 kDa)

Heavy mitochondria

Light mitochondria

B

0 0,05 0,1 0,15 0,2 0,25 0,3

Heavy mitochondria Light mitochondria Absorbtance

at 410 nm

*

* p<0.05

C

BDH activity

Heavy mitochondria

Light mitochondria

D

Fig. 4. SDSPAGE (A), Western blotting (B), ELISA (C) and zymogram gel (D) of the liver BDH from heavy and light mitochondria of the jerboa. Ap-value below 0.05 was considered significant (Student'st-test). SDSPAGE, Western blotting and zymogram gel were assayed with 50μg of protein and ELISA with 10μg of protein. Lanes 1, 2 and 3 represent markers, heavy and light mitochondrial protein fractions respectively. For experimental conditions, see Materials and methods.

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ones. Beside this, the high content of BDH in heavy mitochon- dria partially explains that the specific activity of BDH is twice as high for the heavy mitochondria as for the light mitochondria (Table 3).

In a recent paper, we have reported on the existence of two BDH isoforms from brain and liver mitochondria from active and cold adapted jerboas (Kabine et al., 2003). Indeed, the results obtained reveal: 1) the presence of two distinct enzymatic forms of BDH in liver and brain tissues. This has been also reported for the hepatic glutamate dehydrogenase in Richard- son's ground squirrel (Thatcher and Storey, 2001); 2) that BDH from liver and from brain is subject to differential regulation depending on the hibernation state. This regulation could be a result of post-translational modifications and/or a modification of mitochondrial membrane state, knowing that the BDH activ- ity is phospholipid-dependent. The local environment of the protein may result in an important kinetic change. A change of physical properties of the mitochondrial membrane related to the hibernation process has also been reported in ground squirrel (Raison and Lyons, 1971). Furthermore, post-translational reg- ulation during hibernation was reported for glyceraldehyde-3- phosphate dehydrogenase in jerboa (J. orientalis) (Soukri et al., 1995, 1996).

In conclusion, our results reveal the possible existence of two different BDH populations in jerboa liver associated to either light or heavy mitochondria: one would be associated in great quantity to the heavy mitochondria and the second in small quantity to the light mitochondria. The cause of these distribu- tions may be a difference in the lipid composition of the inner mitochondrial membrane between these mitochondria. This would have two consequences: 1) differences in the targeting of the BDH precursor might lead to a higher import in heavy mitochondria than in light ones; and 2) a modulation of the phospholipid-dependent BDH activation and BDH membrane insertion. Differences in the thermal stability and thermal be- havior as well as changes in the kinetic parameters are a clear illustration. The cellular and physiological roles of the two liver mitochondrial populations associating two BDH subdistribution are unknown. A further cloning of BDH gene(s) will put for- ward the properties and the role of this enzyme. Indeed, the cloning experiment would allow establishing if there are two mRNA splice variants and/or self-specific post-translational modifications.

Acknowledgments

This work was supported by the Regional Council of Bur- gundy and IFR n° 92, and by the « Programme thématique d'Appui à la Recherche Scientifique-Morocco, n° 3 », and by the « Action intégrée franco-marocaine MA 05/134 ». We thank Dr. Mustapha Cherkaoui Malki for his valuable sugges- tions and M. Domminic Batt for english revision.

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