- Microorganisms and growth conditions
Bacteria Pseudomonas aeruginosa (Pasteur Institute, Casablanca, Morocco) were grown aerobically at 37°C without exceeding the exponential phase in nutrient broth (Topley House, Bury, UK). The exponential phase was determined spectrophotometrically at 600 nm. The culture was inoculated with 1% (v/v) overnight preculture in the same medium.
- Buffers
Buffer A: 50 mM potassium phosphate buffer (pH 7.5) containing 2 mM EDTA and 1 mM DTT.
Buffer B: buffer A containing ammonium sulfate at 50% saturation.
- Crude extract preparation
Bacterial culture (5 l) was harvested by centrifugation at 2500 g for 10 min, washed three times with 50 mM potassium phosphate buffer (pH 7.5), and suspended in the same buffer containing 2 mM EDTA and 1 mM DTT (buffer A). Cells were disrupted at 4°C by sonication (30 s, 90% output, 12×) using a Bandelin Sonopuls sonifier. Cellular debris and unbroken cells were removed by centrifugation at 2500 g for 45 min at 4°C. The supernatant obtained constituted the crude bacterial extract (soluble protein fraction).
- BDH purification from the bacterium Pseudomonas aeruginosa
The enzyme was purified from the crude bacterial extract in two steps: ammonium sulfate fractionation and Blue Sepharose CL-6B chromatography. All steps were performed at 4°C.
Ammonium sulfate fractionation
The crude extract of P. aeruginosa was subjected to protein precipitation in the 27–42% saturation range of ammonium sulfate at 4°C. The final pellet was dissolved in a minimal volume of buffer A. The protein solution was dialyzed overnight against 5 l of the same buffer.
Blue Sepharose CL-6B chromatography
The dialyzed enzyme preparation was applied to a Blue Sepharose CL-6B column equilibrated with two bed volumes of buffer A at 4°C. The column was washed with three bed volumes of buffer A. Finally, the enzyme was eluted with buffer A containing 0.1 mM NAD+ at a flow rate of 6 ml/h. Active fractions were collected and conserved in 50% (v/v) glycerol at -20°C until use.
- Production and purification of the anti-BDH antibodies against soluble BDH from Pseudomonas aeruginosa
A 1.5-kg New Zealand white rabbit, grown in the university's animal care facilities, was injected with 1 mg of the BDH purified from P. aeruginosa in aqueous solution (v/v) with incomplete Freund's adjuvant. After 21 days, a second dose of 800 μg of BDH was injected. After the 4th week, a third dose of 500 μg was again injected. One week later, the rabbit was anesthetized and 50 ml of blood were collected. The serum was separated after an overnight coagulation at 4°C and subsequent centrifugation.
Ammonium sulfate precipitation
The resulting serum, containing monospecific anti-BDH polyclonal antibodies, was brought to 40% saturation with solid ammonium sulfate ((NH4)2SO4), stirred for 1 h, and then centrifuged at 2500 g for 45 min. Afterwards, the pellet was dissolved in a minimal volume of phosphate buffer saline (PBS), pH 7.4, containing 137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, and 4.3 mM K2HPO4. The antibody solution was dialyzed overnight against 5 l of the same buffer.
Ion-exchange chromatography
The dialyzed antibody preparation was applied at a flow rate of 6 ml/h to a DEAE-cellulose (Serva, Heidelberg, Germany) column (3 × 12 cm) that had been equilibrated with PBS. The column was extensively washed at the same flow rate with equilibrating buffer solution. Two-milliliter fractions were collected and those containing the anti-BDH antibodies were pooled. Since anti-BDH antibodies are iso-ionic at pH 7.4, they were not retained by the DEAE-cellulose and were generally left with the column's dead volume.
Immunoaffinity chromatography preparation
Sequence alignments from different species, including P. aeruginosa, human, rat, and mouse, revealed that BDHs share an amino acid identity between regions (LVNNAGI, VNI, PG). This property had prompted us to use the antibodies against bacterial BDH to purify the eukaryotic antibody.
We verified the specificity of anti-BDH antibodies by showing that BDH activities were completely inhibited in both P. aeruginosa and jerboa liver using immune serum, which did not inhibit BDH activity in jerboa GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (data not shown). Moreover, preimmune serum had no effect. On the other hand, anti-BDH antibodies reacted with BDHs in western blotting (data not shown).
Immunoaffinity chromatography column (1 × 10 cm) was prepared with CN-Br Sepharose (Pharmacia) coupled with purified BDH from P. aeruginosa according the supplier procedure. After loading total polyclonal antibodies, the specific anti-BDH antibodies were eluted. and subsequently bound to CN-Br Sepharose in order to purify the BDH from jerboa liver with the same procedure as described above.
Purification of mitochondrial membrane-bound BDH from jerboa liver
Jerboa housing: adult greater Egyptian jerboas (Jaculus orientalis, Rodentia, Dipodidae) (120–150 g, 4–6 months old) were captured in the area of Engil Aït Lahcen (in the subdesert eastern Morocco highland). They were adapted to laboratory conditions for 3 weeks at a temperature of 22°C with a diet of lettuce and rat chow and water ad libitum before killing. The light cycle during the experiment was set to 14 h of light and 10 h of darkness. Animal studies were conducted in accordance with the ethical recommendations on Animal Use and Care of the University Hassan II Casablanca.
Remark. We abandoned to purify liver BDH from hibernation Jerboa since hibernation is a complex and very difficult phenomenon to experimentally control and reproduce in a laboratory [9, 21]. The rate of success is only 20% survival in contrast with active Jerboa housing.
Liver mitochondria and mitoplast isolation
The jerboas were decapitated and the livers (75 g total) were rapidly removed for mitochondria purification according to the technique described by Fleischer et al. [41] and as previously used by Mountassif et al. [42]. This method can be used to prepare high-yield mitochondria. The mitoplasts (outer membrane-free mitochondria) were prepared according to Kielley et al. [43]. Briefly, liver mitochondria were swelled in a 20-mM phosphate buffer at 0.5 ml/mg of protein for 30 min at 0°C. The mitoplasts were pelleted by centrifugation at 2500 g for 30 min.
Membrane solubilization and BDH release
The mitoplast fraction was dissolved in an equivalent volume of buffer A containing 0.2% Triton X-100 and then sonicated. The solubilization was complete after 1 h incubation on ice. The mixture was then centrifuged at 2500 g for 1 h and the supernatant containing the solubilized enzyme was collected.
Ammonium sulfate fractionation
The supernatant was subjected to protein precipitation in the 30–50% saturation range of ammonium sulfate. The final pellet was dissolved in a minimal volume of the buffer A containing ammonium sulfate at 50% saturation.
Phenyl Sepharose chromatography
The ammonium sulfate fraction was applied at the low flow rate (12 ml/h) to a phenyl Sepharose HP (Pharmacia Biotech) column (1.6 × 18 cm) pre-equilibrated with buffer B (buffer A containing ammonium sulfate at 50% saturation). After flow-thorough washing, the column was subjected to a decreasing linear gradient of ammonium sulfate (from 50% to 0%) in buffer A. The 5-ml fractions of the activity peak were pooled and dialyzed for 2 h against buffer A after addition of Triton X-100 to the 0.02% final concentration.
Blue Sepharose CL-6B chromatography
The dialyzed enzyme preparation was applied to a Blue Sepharose CL-6B column equilibrated with two bed volumes of buffer A. The column was washed with three bed volumes of buffer A. Finally, the enzyme was eluted with buffer A containing 10 mM NAD+ at a flow rate of 6 ml/h. Active fractions were collected and pooled.
Immunoaffinity chromatography
For preparation (see the section titled "Production and purification of the anti-BDH antibodies against soluble BDH from Pseudomonas aeruginosa" above), BDH from jerboa liver was eluted by 5 M MgCl2, pH 7. Active fractions were selected by measuring the BDH activity level, collected and dialyzed at 4°C for 2 h against 5 l of buffer A containing 5 mM MgCl2 and 50% glycerol.
Phospholipid extraction and preparation of liposomes
Phospholipids were extracted from mitoplasts of jerboa liver according to Rouser and Fleischer [44]. One volume of mitoplast preparation 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 7500 rpm for 3 min. After sedimentation, the organic phase was recovered and methanol/0.8% KCL/chloroform (48/47/3; v/v/v) was added. The chloroform phase was then concentrated in a rotary evaporator. The phospholipids were dissolved and sonicated in buffer A. The solution obtained was left to decant and the supernatant, which contains small liposomes, was stored at -20°C until use [45]. The amount of phospholipids was determined by measuring the phosphorus concentration according to Chen et al. [46]. Before use, the liposome preparation was quickly sonified.
Protein assay
The protein content was measured according to the Bradford procedure, using bovine serum albumin (BSA) as standard [47].
BDH reactivation
Purified BDH (10 μg) was incubated in the buffer containing 6 mM potassium phosphate, pH 8, 0.5 mM EDTA, 0.3 mM dithiothreitol in the presence of 0.2 μg mitochondrial phospholipid (estimated by lipid phosphorus determination). The mixture was incubated for 10 min at room temperature and enzymatic activity was then measured.
BDH activity determination
As described by El Kebbaj and Latruffe [7], BDH activity was measured at 37°C by monitoring NADH production at 340 nm (ε = 6.22 × 103 M-1cm-1) using 100 μg of protein homogenate (or 10 μg of purified enzyme) in a medium containing 6 mM potassium phosphate, pH 8, 0.5 mM EDTA, 0.3 mM dithiothreitol, in the presence of 2 mM NAD+ (Sigma-Aldrich). The reaction was started by adding DL-3-hydroxybutyrate (Sigma-Aldrich) to the 10-mM final concentration.
- Characterization of jerboa membrane-bound BDH
- Denaturing polyacrylamide gel electrophoresis
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by Laemmli [48] on one-dimensional 12% polyacrylamide slab gels containing 0.1% SDS.
- Western blotting
After SDS-PAGE (12%) and subsequent transfer in nitrocellulose [49], the proteins (30 μg) were exposed to 1/100 dilution of monospecific polyclonal anti-BDH antibody and detected with the secondary antibody (anti-rabbit, IgG peroxidase conjugate) (Promega) diluted to 1/2500.
- BDH enzymatic properties
Initial velocities were measured at varying BOH concentrations of (2.5–10 mM) or NAD+ (0.5–2 mM). Michaelis constants (KM), dissociation constants (KD), and maximal velocity of the forward reaction were obtained by mathematical analysis following Cleland [50].
- Determination of optimal pH and temperature-dependent BDH activity
The effect of pH on BDH activity was studied in a range from pH 4 to 10 using a mixture of different buffers (Tris, Mes, Hepes, potassium phosphate, and sodium acetate).
The temperature effects were characterized by activation and denaturation processes. For activation, the buffered medium containing 6 mM potassium phosphate, pH 8, and 0.5 mM EDTA was incubated for 2 min at temperatures from 5 to 80°C. Then, 2 mM of NAD+ and 10 μg of purified BDH were added. The reaction was started immediately by the addition of 10 mM of BOH. For denaturation, 10 μg of purified BDH were incubated at temperatures from 5 to 80°C for 2 min in medium containing 6 mM potassium phosphate, pH 8, and 0.5 mM EDTA. Then 2 mM of NAD+ were added and the enzymatic activity was measured by the later addition of 10 mM of BOH after 2 min of incubation at 37°C.
A BDH Arrhenius plot was obtained by measuring the enzymatic activity at temperatures from 5 to 40°C and interpreted as described by Raison [51].
- RNA isolation and RT-PCR
Total RNAs were obtained from jerboa liver previously frozen in liquid nitrogen and stored at -80°C using Trizol reagent according to the supplier's protocol (Invitrogen).
The primers used were obtained from the alignment between consensus sequences of BDH from human, rat, and mouse.
First-strand cDNA was produced by reverse transcription (RT) using 200 units of Moloney Murine Leukemia Virus Transcriptase (Promega) in conjunction with 2 μg total RNA and the reverse primer; 5'-CCACCAGTAGTAGTCCATG-3' (corresponding to the LPGKALS amino acid sequence starting at amino-acid no. 13 in mouse and human BDH and at no. 14 in rat BDH) in a reaction mixture containing 50 mM Tris-HCl buffer, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, and 0.2 mM of each deoxynucleoside triphosphate for 1 h at 42°C. An aliquot from this template (1/10 of the reaction) was used in a subsequent polymerase chain reaction (PCR) using 1.25 U of GoTaq DNA polymerase (Promega), 0.04 μM of reverse and forward primer (5'-CTCCCAGGAAAA(A/G)C(C/T)CTAAGTG-3') (corresponding to the PMDYYWW amino acid sequence starting at amino acid no. 223 in mouse and human BDH and at no. 224 in rat BDH). PCR was performed for 35 cycles in the following conditions; 92°C for 30 s, 55°C for 30 s, and 72°C for 1 min 30 s.
- Cloning and sequencing of the BDH clone from J. orientalis
The PCR product was purified using QIAEX II Kit (Qiagen) and subcloned into the pGEM-T vector system (Promega), and the nucleotide sequence was determined on both strands using universal primers T7 and SP6 (MWG Biotech, Germany).
The sequence obtained and other sequences were compared using the BioEdit program [36] and ClustalW [52].