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Immunoaffinity purification and characterization of mitochondrial membrane-bound D-3-hydroxybutyrate dehydrogenase from Jaculus orientalis.

Mountassif D, Andreoletti P, El Kebbaj Z, Moutaouakkil A, Cherkaoui-Malki M, Latruffe N, El Kebbaj MS - BMC Biochem. (2008)

Bottom Line: This study applies immunoaffinity chromatography to purify BDH, the membrane-bound and lipid-dependent enzyme, as a 31 kDa single polypeptide chain.In addition, bacterial BDH isolation was achieved in a two-step purification procedure, improving the knowledge of an enzyme involved in the lipid metabolism of a unique hibernating mammal.Sequence alignment revealed conserved putative amino acids for possible NAD+ interaction.

View Article: PubMed Central - HTML - PubMed

Affiliation: INSERM U866 (Institut National de la Santé et de la Recherche Médicale), Université de Bourgogne, LBMC (Biochimie Métabolique et Nutritionnelle), Faculté des Sciences, 6 Bd Gabriel, 21000 Dijon cedex, France. drissmountassif@yahoo.fr

ABSTRACT

Background: The interconversion of two important energy metabolites, 3-hydroxybutyrate and acetoacetate (the major ketone bodies), is catalyzed by D-3-hydroxybutyrate dehydrogenase (BDH1: EC 1.1.1.30), a NAD+-dependent enzyme. The eukaryotic enzyme is bound to the mitochondrial inner membrane and harbors a unique lecithin-dependent activity. Here, we report an advanced purification method of the mammalian BDH applied to the liver enzyme from jerboa (Jaculus orientalis), a hibernating rodent adapted to extreme diet and environmental conditions.

Results: Purifying BDH from jerboa liver overcomes its low specific activity in mitochondria for further biochemical characterization of the enzyme. This new procedure is based on the use of polyclonal antibodies raised against BDH from bacterial Pseudomonas aeruginosa. This study improves the procedure for purification of both soluble microbial and mammalian membrane-bound BDH. Even though the Jaculus orientalis genome has not yet been sequenced, for the first time a D-3-hydroxybutyrate dehydrogenase cDNA from jerboa was cloned and sequenced.

Conclusion: This study applies immunoaffinity chromatography to purify BDH, the membrane-bound and lipid-dependent enzyme, as a 31 kDa single polypeptide chain. In addition, bacterial BDH isolation was achieved in a two-step purification procedure, improving the knowledge of an enzyme involved in the lipid metabolism of a unique hibernating mammal. Sequence alignment revealed conserved putative amino acids for possible NAD+ interaction.

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BDH purification steps from jerboa liver. Proteins (40 μg) were resolved by SDS-PAGE and stained with Coomassie Brilliant Blue (a) or subjected to Western blot (b) using the purified polyclonal anti-BDH antibodies. Lanes M, 1, 2, 3, 4, and 5 represent standard proteins, crude extract, 30–50% ammonium sulphate fraction, phenyl-Sepharose fraction, affinity chromatography fraction, and immunoaffinity chromatography eluate pool (pure protein preparation). Bound antibody was located by immunoreaction combined with peroxidase conjugated goat anti-rabbit IgG. The arrow (b) indicates the band corresponding to the BDH subunit.
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Figure 1: BDH purification steps from jerboa liver. Proteins (40 μg) were resolved by SDS-PAGE and stained with Coomassie Brilliant Blue (a) or subjected to Western blot (b) using the purified polyclonal anti-BDH antibodies. Lanes M, 1, 2, 3, 4, and 5 represent standard proteins, crude extract, 30–50% ammonium sulphate fraction, phenyl-Sepharose fraction, affinity chromatography fraction, and immunoaffinity chromatography eluate pool (pure protein preparation). Bound antibody was located by immunoreaction combined with peroxidase conjugated goat anti-rabbit IgG. The arrow (b) indicates the band corresponding to the BDH subunit.

Mentions: The SDS-PAGE analysis shows that the immunoaffinity step is crucial to eliminate the remaining contaminants of the penultimate fractions. This last purification step shows a single 31 kDa protein (Figure 1), which has been described for other eukaryotic BDH subunits (Figure 1A, lane 5). The 31 kDa jerboa BDH monomer cross-reacts with the purified antibacterial BDH antibodies (Figure 1B).


Immunoaffinity purification and characterization of mitochondrial membrane-bound D-3-hydroxybutyrate dehydrogenase from Jaculus orientalis.

Mountassif D, Andreoletti P, El Kebbaj Z, Moutaouakkil A, Cherkaoui-Malki M, Latruffe N, El Kebbaj MS - BMC Biochem. (2008)

BDH purification steps from jerboa liver. Proteins (40 μg) were resolved by SDS-PAGE and stained with Coomassie Brilliant Blue (a) or subjected to Western blot (b) using the purified polyclonal anti-BDH antibodies. Lanes M, 1, 2, 3, 4, and 5 represent standard proteins, crude extract, 30–50% ammonium sulphate fraction, phenyl-Sepharose fraction, affinity chromatography fraction, and immunoaffinity chromatography eluate pool (pure protein preparation). Bound antibody was located by immunoreaction combined with peroxidase conjugated goat anti-rabbit IgG. The arrow (b) indicates the band corresponding to the BDH subunit.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2572057&req=5

Figure 1: BDH purification steps from jerboa liver. Proteins (40 μg) were resolved by SDS-PAGE and stained with Coomassie Brilliant Blue (a) or subjected to Western blot (b) using the purified polyclonal anti-BDH antibodies. Lanes M, 1, 2, 3, 4, and 5 represent standard proteins, crude extract, 30–50% ammonium sulphate fraction, phenyl-Sepharose fraction, affinity chromatography fraction, and immunoaffinity chromatography eluate pool (pure protein preparation). Bound antibody was located by immunoreaction combined with peroxidase conjugated goat anti-rabbit IgG. The arrow (b) indicates the band corresponding to the BDH subunit.
Mentions: The SDS-PAGE analysis shows that the immunoaffinity step is crucial to eliminate the remaining contaminants of the penultimate fractions. This last purification step shows a single 31 kDa protein (Figure 1), which has been described for other eukaryotic BDH subunits (Figure 1A, lane 5). The 31 kDa jerboa BDH monomer cross-reacts with the purified antibacterial BDH antibodies (Figure 1B).

Bottom Line: This study applies immunoaffinity chromatography to purify BDH, the membrane-bound and lipid-dependent enzyme, as a 31 kDa single polypeptide chain.In addition, bacterial BDH isolation was achieved in a two-step purification procedure, improving the knowledge of an enzyme involved in the lipid metabolism of a unique hibernating mammal.Sequence alignment revealed conserved putative amino acids for possible NAD+ interaction.

View Article: PubMed Central - HTML - PubMed

Affiliation: INSERM U866 (Institut National de la Santé et de la Recherche Médicale), Université de Bourgogne, LBMC (Biochimie Métabolique et Nutritionnelle), Faculté des Sciences, 6 Bd Gabriel, 21000 Dijon cedex, France. drissmountassif@yahoo.fr

ABSTRACT

Background: The interconversion of two important energy metabolites, 3-hydroxybutyrate and acetoacetate (the major ketone bodies), is catalyzed by D-3-hydroxybutyrate dehydrogenase (BDH1: EC 1.1.1.30), a NAD+-dependent enzyme. The eukaryotic enzyme is bound to the mitochondrial inner membrane and harbors a unique lecithin-dependent activity. Here, we report an advanced purification method of the mammalian BDH applied to the liver enzyme from jerboa (Jaculus orientalis), a hibernating rodent adapted to extreme diet and environmental conditions.

Results: Purifying BDH from jerboa liver overcomes its low specific activity in mitochondria for further biochemical characterization of the enzyme. This new procedure is based on the use of polyclonal antibodies raised against BDH from bacterial Pseudomonas aeruginosa. This study improves the procedure for purification of both soluble microbial and mammalian membrane-bound BDH. Even though the Jaculus orientalis genome has not yet been sequenced, for the first time a D-3-hydroxybutyrate dehydrogenase cDNA from jerboa was cloned and sequenced.

Conclusion: This study applies immunoaffinity chromatography to purify BDH, the membrane-bound and lipid-dependent enzyme, as a 31 kDa single polypeptide chain. In addition, bacterial BDH isolation was achieved in a two-step purification procedure, improving the knowledge of an enzyme involved in the lipid metabolism of a unique hibernating mammal. Sequence alignment revealed conserved putative amino acids for possible NAD+ interaction.

Show MeSH