Abstract
BackgroundThe 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.ResultsPurifying 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.ConclusionThis 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.
Highlights
The interconversion of two important energy metabolites, 3-hydroxybutyrate and acetoacetate, is catalyzed by D-3-hydroxybutyrate dehydrogenase (BDH1: EC 1.1.1.30), a NAD+-dependent enzyme
The NAD+-dependent D-3-hydroxybutyrate dehydrogenase (BDH: EC 1.1.1.30), which has been studied by our group for several years [1,2,3,4,5,6,7,8,9], plays a key role in redox balance and energy metabolism since it reversibly converts 3hydroxybutyrate into acetoacetate
BDH is a mitochondrial inner membrane-bound enzyme [1,10,11] and its active site is located on the matrix side [2,12]
Summary
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 NAD+-dependent D-3-hydroxybutyrate dehydrogenase (BDH: EC 1.1.1.30), which has been studied by our group for several years [1,2,3,4,5,6,7,8,9], plays a key role in redox balance and energy metabolism since it reversibly converts 3hydroxybutyrate into acetoacetate (the two major ketone bodies largely produced under high lipolysis, diabetes, or fasting). A catalytic mechanism involving cystenyl and histidyl residues of the BDH active site for the interconversion of D-3hydroxybutyrate and acetoacetate in both liver and peripheral tissues has been previously proposed by our group [7]
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