Purification and characterization of the D-beta-hydroxybutyrate dehydrogenase from dromedary liver mitochondria.
D-beta-hydroxybutyrate dehydrogenase (BDH) (EC 1.1.1.30), a membrane enzyme, has been purified to homogeneity from dromedary (Camelus dromedarius) liver mitochondria. Our new purification method consisted of the solubilization of mitochondrial membranes by Triton X 100 and purification of BDH by two steps: DEAE-Sephacel and Phenyl-Sepharose. The molecular mass of the enzyme subunit size was 67 kDa. The purified enzyme is recognized by anti rat liver mitochondrial BDH antibodies. Furthermore, BDH activity was absolutely dependent upon phospholipids. BDH is also characterized by specific enzymatic parameters: an optimum pH of approximately 8 for the oxidation reaction, and approximately 7 for the reduction reaction and kinetic constant (Michaelis and dissociation constants) values of 1.07+/-0.13 mM for K(MBOH), 0.21+/-0.01 mM for K(MNAD(+)), 1.04+/-0.20 mM for K(DNAD(+)), 0.29+/-0.01 mM for K(MAcAc), 0.27+/-0.03 mM K(MNADH) and 1.12+/-0.18 mM for K(DNADH).
Nasser
Boubker
B
Laboratoire de Biochimie de la Faculté des Sciences et Techniques BP, 577, Settat, Morocco.
El Kebbaj
M'hammed Said
MS
Cottin
Patrick
P
Latruffe
Norbert
N
D000818:Animals; D015153:Blotting, Western; D002162:Camelus; D002852:Chromatography, Ion Exchange; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D006884:Hydroxybutyrate Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D007700:Kinetics; D008930:Mitochondria, Liver / Q000737:chemistry / Q000201:enzymology*; D010743:Phospholipids / Q000378:metabolism; D013696:Temperature
D010743:Phospholipids; D006884:Hydroxybutyrate Dehydrogenase