A rapid and efficient procedure for the purification of mitochondrial beta-hydroxybutyrate dehydrogenase.
A new, rapid and efficient procedure for the purification of the mitochondrial enzyme beta-hydroxybutyrate dehydrogenase (EC 1.1.1.30) to homogeneity is described. It involves the following steps. The mitochondria are solubilized with potassium cholate and the 100 000 X g supernate is fractionated with ammonium sulfate. This is followed by precipitation of the enzyme at pH 5.2 and then selective solubilization at pH 8.8. This key step removes eighty percent of the contaminating proteins and allows subsequent DEAE-Sepharose and glass bead column chromatography to be performed in the absence of detergents. The overall yield is consistently around 35% and the purified protein is homogeneous on polyacrylamide gel electrophoresis. The purified enzyme is absolutely dependent upon phosphatidylcholine for activity.
D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D006884:Hydroxybutyrate Dehydrogenase / Q000302:isolation & purification*; D008929:Mitochondria, Heart / Q000201:enzymology*; D012995:Solubility
D006884:Hydroxybutyrate Dehydrogenase