Preparation of a homogeneous soluble D-beta-hydroxybutyrate apodehydrogenase from mitochondria.
D-beta-Hydroxybutyrate dehydrogenase of bovine heart mitochondria has been purified to apparent homogeneity. The membrane-bound enzyme is first released by phospholipase A digestion of the mitochondria. Lithium bromide, 0.4 M, is used to aid release, and dithiothreitol is required to stabilize the enzyme. The membranous material is removed by centrifugation, and the apoenzyme is recovered in the supernatant and precipitated with ammonium sulfate to 50 percent of saturation. The main purification (100-fold) is achieved by selective adsorption and elution on controlled pore glass beads. The purified enzyme has been purified approximately 250-fold from the mitochondria. The purified enzyme is homogeneous as shown by poly-acrylamide gel electrophoresis in sodium dodecyl sulfate or acid-urea systems; a sharp band is obtained which is equivalent to a subunit molecular weight of 31,500. The apoenzyme is devoid of lipid and is completely inactive as isolated. It can be reactivated by adding aqueous microdispersions of lecithin or phospholipids containing lecithin. The apoenzyme is stable, i.e. it has a half-life of about 450 hours at 0-2 degrees in 0.4 M lithium bromide, containing 5 mM dithiothreitol at pH 7, and is soluble at these conditions, existing mainly as a monomer and dimer in dilute solution. It has a tendency to associate into larger aggregates when the salt concentration is lowered. The enzyme does not have a distinctive amino acid composition as compared with other proteins or soluble dehydrogenases. The purified apodehydrogenase is well suited for study of specific protein-lipid interaction, as well as the molecular basis for the role of phospholipid in this lipid-requiring enzyme.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001051:Apoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D001059:Apoproteins / Q000302:isolation & purification*; D002417:Cattle; D002845:Chromatography; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005898:Glass; D006884:Hydroxybutyrate Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism; D008566:Membranes / Q000201:enzymology; D008931:Mitochondria, Muscle / Q000201:enzymology*; D009206:Myocardium; D014462:Ultrafiltration
D000596:Amino Acids; D001051:Apoenzymes; D001059:Apoproteins; D006884:Hydroxybutyrate Dehydrogenase