Preparation of a homogeneous soluble D-beta-hydroxybutyrate apodehydrogenase from mitochondria.
Preparation of a homogeneous soluble D-beta-hydroxybutyrate apodehydrogenase from mitochondria.
复制标题
从线粒体制备均质可溶性 D-β-羟基丁酸脱氢酶。
DOI：
10.1016/s0021-9258(19)41121-6
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发表时间：
1975
期刊：
影响因子：
--
通讯作者：
S. Fleischer
中科院分区：
文献类型：
--
作者：
H. Bock;S. Fleischer
牛心脏线粒体D-β-羟丁酸脱氢酶已纯化到明显的均一状态。膜结合的酶首先通过磷脂酶A消化线粒体而释放。0.4M的溴化锂用于辅助释放，需要二硫苏糖醇来稳定酶。用离心法除去膜物质，在上清液中回收酶，并用硫酸铵沉淀至饱和50%。主要的纯化(100倍)是通过在可控孔玻璃微珠上的选择性吸附和洗脱实现的。纯化的酶已从线粒体中纯化了约250倍。经十二烷基硫酸钠或酸-尿素体系的聚丙烯酰胺凝胶电泳法测定，纯化后的酶均一条带，其亚基相对分子质量为31,500。脱辅酶不含脂类，分离后完全失活。它可以通过加入卵磷脂或含有卵磷脂的微分散体水溶液来重新激活。酶是稳定的，即在0.4M溴化锂中0-2度，含5 mM二硫苏糖醇，在0-2℃下半衰期约为450小时，在此条件下可溶，在稀溶液中主要以单体和二聚体形式存在。当盐浓度降低时，它有形成更大聚集体的趋势。与其他蛋白质或可溶性脱氢酶相比，该酶没有独特的氨基酸组成。纯化的脱氢酶非常适合于研究特定的蛋白质-脂肪相互作用，以及磷脂在这种需要脂肪的酶中作用的分子基础。
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.