单峰驼肝脏线粒体中D-β-羟丁酸脱氢酶的纯化与特性分析

Purification and characterization of the D-beta-hydroxybutyrate dehydrogenase from dromedary liver mitochondria.

作者信息

Nasser Boubker, El Kebbaj M'hammed Said, Cottin Patrick, Latruffe Norbert

机构信息

Laboratoire de Biochimie de la Faculté des Sciences et Techniques BP, 577, Settat, Morocco.

出版信息

Comp Biochem Physiol B Biochem Mol Biol. 2002 Jan;131(1):9-18. doi: 10.1016/s1096-4959(01)00461-4.

DOI:10.1016/s1096-4959(01)00461-4

PMID:11742753

Abstract

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).

摘要

D-β-羟丁酸脱氢酶（BDH）（EC 1.1.1.30）是一种膜酶，已从单峰骆驼（骆驼属单峰驼）肝脏线粒体中纯化至同质。我们的新纯化方法包括用Triton X 100溶解线粒体膜，并通过两步纯化BDH：DEAE-琼脂糖凝胶和苯基琼脂糖凝胶。该酶亚基大小的分子量为67 kDa。纯化后的酶可被抗大鼠肝脏线粒体BDH抗体识别。此外，BDH活性绝对依赖于磷脂。BDH还具有特定的酶学参数：氧化反应的最适pH约为8，还原反应的最适pH约为7，动力学常数（米氏常数和解离常数）值为：K(MBOH)为1.07±0.13 mM，K(MNAD(+))为0.21±0.01 mM，K(DNAD(+))为1.04±0.20 mM，K(MAcAc)为0.29±0.01 mM，K(MNADH)为0.27±0.03 mM，K(DNADH)为1.12±0.18 mM。