Purification and characterization of a rat liver bile acid coenzyme A ligase from rat liver microsomes.
Purification and characterization of a rat liver bile acid coenzyme A ligase from rat liver microsomes.
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从大鼠肝微粒体中纯化和表征大鼠肝胆汁酸辅酶 A 连接酶。
DOI：
10.1006/abbi.1997.0391
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Barnes,S
中科院分区：
文献类型：
--
作者：
Wheeler,JB;Shaw,DR;Barnes,S
在本研究中，使用C24胆汁酸鹅去氧胆酸作为底物，大鼠肝胆汁酸CoA连接酶活性（rBAL）纯化200倍，从洗涤剂溶解的微粒体使用Q-Sepharose阴离子交换，羟基磷灰石，和CM-Sepharose色谱的组合。经SDS-PAGE分析，rBAL的分子量约为65 kDa。纯化的rBAL的凝胶过滤表明，rBAL活性与其他蛋白质形成复合物，其表观聚集分子量为243 kDa。单克隆抗体提出了对65 kDa的蛋白质和共价偶联到6 B-琼脂糖完全吸收rBAL活性从大鼠肝微粒体的半纯化制剂。蛋白质印迹分析证实了洗脱的65-kDa的蛋白质从亲和相在低pH值。最佳rBAL活性被发现在pH 8.5，和活性依赖于二价阳离子Mg 2+。在50 μM CoA和2.5 mM MgCl 2存在下，动力学分析表明，纯化酶的ATP和鹅去氧胆酸的表观Km分别为548 ± 247和18.0 ± 6.2 μM，表观Vmax为9.53 ± 2.0 nmol min-1 mg protein-1。疏水性胆汁酸（C24单羟基胆汁酸石胆酸和3α，7 α，12 α-三羟基-5 β-胆甾烷酸，胆酸的C27同系物）强烈抑制rBAL形成鹅脱氧胆酰辅酶A，而胆酸仅弱抑制rBAL形成鹅脱氧胆酰辅酶A。经高效液相色谱-电喷雾电离质谱（HPLC-ESI-MS）鉴定，rBAL的反应产物为鹅去氧胆酰辅酶A和3α，7 α，12 α-三羟基-5 β-胆甾烷-27-酰辅酶A。
In the present study, using the C24bile acid chenodeoxycholic acid as substrate, rat liver bile acid CoA ligase activity (rBAL) was purified 200-fold from detergent-solubilized microsomes using a combination of Q-Sepharose anion exchange, hydroxyapatite, and CM-Sepharose chromatography. Purified rBAL had a molecular weight of 65 kDa by SDS–PAGE analysis. Gel filtration of purified rBAL indicated that rBAL activity forms a complex with other proteins with an apparent aggregate molecular weight of 243 kDa. A monoclonal antibody raised against the 65-kDa protein and covalently coupled to 6B-Sepharose completely absorbed rBAL activity from a semipurified preparation of rat liver microsomes. Western blot analysis confirmed the elution of the 65-kDa protein from the affinity phase at low pH. Optimum rBAL activity was found at pH 8.5, and activity was dependent on the divalent cation Mg2+. In the presence of 50 μM CoA and 2.5 mM MgCl2, kinetic analysis revealed that the apparentKms of ATP and chenodeoxycholic acid of the purified enzyme were 548 ± 247 and 18.0 ± 6.2 μM, respectively, and the apparentVmaxwas 9.53 ± 2.0 nmol min−1mg protein−1. The formation of chenodeoxycholyl-CoA by rBAL was strongly inhibited by hydrophobic bile acids (the C24monohydroxy bile acid lithocholic acid and 3α,7α,12α-trihydroxy-5β-cholestanoic acid, the C27homolog of cholic acid), but only weakly by cholic acid. Chenodeoxycholyl-CoA and 3α,7α,12α-trihydroxy-5β-cholestan-27-oyl-CoA were confirmed as reaction products of purified rBAL by HPLC-electrospray ionization mass spectrometry.