Wheeler J B, Shaw D R, Barnes S
Department of Pharmacology and Toxicology, University of Alabama at Birmingham 35294, USA.
Arch Biochem Biophys. 1997 Dec 1;348(1):15-24. doi: 10.1006/abbi.1997.0391.
In the present study, using the C24 bile 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 microM CoA and 2.5 mM MgCl2, kinetic analysis revealed that the apparent K(m)s of ATP and chenodeoxycholic acid of the purified enzyme were 548 +/- 247 and 18.0 +/- 6.2 microM, respectively, and the apparent Vmax was 9.53 +/- 2.0 nmol min-1 mg protein-1. The formation of chenodeoxycholyl-CoA by rBAL was strongly inhibited by hydrophobic bile acids (the C24 monohydroxy bile acid lithocholic acid and 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid, the C27 homolog of cholic acid), but only weakly by cholic acid. Chenodeoxycholyl-CoA and 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-27-oyl-CoA were confirmed as reaction products of purified rBAL by HPLC-electrospray ionization mass spectrometry.
在本研究中，以C24胆汁酸鹅去氧胆酸为底物，通过Q-Sepharose阴离子交换、羟基磷灰石和CM-Sepharose层析相结合的方法，从去污剂增溶的微粒体中纯化出大鼠肝脏胆汁酸CoA连接酶活性（rBAL），纯化倍数达200倍。SDS-PAGE分析显示纯化后的rBAL分子量为65 kDa。对纯化后的rBAL进行凝胶过滤表明，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。针对65-kDa蛋白制备的单克隆抗体与6B-Sepharose共价偶联，可从大鼠肝脏微粒体的半纯化制剂中完全吸收rBAL活性。蛋白质印迹分析证实，在低pH条件下，65-kDa蛋白从亲和相中洗脱。发现rBAL的最佳活性在pH 8.5，且活性依赖于二价阳离子Mg2+。在存在50 μM CoA和2.5 mM MgCl2的情况下，动力学分析表明，纯化酶对ATP和鹅去氧胆酸的表观K(m)值分别为548±247和18.0±6.2 μM，表观Vmax为9.53±2.0 nmol min-1 mg蛋白-1。rBAL催化生成鹅去氧胆酰-CoA的过程受到疏水胆汁酸（C24单羟基胆汁酸石胆酸和3α,7α,12α-三羟基-5β-胆甾烷酸，即胆酸的C27同系物）的强烈抑制，但仅受到胆酸的微弱抑制。通过HPLC-电喷雾电离质谱法确认鹅去氧胆酰-CoA和3α,7α,12α-三羟基-5β-胆甾-27-酰-CoA为纯化后rBAL的反应产物。
