Evidence for a new physiological role of hepatic NADPH:ferricytochrome (P-450) oxidoreductase. Direct electron input to the microsomal fatty acid chain elongation system.
Evidence for a new physiological role of hepatic NADPH:ferricytochrome (P-450) oxidoreductase. Direct electron input to the microsomal fatty acid chain elongation system.
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DOI：
10.1016/s0021-9258(19)68742-9
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发表时间：
1981-10
期刊：
影响因子：
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通讯作者：
Z. Ilan;R. Ilan;D. L. Cinti
中科院分区：
文献类型：
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作者：
Z. Ilan;R. Ilan;D. L. Cinti
材料和方法：微粒体的分离-从雄性Sprague-Dawley大鼠的肝脏中提取微粒体，150-200 g，饥饿24小时，高碳水化合物，无脂肪饮食2-3天，如前所述（37）。用Bio-Rad试剂（Bio-Rad, Richmond， CA）测定蛋白浓度，用牛y-球蛋白（Sigma）作为标准曲线。NADPH细胞色素P-450还原酶-NADPH -450还原酶按照Yasukochi和Masters（19）的程序从大鼠肝微粒体中纯化，并进行了以下修改：(a)用非离子洗涤剂Emulgen 911 （Kao-Atlas Co., Ltd, Tokyo, Japan）代替Renex 690；(6)将甘油（20%）加入25 m Tris缓冲体系中，用于deae -纤维素柱层析（Whatman DE52）的初始纯化步骤；(c)用2 ',5 ' - adp琼脂糖代替ADP-Sepharose， type 2 (PL Biochemicals Inc., Milwaukee， WI)；(d)从DE52柱中收集的NADPH-cvtochrome c还原活性的部分汇集，透析，并在不减少体积的情况下应用于adp -琼脂糖。最终制剂的比活性为22 pmol细胞色素c还原/min/mg蛋白质；用0.05 M磷酸钾缓冲液，pH 7.5，在20℃下测量了该活性，与Yasukochi和Masters（19）报道的结果比较有利。细胞色素bs的纯化-细胞色素b5的纯化可以从上面描述的还原酶纯化的初始DE52步骤中获得的含有细胞色素bs的部分中纯化，或者从AH-Sepharose 4b -睾酮亲和柱中洗脱的含有bs的部分中纯化。制备AH-Sepharose 4b -睾酮柱时，将1 g睾酮和3 g丁二酸酐溶解于20 ml吡啶中。将反应混合物在室温下轻轻搅拌3天，然后倒入100 ml的6 M HC1中。收集形成的白色沉淀物，用1 M盐酸洗涤，再溶解于0.2 M Na3P04中，pH值为7。收集最终沉淀物，用醋酸乙酯：丙酮（1:1）混合物重结晶。将AH-Sepharose 4B （15 g）搅拌到100 d的水：二甲基甲酰胺（1:1）中，然后加入0。3克
MATERIALS AND METHODSIsolation of Microsomes-Microsomes were prepared from the livers of male Sprague-Dawley rats, 150-200 g, following 24 h of starvation and 2-3 days on a high carbohydrate, fat-free diet, as described previously (37). The protein concentration was measured with the Bio-Rad reagent (Bio-Rad, Richmond, CA) using bovine y-globulin (Sigma) for the standard curve. Purification of NADPH Cytochrome P-450 Reductase-NADPHP-450 reductase was purified from rat liver microsomes according to the procedure of Yasukochi and Masters (19) with the following modifications:(a) the non-ionic detergent Emulgen 911 (Kao-Atlas Co., Ltd., Tokyo, Japan) was used instead of Renex 690;(6) glycerol (20%) was included in the 25 m Tris buffer system used for the initial purification step involving DEAE-cellulose column chromatography (Whatman DE52);(c) ADP-Sepharose was replaced by 2’, 5’-ADP-agarose, type 2 (PL Biochemicals Inc., Milwaukee, WI); and (d) fractions collected from the DE52 column and active in NADPH-cvtochrome c reduction were pooled, dialyzed, and applied to the ADP-agarose without reducing the volume. The specific activity of the final preparation was 22 pmol of cytochrome c reduced/min/mg of protein; this activity was measured with 0.05 M potassium phosphate buffer, pH 7.5, at 20 “C and compares favorably with that reported by Yasukochi and Masters (19). Purification of Cytochrome bs-Cytochrome b5 was purified either from the cytochrome bs-containing fractions obtained from the initial DE52 step of the reductase purification described above, or from bs-containing fractions eluted from an AH-Sepharose 4B-testosterone affinity column.For the preparation of the AH-Sepharose 4B-testosterone column, 1 g of testosterone and 3 g of succinic anhydride were dissolved in 20 ml of pyridine. The reaction mixture was gently stirred at room temperature for 3 days and then poured into 100 ml of ice-cold 6 M HC1. The white precipitate formed was collected, washed with 1 M HCL, redissolved in 0.2 M Na3P04, and brought to pH 7. The final precipitate was collected and recrystallized with an ethyl acetate: acetone (1: l) mixture. AH-Sepharose 4B (15 g) was stirred into 100 d of water: dimethylformamide (l: l), followed by addition of0. 3 g of