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Cytosokic Acetyl CoA Hydrolase and Induction by Clofibrate

胞质乙酰辅酶A水解酶和氯贝特诱导

基本信息

批准号：
03670123
负责人：
ISOHASHI Fumihide
金额：
$ 1.28万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1993
项目状态：
已结题

项目摘要

The hypolipidemic drug ethyl chlorophenoxyisobutyrate (clofibrate) is known to induce peroxisome proliferation and to ve carcinogenic after long term administration to rats and mice. We examined the effects of treatment with this drug for periods of up to 18 months on cytosolic ATP-stimulated and ADP-inhibited acetyl-CoA hydrolase in rat liver. In male donryu albino rats on a diet containing 0.5% clofibrate, the enzyme activity increased to about 2- and 3-fold the initial level permilligram liver protein and cytosolic protein, respectively, and 2-fold per milligram DNA in 3 days, and then remained at this level for up to 18 months. The increased activity in rats receiving clofivrate for 3 months returned to control level within a week when clofibrate was sithdrown. The change in enzyme activity paralleled the change in the amount of enzyme protein determined by immunoblotting with anyibody against purified acetyl-CoA hydrolasa from rat liver cytosol. No liver tumors were detected macro … More scopically after administration of clofibrate for 18 months. However, our results suggest that cytosolic acetyl-CoA hydrelase could be an extraperoxisomal marker enzyme induced by this type of drug.An extramitochondrial acetyl-CoA hydrolase [EC 3.1.2.1] in the rat liver, which is atimulated by ATP and inhibited by ADP, is known to ve extremely cold-labile. During subcellular fractionations at low temperatures (2-4*C), most of the enxyme activity was lost ; however, most could be recovered by rewarming at 37*C in the presence of a high concentration of potassium phosphate. This enabled us to measure the activities of cold-treated samples. The majority of the ATP-stimulated and ADP-imhibited acctyl-Coa hydrolase activity in rat livers was detected in the cytosolic fraction and small amounts were detected in the peroxixomal fraction. The activity of peroxisomal ATP-stimulated acetyl-CoA hydrolasa was not noticeably increased after clofibrate-treatment. However, the cytosolic activity greatly increased after clofibrate treatment. The activity in the isolated peroxisomal fraction per g of liver was about 5% of that in the cytosolic fraction of liver from the control and about 2% in that from clofibrate-treated rats. Vesides having similar nucleotide (ATP and ADP) sensitivity and cold lability, the enzyme protein in the peroxisomal fraction migrated to the same position as the cytosolic acetyl-CoA hydroiase based on Western blot analysis with antibody against purified acetyl-CoA gydrolase from rat liver cytosol. These results suggest that the peroxisomal enzyme and cytosolic enzyme may be the same extity. Less

已知降血脂药物氯苯氧基异丁酸乙酯（氯贝丁酯）会诱导过氧化物酶体增殖，长期给大鼠和小鼠服用后会致癌。我们研究了该药物长达 18 个月的治疗对大鼠肝脏中胞质 ATP 刺激和 ADP 抑制的乙酰辅酶 A 水解酶的影响。在食用含有 0.5% 氯贝特的饮食的雄性 Donryu 白化大鼠中，酶活性在 3 天内分别增加到每毫克肝蛋白和胞质蛋白初始水平的约 2 倍和 3 倍，每毫克 DNA 增加 2 倍，然后保持在该水平长达 18 个月。接受氯贝特治疗 3 个月的大鼠的活动增加在注射氯贝特后一周内恢复到对照水平。酶活性的变化与通过用任何抗体针对来自大鼠肝细胞质的纯化乙酰辅酶A水解酶进行免疫印迹测定的酶蛋白量的变化平行。服用安妥明 18 个月后，宏观上没有检测到肝脏肿瘤。然而，我们的结果表明，胞质乙酰辅酶A水解酶可能是此类药物诱导的过氧化物酶体外标记酶。大鼠肝脏中的线粒体外乙酰辅酶A水解酶[EC 3.1.2.1]受ATP刺激并被ADP抑制，已知其对冷极不稳定。在低温（2-4*C）的亚细胞分级分离过程中，大部分酶活性丧失；然而，大多数可以通过在高浓度磷酸钾存在下在 37°C 下重新加热来恢复。这使我们能够测量冷处理样品的活性。大鼠肝脏中大部分 ATP 刺激和 ADP 抑制的乙酰辅酶阿水解酶活性在胞质部分中检测到，少量在过氧化物酶体部分中检测到。过氧化物酶体 ATP 刺激的乙酰辅酶 A 水解酶的活性在安妥明治疗后没有明显增加。然而，氯贝特处理后细胞质活性大大增加。每克肝脏中分离的过氧化物酶体部分的活性约为对照肝脏胞质部分活性的5%，约为安妥明治疗大鼠肝脏细胞质部分活性的2%。过氧化物酶体部分中的酶蛋白具有相似的核苷酸（ATP 和 ADP）敏感性和冷不稳定性，根据使用来自大鼠肝细胞溶胶的纯化乙酰辅酶 A 水解酶抗体进行的蛋白质印迹分析，过氧化物酶体级分中的酶蛋白迁移到与胞质乙酰辅酶A水解酶相同的位置。这些结果表明过氧化物酶体酶和胞质酶可能具有相同的性质。较少的