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Radioactive Assay AST IV activity in lung and STa activity in lung and liver cytosol were determined using the radioactive assay method as previously described . Two hundred micrograms protein from lung cytosol was used to assay the AST IV activity. Fifty micrograms protein from liver cytosol and 200 ug protein from lung cytosol were used to assay the STa activity. For radioactive 2-naphthol sulfation activity, 2-naphthol (4.7 mCi/mmol; 0.1 mM final concentration) was used as substrate. To determine STa (DHEA sulfation) activity, [3H] DHEA (diluted to 0.4 Ci/mmol; 2 M final concentration) was used as substrate. For all assays, 20 uM PAPS was used. All enzymatic reactions were performed in a total reaction volume of 250 L. After 30-min incubation at 37◦C in a shaking water bath, the reaction was stopped by adding 250 L of 0.25 M Tris, pH 8.7. Extraction was performed twice by addition of 0.5 mL of water-saturated chloroform. After the final extraction, 50 uL of aqueous phase was used for scintillation counting. For the controls, PAPS was omitted in both assays. Assays were run in duplicate and the average of the results was used for enzyme activity calculations. The data are expressed as means±SEM from three rats. |
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zuodg
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sltmac(金币+1): 谢谢交流，欢迎常来~~ 2011-05-25 15:41:32
wenjie(金币+5, 翻译EPI+1): 2011-05-25 21:08:22
sltmac(金币+1): 谢谢交流，欢迎常来~~ 2011-05-25 15:41:32
wenjie(金币+5, 翻译EPI+1): 2011-05-25 21:08:22
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放射性测试 采用前述放射性测试方法测定肺AST IT活性以及肝细胞、肺细胞细胞质STa活性。200 ug肺细胞细胞质蛋白用以测定AST IT活性。50 ug肝细胞细胞质蛋白、200 ug肺细胞细胞质蛋白用以测定STa活性。关于硫酸萘酚活性，使用2-萘酚（放射强度是4.7mCi/mmol; 终浓度0.1mM）作为底物。关于STa（硫酸DHEA）活性, 使用[3H]DHEA（稀释至0.4 Ci/mmol, 终浓度是2M）作为底物。所有的测试都是用20 uM的PAPS。所有的没血反应体系都是250 Ul。酶反应在37℃震荡水浴中温育30min之后，加入250 ul 0.25M PH8.7的Tris。使用0.5 ml水饱和氯仿提取两次。最后一次提取后，取50 ul水相进行闪烁计数。作为对照，两个测试中的PAPS全部忽略不计。测试平行进行两次，结果的平均值用来计算酶活。结果根据3只小鼠的数据表示成“结果±标准差”的形式。 |
2楼2011-05-23 13:33:01
