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| Extracts for ACC synthase (ACS) activity assays were prepared according to the method of Boller et al. (1979), and determined by the method of Lizada and Yang (1979) with slight modifications. One g tomato pericarp tissue was homogenized using a cold pestle and mortar in 5mL of extraction buffer which consisting of 4 mM dithiothreitol, 10mM pyridoxal phosphate and 1.0mM EDTA in 100mM pH 8.0 Hepes buffer. After centrifugation of the homogenate at 10,000×g for 20min, the supernatant was used for enzyme assays. All steps in the enzyme extraction were performed at 4◦C. ACC synthase activity was assayed in a 0.8mL reaction mixture containing 0.25mM S-adenosylmethionine, 50mM Hepes buffer and 10mM pyridoxal phosphate with 0.2mL of enzyme extract in test tubes. After incubation of the reaction mixture at 30 ◦C for 1 h, the tubes were sealed with a stopper and rubber septum, 0.1mL HgCl2 (80mM) and 0.1mL 5% NaOH–NaClO solution (1:2, v/v) were injected into the tubes, shaken and incubated for 2min. One mL of the gaseous portion was removed and assayed for ethylene as described above. ACS activity was expressed as nmol g−1 FWh−1. |
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After centrifugation of the homogenate at 10,000×g for 20min, the supernatant was used for enzyme assays. All steps in the enzyme extraction were performed at 4◦C. ACC synthase activity was assayed in a 0.8mL reaction mixture containing 0.25mM S-adenosylmethionine, 50mM Hepes buffer and 10mM pyridoxal phosphate with 0.2mL of enzyme extract in test tubes. 将上述匀浆在10000转20分钟离心后，取上清液用于酶检测。整个酶提取过程都应该在4度环境下进行。 ACC合成酶的活性需要放入装有0.8mL的反应复合物的试管中进行测定，此复合物含0.25毫摩S -腺苷甲硫氨酸，50毫摩HEPES缓冲液和10毫摩带有0.2ml酶提取物的磷酸吡哆醛。 |
5楼2011-03-20 22:27:26
昔年残梦(金币+10, 翻译EPI+1): 2011-03-20 22:44:44
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Extracts for ACC synthase (ACS) activity assays were prepared according to the method of Boller et al. (1979), and determined by the method of Lizada and Yang (1979) with slight modifications. One g tomato pericarp tissue was homogenized using a cold pestle and mortar in 5mL of extraction buffer which consisting of 4 mM dithiothreitol, 10mM pyridoxal phosphate and 1.0mM EDTA in 100mM pH 8.0 Hepes buffer 根据1979年Boller 等进行的ACC合成酶的提取物（ACS）的活性测定并经Lizada和杨（1979）修改过后的准备方法如下：将1g番茄果皮组织通过一个冷的杵和研钵进行研磨，直到将其研磨成匀浆。在研磨过程中加入5毫升的提取缓冲液，其中包含有4毫摩二硫苏糖醇，10毫摩磷酸吡哆醛，加有1毫摩EDTA的100毫摩、PH为8的HEPES缓冲液。 |
2楼2011-03-20 22:20:14
3楼2011-03-20 22:20:33
6楼2011-03-20 22:28:10