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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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鐵桿木蟲 (職業(yè)作家)
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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. 將上述勻漿在10000轉(zhuǎn)20分鐘離心后，取上清液用于酶檢測。整個酶提取過程都應(yīng)該在4度環(huán)境下進(jìn)行。 ACC合成酶的活性需要放入裝有0.8mL的反應(yīng)復(fù)合物的試管中進(jìn)行測定，此復(fù)合物含0.25毫摩S -腺苷甲硫氨酸，50毫摩HEPES緩沖液和10毫摩帶有0.2ml酶提取物的磷酸吡哆醛。 |
5樓2011-03-20 22:27:26
Mally89
鐵桿木蟲 (職業(yè)作家)
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昔年殘夢(金幣+10, 翻譯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 根據(jù)1979年Boller 等進(jìn)行的ACC合成酶的提取物（ACS）的活性測定并經(jīng)Lizada和楊（1979）修改過后的準(zhǔn)備方法如下：將1g番茄果皮組織通過一個冷的杵和研缽進(jìn)行研磨，直到將其研磨成勻漿。在研磨過程中加入5毫升的提取緩沖液，其中包含有4毫摩二硫蘇糖醇，10毫摩磷酸吡哆醛，加有1毫摩EDTA的100毫摩、PH為8的HEPES緩沖液。 |
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Mally89
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6樓2011-03-20 22:28:10
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