液泡分离及V-PPase酶活和H+转运检测(1).doc

Isolation of Tonoplast Membranes in Rice 10g 14-day seedlings and 5-DAF seeds were homogenized in a total of 100 ml of grinding medium containing 0.25 M sorbitol (山梨醇) + 5 mM EGTA+1.5% polyvinylpyrrolidone (PVP,聚乙烯吡咯烷酮), 1% ascorbic acid (Vc), and 50 mM Mops/KOH (or Tris-acetate)+用前加入1 mM phenylmethylsulfonyl fluoride (PMSF，苯甲基磺酰氟)和DTT 1 mM (常用还原剂，有抗氧化作用，它能保护酶分子上的还原性基团，维持还原性环境，稳定酶的活性) buffer, pH 7.6，按材料?匀浆缓冲液=1 ? 3 (w/v) 加入预冷匀浆缓冲液. The homogenate was filtered through four layers of cheesecloth and centrifuged at 3,600 g for 10 min. The 100ml supernatant was centrifuged at 120,000 g for 20 min in a Beckman Type 70 Ti rotor. The precipitate was suspended in 3 ml of 10 mM potassium phosphate (K3PO4, pH 7.8), 10% sucrose, 1 mM EGTA and 2 mM DTT, and carefully layered on the 10% (w/w), 25% (w/w) and 40% (用上述3中该悬浮液配置) of continuous sucrose density gradient. 蔗糖梯度制备方法：在26.8ml超速离心机中先加5ml 10％蔗糖，再吸取5ml 25%蔗糖伸到底部缓慢加入，最后一滴不加，防止起泡产生，再同样方法加10ml 40%的蔗糖，并在4℃冰箱放置过夜（12小时后）变成连续梯度即可用； After centrifugation at 120,000g for 40 min, the interface portion (5–8ml) between the 15% and 25% sucrose solutions was collected and diluted in two-fold volumes of ice-cold buffer containing 5 mM Mops/KOH (pH 7.3), 0.25 M sorbitol, 1 mM EGTA, and 2 mM DTT. The suspension was then centrifuged at 130,000 g for 30 min, and the resulting pellet (tonoplast vesicles) was suspended in 2ml of 20 mM Tris acetate (Tris-Ac, pH 7.5), 20% glycerol(甘油), 1 mM DTT, 1 mM EGTA and 2 mM MgCl Enzyme Purification：To the tonoplast suspension were added solid KCl and deoxycholate (脱氧胆酸) or TritonX-100 at final concentrations of 50 mM and 0.6%（w/v）or 0.2%, respectively, to remove peripheral proteins, and the suspension was centrifuged at 150,000 g for 30 min. The pellet was suspended in Tris/GDEM buffer containing 0.4% lysophosphatidylcholine (溶血卵磷脂、溶血磷脂胆碱, egg yolk蛋黄, type I,使酶可溶解) and 0.1% TritonX-100 to bring the volume to 0.5 of the original tonoplast suspension. The suspension was centrifuged at 150,000 g for 40 min at