LOCALIZATION OF RNA-DEGRADING ENZYME-ACTIVITY WITHIN VACUOLES OF CULTURED TOMATO CELLS
LOCALIZATION OF RNA-DEGRADING ENZYME-ACTIVITY WITHIN VACUOLES OF CULTURED TOMATO CELLS
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DOI：
10.1111/j.1399-3054.1986.tb01237.x
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发表时间：
1986-01-01
影响因子：
6.4
通讯作者：
GLUND, K
中科院分区：
文献类型：
--
作者：
ABEL, S;GLUND, K
从番茄（Lycopersicon esculentum Mill. CV. Lukullus）悬浮培养并纯化以消除用于细胞壁消化的酶混合物中存在的高活性外源RNA酶。纯化的原生质体用于确定内源RNA酶活性的位置（在pH 5下用酵母RNA作为底物测量）。通过在含有EDTA的碱性缓冲液中振荡纯化的原生质体来释放异戊醇。通过（i）RNA酶以不连续梯度与液泡共纯化和（ii）RNA酶和α-RNA酶的共迁移，明确显示RNA酶位于液泡内。甘露糖苷酶（EC 3.2.1.24），一种液泡标记物，在液泡的重复离心期间。该酶对EDTA、Mg ~（2+）、Mn ~（2+）和Ca ~（2+）不敏感，但对柠檬酸盐和磷酸二氢钾有激活作用。它在pH 5-6范围内表现出最适pH。凝胶电泳分析显示，一个单一的条带的核糖核酸酶的分离液泡。这种活性与来自在温和条件下提取的细胞的RNA酶共迁移。因此，有可能区分空泡RNA酶和细胞外来源的RNA酶。RNA降解活性存在于整个培养物生长的液泡中。液泡中的活性在指数生长期间逐渐增加，随后在稳定期急剧增加。
Protoplasts were prepared from cells of a tomato (Lycopersicon esculentum Mill. cv. Lukullus) suspension culture and purified to eliminate the highly active exogenous RNase present in the enzyme mixture used for cell wall digestion. The purified protoplasts were used to determine the location of the endogenous RNase activity (measured at pH 5 with yeast RNA as the substrate). Vacuoles were released by shaking the purified protoplasts in alkaline buffer containing EDTA. RNAse was unambiguously shown to be located within the vacuoles by (i) its co-purification with the vacuoles in a discontinuous gradient and by (ii) the co-migration of RNase and .alpha.-mannosidase (EC 3.2.1.24), a vacuolar marker, during repeated centritugation of the vacuoles. Vacuolar RNase was insenstive to EDTA, Mg2+, Mn2+ and Ca2+ but was stimulated by citrate or KH2PO4. It exhibited a pH-optimum in the range of pH 5-6. Gel electrophoretic analysis revealed one single band for RNase of isolated vacuoles. This activity co-migrated with an RNase from cells extracted under mild conditions. Thus, it was possible to distinguish the vacuolar RNase from the RNase of extracellular origin. RNA-degrading activity was present in vacuoles throughout the growth of the culture. The activity in vacuoles gradually increased during exponential growth followed by a dramatic increase in the stationary phase.