HYDROLYTIC ENZYMES IN THE CENTRAL VACUOLE OF PLANT-CELLS
HYDROLYTIC ENZYMES IN THE CENTRAL VACUOLE OF PLANT-CELLS
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DOI：
10.1104/pp.63.6.1123
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发表时间：
1979-01-01
期刊：
影响因子：
7.4
通讯作者：
KENDE, H
中科院分区：
文献类型：
--
作者：
BOLLER, T;KENDE, H
本文研究了悬浮培养的烟草细胞[威斯康星州38号烟草]、郁金香花瓣[郁金香属种（Tulipa sp. cv. Paul Richter]和菠萝[Ananas comosus cv.研究了白色卡宴叶片的叶片形态和烟草液泡膜的沉降行为。对液泡水解酶和液泡膜的分析，有三个注意事项是重要的。需要在Ficoll梯度中纯化原生质体，以去除含有从真菌细胞壁降解酶制剂中吸附的污染性水解酶的细胞碎片。比较使用的完整细胞或组织的匀浆和纯化的原生质体的匀浆中的水解酶活性，以验证原生质体制备物中不存在污染的水解酶。在Ficoll梯度中从裂解物中纯化通过渗透压休克从原生质体获得的异戊醇。由于中央液泡的密度接近原生质体的密度，因此不能消除约10%的存活原生质体对液泡制剂的污染，并且在计算酶和放射性分布时必须考虑到这一点。下列酸性水解酶的细胞内活性主要位于烟草细胞的液泡中：甘露糖苷酶，β- N-乙酰氨基葡糖苷酶，β-果糖苷酶、核酸酶、磷酸酶和磷酸二酯酶。在郁金香花瓣原生质体的液泡中也发现了类似的酸性水解酶。蛋白酶，一种水解酶，在烟草细胞和郁金香花瓣中活性低，因此难以明确定位，在菠萝叶中，含有高水平的这种酶的组织是液泡。高等植物细胞的中央液泡具有类似于动物溶酶体的酶组成的假设得到了支持。研究的液泡酶没有一个与液泡膜结合。当空泡从放射性胆碱标记的细胞中分离时，空泡膜含有放射性。在蔗糖梯度上，掺入液泡膜中的标记围绕1.10 g/cm 3的密度（24%蔗糖，wt/wt）成带。
The hydrolase content of vacuoles isolated from protoplasts of suspension-cultured tobacco cells [Nicotiana tabacum cv Wisconsin 38], of tulip petals [Tulipa sp. cv. Paul Richter] and of pineapple [Ananas comosus cv. White Cayenne] leaves and the sedimentation behavior of tobacco tonoplasts, were studied. Three precautions were important for the analysis of vacuolar hydrolases and of the tonoplast. Purification of protoplasts in a Ficoll gradient was necessary to remove cell debris which contained contaminating hydrolases adsorbed from the fungal cell-wall-degrading enzyme preparation. Hydrolase activities in the homogenates of the intact cells or the tissue used and of the purified protoplasts were compared to verify the absence of contaminating hydrolases in the protoplast preparation. Vacuoles obtained from the protoplasts by an osmotic shock were purified from the lysate in a Ficoll gradient. Since the density of the central vacuole approximates that of the protoplasts, about a 10% contamination of the vacuolar preparation by surviving protoplasts could not be eliminated and had to be taken into account when the distribution of enzymes and of radioactivity was calculated. The intracellular activities of the following acid hydrolases were primarily localized in the vacuole of tobacco cells: .alpha.-mannosidase, .beta.-N-acetylglucosaminidase, .beta.-fructosidase, nuclease, phosphatase and phosphodiesterase. A similar composition of acid hydrolases was found in vacuoles obtained from protoplasts of tulip petals. Proteinase, a hydrolase with low activity in tobacco cells and tulip petals and therefore difficult to localize unequivocally, was vacuolar in pineapple leaves, a tissue containing high levels of this enzyme. The hypothesis that the central vacuole of higher plant cells has an enzyme composition analogous to that of the animal lysosome was supported. None of the vacuolar enzymes investigated was bound to the tonoplast. When vacuoles were isolated from cells labeled with radioactive choline, the vacuolar membrane contained radioactivity. On sucrose gradients, the label incorporated into tonoplasts banded aound a density of 1.10 g/cm3 (24% sucrose, wt/wt).