Carqueijeiro Inês, Noronha Henrique, Bettencourt Sara, Guedes Joana G, Duarte Patrícia, Gerós Hernâni, Sottomayor Mariana
EA2106 Biomolécules et Biotechnologies Végétales, Département de Biologie et Physiologie Végétales, UFR Pharmacologie, Université François-Rabelais de Tours, Tours, France.
Centro de Investigação e de Tecnologias Agro-Ambientais e Biológicas, Vila Real, Portugal.
Methods Mol Biol. 2018;1789:81-99. doi: 10.1007/978-1-4939-7856-4_7.
The isolation of vacuoles is an essential step to unravel the important and complex functions of this organelle in plant physiology. Here, we describe a method for the isolation of vacuoles from Catharanthus roseus leaves involving a simple procedure for the isolation of protoplasts, and the application of a controlled osmotic/thermal shock to the naked cells, leading to the release of intact vacuoles, which are subsequently purified by density gradient centrifugation. The purity of the isolated intact vacuoles is assayed by microscopy, western blotting, and measurement of vacuolar (V)-H-ATPase hydrolytic activity. Finally, membrane functionality and integrity is evaluated by measuring the generation of a transtonoplast pH gradient by the V-H-ATPase and the V-H-pyrophosphatase, also producing further information on vacuole purity.
液泡的分离是揭示该细胞器在植物生理学中重要而复杂功能的关键步骤。在此，我们描述了一种从长春花叶片中分离液泡的方法，该方法包括一个简单的原生质体分离程序，以及对裸露细胞施加可控的渗透/热激处理，从而导致完整液泡的释放，随后通过密度梯度离心对其进行纯化。通过显微镜检查、蛋白质免疫印迹以及液泡（V）-H-ATP酶水解活性的测定来分析所分离完整液泡的纯度。最后，通过测量V-H-ATP酶和V-H-焦磷酸酶产生的跨液泡膜pH梯度来评估膜的功能和完整性，这也能提供有关液泡纯度的更多信息。
