Publicação
Isolation of vacuoles from the leaves of the medicinal plant catharanthus roseus
| Resumo: | 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. |
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| Autores principais: | Carqueijeiro, Ines |
| Outros Autores: | Noronha, Henrique Luís Silva; Bettencourt, Sara; Guedes, Joana G.; Duarte, Patricia; Gerós, H.; Sottomayor, Mariana |
| Assunto: | Plant vacuoles Vacuole isolation Protoplasts Catharanthus roseus ATPase hydrolytic activity Vacuolar (V)-H+-ATPase Vacuolar (V)-H+-pyrophosphatase Proton pumping activity Ciências Naturais::Ciências Biológicas |
| Ano: | 2018 |
| País: | Portugal |
| Tipo de documento: | capítulo de livro |
| Tipo de acesso: | acesso restrito |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | 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. |
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