Vacuoles isolation

Walker RR, Leigh RA. Mg2+-dependent, cation-stimulated inorganic pyrophosphatase associated with vacuoles isolated from storage roots of red beet (Beta vulgaris L.). Planta 1981 133 150-155. 

Darwen, C.W.E. and John, P., Localization of the enzymes of fructan metabolism in vacuoles isolated by a mechanical method from tubers of Jerusalem artichoke Helianthus tuberosus L.), Plant Physiol., 89, 58-663, 1989. 

Frehner, M., Keller, F., and Wiemken, A., Localisation of fructan metabolism in the vacuoles isolated from protoplasts of Jerusalem artichoke tubers Helianthus tuberosus L.), J. Plant Physiol., 116, 197-208, 1984. 

By separating vacuoles isolated from mesophyll and epidermal tissue of Sorghum leaves, Kojima and co-workers (1979) showed that the cyanogenic glucoside, dhurrin, is preferentially localized in the epidermal layer. The epidermal localization of leucine-derived cyanogenic glucosides was also reported for barley leaves (Pourmohseni et al, 1993). 

Burris,R.H. Organic acids in plant metabolism. Ann. Rev. Plant Physiol. 4, 91-114 (1953) Buser,Ch., Matile,Ph. Malic acid in vacuoles isolated from Bryophyllum leaf cells. Z. Pflanzenphysiol. 82,462-A66 (1977)... 

Fig. 5. Renografin density gradient profile of dopamine. Vacuole isolation was performed as described in Fig. 4 with pectin-ase from Aspergillus niger in the cell digestion medium. Gradient fractions were analyzed for dopamine content as described in Fig. 1...

In higher plants, the lipolytic enzymes and their physiological functions are not well characterized [1]. iMost reports demonstrated that phospholipid catabolism in plants is achieved by the concerted actions of membrane-bound enzymes including phospholipase D, phosphatidate phosphatase, lipolytic acyl hydrolases and lipoxygenases [1,2]. With the exception of the phospholipase D, the literature on plant phospholipases is still very limited. We previously reported that tonoplast from Acer pseudoplatanus cells contains small amounts of phosphatidic acid and lysophospholipids, which were produced together with free fatty acids, particularly after addition of Ca " [31. These data suggested the possible involvement of phospholipase D and phospholipase A in the metabolism of vacuolar membrane lipids. The phospholipase activities were studied by following the hydrolysis of added sn-2-[14c]linoleyl-PC to tonoplast vesicles. Tonoplast was obtained by osmotic lysis of pure preparations of vacuoles isolated from protoplasts derived from Acer pseudoplatanus cells [4]. This present work demonstrated clearly the presence of phospholipase D and phospholipase Ai activities associated with the tonoplast of Acer, The phospholipase Ai showed an optimal activity at pH about 6-6.5, did not necessarily require divalent cations, but was stimulated by Mg- and particularly by Ca. This work presents the first evidence for the presence of phospholipases A in plant cells. 

Similar problems are apparent when using isolated cellular structures such as vacuoles. The use of vacuoles in furthering our understanding of the role of compartmentation of osmotica has to be tempered with the frequent... 

Leigh, R.A. (1983). Methods, progress and potential for use of isolated vacuoles in studies of solute transport in higher plant cells. Physiologic Plantarum, 57,390-6. 

Wagner, G.J. (1983). Higher plant vacuoles and tonoplasts. In Isolation of Membrane and Organelles from Plant Cells, ed. J.L. Hall and A.L. Moore, pp. 83-118. London Academic Press. 

Membrane Transport in Organelles Metabolite levels in specific cells and subcellular compartments of plant leaves, 174, 518 isolation of plant vacuoles and measurement of transport, 174, 552 transport in fungal cells kinetic studies of transport in yeast,... 

Salem, S., D. Linstedt, and]. Reinert. The cytokinins of cultured carrot cells. Protoplasma 1979 101 526-534. Sasse, F., D. Backs-Husemann, and W. Barz. Isolation and characterization of vacuoles from cell suspension cultures of Daucus carota. Z Naturforsch 1979 Ser C 34 103-109. 

There are few studies on vacuolar importation of flavonoids other than anthocyanins and PAs. Klein et al. reported uptake of flavone glycosides by isolated H. vulgare primary leaf vacuoles via a vacuolar H -ATPase linked mechanism,and by vacuoles from Secale cereale (rye) mesophyll via a possible ABC transporter mechanism. Li et al. "" found medicarpin conjugated to glutathione was also sequestered by an ABC transporter mechanism. 

Nozzolillo, C. and Ishikura, N., An investigation of the intracellular site of anthocyanoplasts using isolated protoplasts and vacuoles. Plant Cell Rep., 7(6), 389, 1988. 

Returning once again to the questions of function and uses, the old concept of flavonoids being merely the by-products of cellular metabolism, which are simply compartmentalized in solution in the cell vacuole, is well and truly past its use-by date. For a start, studies have revealed that flavonoids are also commonly found on the outer surfaces of leaves and flowers, albeit only the aglycone form. Additionally, flavonoids have been shown over the past few years to be found in the cell wall, the cytoplasm, in oil bodies, and associated with the nucleus and cell proteins, as well as in the vacuole. Even in the vacuole, flavonoids are not necessarily found free in solution. For example, protein-bound flavonoids have been isolated from lisianthus and other flowers in which a structurally specific binding has been identified (in anthocyanic vacuolar inclusions). It is probable that flavonoid location and specific protein binding properties will ultimately prove to relate directly to their function in plants. 

Both proteins have been shown to be translated vitro In a reticulocyte lysate system as preinhibitors, 2000-3000 daltons larger than those synthesized and accumulated vivo (11). The preinhibitors may be Important In the compartmentall-zatlon of the Inhibitors as they are stored In the central vacuole, or plant lysosome, of the plant cells (12). We have now studied the time course of the Increase In translatable mRNA In leaves of wounded plants utilizing poly(A) mRNA Isolated at various times following wounding. 

Inhalation exposure of male B6C3Fi mice to dichloromethane (6 h, once) led to vacuolation of bronchiolar cells at exposure levels > 2000 ppm [6940 mg/m ], while no effect was obsened at levels < 1000 ppm [3470 mg/m (Foster et al., 1994). Pretreatment with the cytochrome P450 inhibitor piperonyl butoxide (300 mg/kg intraperi-toneally) 1 h before the exposure practically abolished the toxic effect upon bronchiolar cells, while buthionine sulfoximine (1 g/kg intraperitoneally), which decreased the pulmonary glutathione content by 50%, had no such protective effect. In Clara cells isolated after exposure to dichloromethane exposure (> 1000 ppm), the proportion of cells in tlie S-phase was increased. 

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