Isolation and Characterization of Vacuoles from Cell Suspension Cultures of Daucus carota

A reliable procedure for the isolation o f vacuoles from anthocyanin-containing cells o f Daucus carota cell suspension cultures has been developed. From cells o f the late linear growth phase, protoplasts were prepared and purified in a sucrose/sorbitol gradient. Vacuoles were liberated from these protoplasts by osmotic shock and purified in a Metrizamide step gradient. The vacuolecontaining fractions were analysed for their anthocyanin content as a measure for the yield o f vac­ uoles. The purity o f vacuoles was examined by assaying various marker enzymes in both proto­ plasts and vacuoles. The purest vacuolar fraction had 8% o f the total activity o f glucose-6-phosphate dehydrogenase (marker for cytosol), 8% o f cytochrome oxidase (mitochondria) and 10% of NADPH-cytochrome c reductase (ER). 55% o f the acid phosphatase activity o f the protoplasts and 35% o f the total malate were recovered in the vacuoles. The vacuolar pool o f amino acids is quite large. Data for 15 amino acids show that 44 to 73% are being located in the vacuole.