termination of phenolic compounds, sugars, acids, and cations, the skins were removed manually from the grapes, rinsed briefly with distilled H20, frozen in N2, and freeze-dried. To prevent oxidation of phenolic compounds, vacuum was released with N2, and samples were stored in N2 atmosphere., Preparation of Vacuoles. Grape berries (1 kg) were rinsed briefly in 70%o aqueous ethanol and dried on paper towels, and the skins were manually removed by gentle squeezing and cut in half. Batches of about 30 g of skins were rinsed with 100 ml buffer (17 mm citric acid, 70 mim Na2HPO4, and 1.2 M sorbitol at pH 6.0) for 30 min in 1,000-ml beakers on a reciprocal shaker (100 rpm). The buffer was replaced with a Cellulysin (1.5%; Calbiochem) and Macerase (1%; Calbiochem) solution in the same volume as the above buffer. The grape skins were digested for 7 h at room temperature, the incubation medium decanted, and the protoplasts sedimented at 100g in a centrifuge. The sediment was resuspended in the rinsing buffer from above (1,500 ml) and filtered through a glass wool layer in a Buchner funnel under mild suction (aspirator). This treatment produced vacuoles from protoplasts. The vacuoles were sedimented at 100g, resuspended in 225 ml rinsing buffer, and sedimented by gravity twice. The number of vacuoles in a preparation was determined in a well slide using 5 ,Al aliquot and 20 ul. buffer.Disruption of Vacuoles. The vacuole preparations were transferred into 40-ml centrifuge tubes and the vacuoles sedimented at 50g. The supernatant buffer was decanted, the volume made up to 40 ml with distilled H20, and the preparation homogenized by sonication. Microscopic examination of this preparation showed that all vacuoles had lysed. The homogenate was centrifuged at 180,000g for 2 h to remove membranous material. The supernatant (37 ml) was decanted, frozen in N2, and stored at -40 C.Identification of Anthocyanins. The anthocyanins in DeChaunac grapes have been previously identified (28).Determination of Total Anthocyanin Content in Grape Skins.Freeze-dried grape skin powder (1.0 g) was extracted with 0.1% aqueous HCI (100 ml), the absorbance of this solution measured at 520 nm, and the anthocyanin concentration determined using E = 33,000 1-mol-1cm-' (27).Determination of the Total Anthocyanin Content in Vacuole Preparations. One ml of the vacuole lysate was used for the determination, as described above.Quantitative Determination of the Individual Anthocyanins. The individual anthocyanin content of grape skins and vacuole preparations was determined by quantitative adaptation ofa previously described HPLC method (28). Only pigments present in significant amounts were quantitated. For the establishment of anthocyanin standard curves, the following pigment stock solutions were pre-
