Hydrolytic Enzymes in the Central Vacuole of Plant Cells

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Abstract
The hydrolase content of vacuoles isolated from protoplasts of suspension-cultured tobacco cells [Nicotiana tabacum cv Wisconsin 38], of tulip petals [Tulipa sp. cv. Paul Richter] and of pineapple [Ananas comosus cv. White Cayenne] leaves and the sedimentation behavior of tobacco tonoplasts, were studied. Three precautions were important for the analysis of vacuolar hydrolases and of the tonoplast. Purification of protoplasts in a Ficoll gradient was necessary to remove cell debris which contained contaminating hydrolases adsorbed from the fungal cell-wall-degrading enzyme preparation. Hydrolase activities in the homogenates of the intact cells or the tissue used and of the purified protoplasts were compared to verify the absence of contaminating hydrolases in the protoplast preparation. Vacuoles obtained from the protoplasts by an osmotic shock were purified from the lysate in a Ficoll gradient. Since the density of the central vacuole approximates that of the protoplasts, about a 10% contamination of the vacuolar preparation by surviving protoplasts could not be eliminated and had to be taken into account when the distribution of enzymes and of radioactivity was calculated. The intracellular activities of the following acid hydrolases were primarily localized in the vacuole of tobacco cells: .alpha.-mannosidase, .beta.-N-acetylglucosaminidase, .beta.-fructosidase, nuclease, phosphatase and phosphodiesterase. A similar composition of acid hydrolases was found in vacuoles obtained from protoplasts of tulip petals. Proteinase, a hydrolase with low activity in tobacco cells and tulip petals and therefore difficult to localize unequivocally, was vacuolar in pineapple leaves, a tissue containing high levels of this enzyme. The hypothesis that the central vacuole of higher plant cells has an enzyme composition analogous to that of the animal lysosome was supported. None of the vacuolar enzymes investigated was bound to the tonoplast. When vacuoles were isolated from cells labeled with radioactive choline, the vacuolar membrane contained radioactivity. On sucrose gradients, the label incorporated into tonoplasts banded aound a density of 1.10 g/cm3 (24% sucrose, wt/wt).