Distribution of Sorbitol, Neutral Sugars, Free Amino Acids, Malic Acid and Some Hydrolytic Enzymes in Vacuoles of Apple Cotyledons

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Vacuoles of apple cotyledons (Malus pumila Mill. var. domestica Schneid.) were obtained by purification with Ficoll density gradient centrifugation after the protoplasts were lysed by both osmotic shock and the addition of EDTA. High levels of acid protease and carboxypeptidase activity were detected in the vacuoles along with acid phosphatase, phosphodiesterase and ATPase. The distribution of sorbitol and other sugars in the vacuoles, the protoplast and extracellular free space was determined. About 45, 60 and 90% of the sorbitol, glucose and sucrose, respectively, contained in whole tissue were found in the extracellular free space, and 54% of the sorbitol in the protoplast was detected in the vacuole. The sorbitol in the vacuoles and the extra-vacuole of the protoplast was about 80 and 70%' respectively, of the total sugar content of the cell. Arginine was the most abundant free amino acid in the protoplast, and about 90% of it was contained in the vacuole. More than 80% of the total amino acids and 50% of the protein were also located in the vacuole, as well as most of the malate. The amounts of the total sugars, total amino acids, protein and malate in the vacuoles were to 250, 400, 48 and 9 μg/10^6 cells, respectively. The results suggest that the vacuoles of apple cotyledons contain a large pool of amino acids and proteins rather than sugars, and have a close connection with protein body degradation.
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