Subcellular Localization of Spermidine Synthase in the Protoplasts of Chinese Cabbage Leaves

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Abstract
Previous studies on the presence of spermidine synthase (EC 2.5.1.16) in the protoplasts of Chinese cabbage (B. pekinensis cv. Pak Choy) leaves had detected a small but significant fraction of the enzyme in a crude chloroplast fraction (Cohen, Balint, Sindhu, 1981). To establish whether this enzyme is truly a chloroplast component, purified intact chloroplasts were isolated from protoplasts by density gradient centrifugation in silica sols (Ludox AM). Such chloroplasts contained all of the diaminopimelate decarboxylase (EC 4.1.1.20) of the protoplasts, but were essentially devoid of spermidine synthase. Control experiments showed that the latter had not been inactivated under conditions of isolation, purification and assay of the intact chloroplasts. Isolation and assay of protoplasts vacuoles in a further examination of the supernatant fluid containing the enzyme revealed a significant fraction of the enzyme in the vacuole fraction. However, this fraction contained similar proportions of a soluble enzyme, glucose 6-phosphate dehydrogenase. Vacuolar fractions are difficult to separate from soluble cytoplasmic material, which is probably the only compartment containing spermidine synthase.