Intracellular localisation of some peptidases and α-mannosidase in cotyledons of resting kidney bean, Phaseolus vulgaris

Cotyledons of resting kidney beans ( Phaseolus vulgaris , L., cv. "Processor") contain high activities of two alkaline peptidases, an aminopeptidase (EC 3.4.11) acting on Leu-Tyr and Leu-Gly-Gly and a dipeptidase (EC 3.4.13) hydrolysing Ala-Gly together with low activities of neutral naphthyiamidases (marker substrate Leu-β-NA) and of acid carboxypeptidases (EC 3.4.16; marker substrate Z-Phe-Ala). The intracellular localisation of these peptidases and that of α-mannosidase (EC 3.2.1.24) was studied by subcellular fractionations in different media. In density gradient centrifugations in non-aqueous glycerol-potassium iodide media the alkaline peptidases remained mainly in the application zone suggesting localisation in the cytosol. The carboxypeptidase and α-mannosidase activities banded mainly in the protein body zone, but about 15–30% of each activity was found in the cell wall zone. Results obtained by short centrifugation in glycerol or high-density sucrose solutions (65/70%) and by the isolation of essentially pure cell wall fractions confirmed these assignments. The results are in accordance with previous suggestions that the abundant alkaline peptidases may play a role in the mobilization of reserve proteins in germinated seeds by hydrolysing peptides which are produced initially within the protein bodies by acid proteinases and carboxypeptidases and which subsequently leak out or are transported from the autolyzing protein bodies to the cytosol.