Occurrence and properties of proteolytic enzymes in germinating legume seeds

Abstract

Storage proteins in legume seeds are almost totally located in the protein bodies of the cotyledonary cells. The proteins are hydrolyzed during germination into free amino acids which are transported to the growing seedling for use in protein synthesis. This hydrolysis is catalyzed by proteolytic enzymes, both proteinases and peptidases. Cotyledons of resting kidney bean (Phaseolus vulgaris L.) contained low activities of neutral aminopeptidases (marker substrate Leu-β-NA) and acid carboxypeptidases (marker substrate Z-Phe-Ala) but high activities of two alkaline peptidases: an aminopeptidase liberating most rapidly Leu and Ala residues from N-termini of di- and oligopeptides optimally near pH 9 and a dipeptidase hydrolysing Ala-Gly with a pH optimum at 8.5. Both of these alkaline peptidases were separated from neutral aminopeptidases by ion exchange and gel permeation hromatography. The purified aminopeptidase had a large molecular weight, 360 000 which distinguishes it from all the other peptidases known in legume seeds. It apparently belongs to the universal group of enzymes called leucine aminopeptidases. Subcellular fractionation studies carried out by ultracentrifugation in aqueous and non-aqueous media showed that the alkaline peptidases of resting beans were located in the cytosol. Most of the carboxypeptidase activity was associated with protein bodies (70%) and a minor but significant proportion (30%) with the cell wall fraction. The activities of naphthylamidase and alkaline peptidase decreased during germination, whereas the carboxypeptidase and endoproteinase activities increased, reaching their maximal values when the mobilization of nitrogen was highest. However, during the period of rapid mobilization of nitrogen, the alkaline peptidase and carboxypeptidase activities were at approximately the same level, indicating that the alkaline peptidases may also have an important role in the mobilization of reserve proteins. These results suggest that the alkaline peptidases may complete the hydrolysis of oligopeptides which are produced initially within the protein bodies by acid proteinases and carboxypeptidases and passed to the cytosol. The free amino acids formed are then transported to the growing seedling.