Purification and properties of an endopeptidase of the dormant lupin seed

Abstract

An endopeptidase present in the albumin fraction of the dormant lupin seed has been purified over 3000-fold. The preparation was virtually homogeneous on gel filtration, ion exchange chromatography, non-denaturing PAGE and SDS-PAGE. The enzyme consists of one polypeptide chain of M r ca 70 000. It acts on legumin-like proteins of lupin and pea seeds and causes limited proteolysis of their acidic polypeptide (s). The activity becomes apparent after a 24-36 hr incubation at 37°. The enzyme cleaves the α III carboxymethylated subunit of pea legumin stepwise to fragments part of which are below the detection limit of SDS-PAGE. Activity on this substrate and on benzoyl- d,l-arginine- p-nitroanilide is manifest straightaway. The enzyme has no effect on other substituted aminoacids, including Leu- p-nitroanilide and Ala- p-nitroanilide, on synthetic peptides or on aminoacid esters. Its optimal pH is 7.4 with the protein substrate and 8.4 with the synthetic substrate. The enzyme is inhibited by the sulphydryl reagents, leupeptin and salmin.