Characterization of a soybean β-conglycinin-degrading protease cleavage site

Abstract

Protease C1, an enzyme from soybean (Glycine max [L] Merrill cv Amsoy 71) seedling cotyledons, was previously determined to be the enzyme responsible for the initial degradation of the α′ and α subunits, but not the 0 subunit, of β-conglycinin storage protein. The sizes of the proteolytic products generated by the action of protease C1 suggest that the cleavage sites on the α′ and α subunits of β-conglycinin may be located in their N-terminal domain, which is not found in the β subunit of β-conglycinin. To check this hypothesis, storage proteins from other plant species that are homologous to either the α′/α or the β subunit of β-conglycinin were tested as substrates. As expected, the convicilin from pea (Pisum sativum), a protein homologous to the α′ and α subunits of β-conglycinin, was digested by protease C1. The vicilins from pea as well as vicilins from adzuki bean ( Vigna angularis), garden bean (Phaseolus vulgaris), black-eyed pea (Vigna unguiculata), and mung bean (Vigna radiata), storage proteins that are homologous to the β subunit of soybean β-conglycinin, were not degraded by protease C1. Degradation of soybean β-conglycinin involves a sequential attack of the α subunit at multiple sites, culminating in the formation of a stable intermediate of 53.5 kD and a final product of 48.0 kD. The cleavage sites resulting in this formation of the intermediates and final product were determined by N-terminal analysis. These were compared to the known amino acid sequences of the three β-conglycinin subunits. Results showed these two polypeptides to be generated by proteolysis of the α subunit at regions bearing long strings of acidic amino acid residues.